Giardia secretome highlights secreted tenascins as a key component of pathogenesis

Giardia secretome highlights secreted tenascins as a key component of pathogenesis Background: Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood. Results: The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. Conclusions: We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators. Keywords: Giardia; secretion; proteomics; quantitative proteomics; tenascin; cysteine protease; enteric pathogen Received: 12 June 2017; Revised: 23 October 2017; Accepted: 17 January 2018 The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Giardia secretome of the protective mucous afforded by the action of a secreted Background nuclease and GCATB, and damage to cellular junctions by With some 280 million symptomatic cases, giardiasis causes GCATB. Tenascins act by means of epidermal growth factor more bouts of human illness than any other parasitic disease (EGF) receptor ligation, to prevent repair to those damaged [1]. The mechanism and mediators of pathogenesis by Giardia, junctions. however, remain largely unknown. Thanks to human volunteer studies, the association of Giardia infection itself, and the sig- nificance of the virulence of the infecting Giardia strain, is ex- Data Description perimentally unambiguous [2]. The molecular definition asso- Soluble and cytosolic fractions from in vitro grown assemblage ciated with strain virulence, though, is largely unexplored. It is A and B trophozoites, the aetiologic agents of human giardiasis, clear that the majority of Giardia infections are asymptomatic. were extracted in order to establish which proteins are secreted It is also clear that infection is primarily localized to the duode- in the steady state by healthy, growing trophozoite populations. num and that some localized damage, close to the sites of col- We reasoned that secreted proteins would be overrepresented in onization, causes villus atrophy and apoptosis of surrounding the medium in which parasites were incubated compared with cells. However, this localized damage cannot be the sole cause the trophozoites that produced them. This ostensibly straight- of the profound diarrhoea that is often characteristic of the dis- forward assessment relied on the sensitive, specific, and quanti- ease and that appears to affect absorption over a much wider tative detection of the proteins expressed by Giardia trophozoites area of the digestive tract than the site of infection alone. in whole cells and in the medium in which the trophozoites were One of the secreted mediators of damage to the duodenum incubated. is believed to be cathepsin B protease [3]. Cathepsin B-like pro- The WB (assemblage A: ATCC 50803) and GS (assemblage B: teases compose one of the superfamilies belonging to the CA ATCC 50581) reference strains were utilized to facilitate ease clan of cysteine peptidases [4]. Compared with other cathepsins, of comparison between genetically divergent human infective cathepsin B proteases possess an additional 20 amino acid inser- isolates with the available reference genomes. For each ex- tions, named the occluding loop, that enable their function as an periment, trophozoites were harvested from mid–log growth endo- or exopeptidase [5]. Although 27 genes encoding cathep- and incubated in nonsupplemented Dulbecco’s Modified Eagle sin proteases have been identified in Giardia, for the majority medium (DMEM) for 45 minutes at 37 C before supernatants and of these proteases, function remains elusive [6]. While some pellets were collected for proteomic and other analyses includ- parasites may secrete cathepsin B proteases to either evade or ing validation of their viability by flow cytometry (Additional modulate their host’s immune responses [7], a recent study has file 1: Fig. S1). Proteomic analyses were based on samples from demonstrated that Giardia trophozoites secrete cathepsin B–like 3 distinct biological replicates. Each sample was analysed us- proteases, degrading intestinal IL-8 and thereby reducing the in- ing 2 quantitative proteomic platforms, the Orbitrap MS and the flammation reaction by the host [ 3]. Secreted Giardia cathepsin B Q-Exactive MS. Thus, in total, the results from 24 (2 × 2 × 2 × 3) protease (GCATB) may also contribute to degradation of intesti- proteomic analyses are reported. nal mucin and facilitate trophozoite attachment to intestinal ep- The identification of abundant, secreted, Giardia virulence ithelia [8, 9]. factors led us to consider whether the secretions from Gia- Most of the proteomic studies so far reported for rdia alone could affect changes in the behaviour of enteric Giardia were undertaken in trophozoites undergoing encystation epithelia—even in the absence of the trophozoites themselves. [10–12]. Only a few studies have focused on proteins secreted In order to determine the effect of Giardia trophozoite–secreted by Giardia and their role in the host-pathogen interaction [3, factors on the intestinal epithelia, chopstick-type electrodes 13–15]. These studies were focused on parasite interaction connected to a voltmeter were used to measure the trans- with intestinal cell lines. No studies have yet attempted to epithelial electrical resistance (TEER) of polarized CaCo-2 epithe- quantify proteins that are the product of steady state secretion lial cells grown on permeable supports. CaCo-2 cells were cul- by healthy, growing Giardia trophozoites, which we hypothesize tured over 6 days until confluent. TEER across the developing as the primary mediators of giardiasis pathology. In this study, CaCo-2 monolayer was measured on a daily basis, as shown in we have identified, to the limit of existing technology, the Fig. 2A. Once confluence was established, Giardia trophozoites proteins expressed by populations of healthy, growing human were added to the apical side of the confluent epithelium, and infective Giardia trophozoites. We have provided quantitation after 24 hours’ incubation, the trophozoites were washed from of the relative abundance of retained and released trophozoite the apical surface. In order to determine whether co-cultures of proteins from 2 human infective assemblages, affording cal- Giardia trophozoites or diluted Giardia supernatants affected the culation of the specific enrichment of released proteins and ion channels responsible for secretory movement across the ep- thereby the description of which proteins are most likely to be ithelium, an Ussing chamber system was utilized with different secreted by trophozoites of each assemblage. Thereafter, we chloride secretion inhibitors and activators. compared the profile of enrichment between the 2 assemblages Further details about sample collection, secretome analysis, in order to identify conserved as well as assemblage-specific and electrophysiology can be found in the Methods section and secreted proteins. We provide electrophysiological analysis that protocols provided. confirms that trophozoite-secreted molecules adversely affect the homeostasis of enteric epithelia, and our analysis of the heterogeneity of encoding genes between lineages demon- Analyses strates the direct selective pressure on these virulence factors Protein expression in Giardia trophozoites and affords their use in discriminating clinically important strains and outbreaks. Finally, the discovery of tenascins as a To describe definitive Giardia secretomes under a standard set highly represented and variable group of proteins secreted by of conditions with high confidence and based on a robust trophozoites strongly implicates this new class of virulence dataset and to reduce the potential for technical artefact, the factors in a novel model for the mechanism of Giardia patho- 2 MS techniques, Q-Exactive and Orbitrap MS, were used with genesis. We propose that tenascin action follows degradation similar settings on the same 3 independent replicates to Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 3 Table 1: The proteins which comprise the Giardia secretome ranked by abundance. Protein abundance A:B Protein GI number GI number iden- Signal Pellet Supernatant Abundance description assemblage A assemblage B tity dN/dS peptide (P) iBAQ (SP) iBAQ SP/P ratio ranking PNPO GL50803 5810 GL50581 4133 99.2 0.038 NP 5.71E+07 1.18E+08 2.063091 1 Tenascin GL50803 95162 GL50581 1982 76.2 1.597 0.99 2.97E+07 4.77E+07 1.607024 2 Tenascin GL50803 10330 GL50581 4057 73.5 0.347 0.99 4.66E+06 2.02E+07 4.342293 3 Cathepsin B GL50803 16468 GL50581 438 83.6 0.1072 0.78 9.63E+06 1.79E+07 1.861309 4 Tenascin GL50803 8687 GL50581 4316 77.6 44.176 0.98 6.22E+06 1.10E+07 1.770526 5 Uncharacterized GL50803 5258 GL50581 2767 91.2 0.029 NP 3.93E+06 1.09E+07 2.780918 6 Extracellular GL50803 8742 GL50581 3607 83.1 0.234 1 1.03E+06 4.57E+06 4.436193 7 nuclease Tenascin-37 GL50803 16477 GL50581 3575 79.8 0.1256 0.99 8.35E+05 4.03E+06 4.830956 8 Cathepsin B GL50803 15564 GL50581 2036 79.1 1 1.10E+06 3.95E+06 3.589608 9 26.5782 CKS1 GL50803 2661 GL50581 3484 100 0.001 NP 1.14E+06 3.20E+06 2.803062 10 Tenascin GL50803 113038 GL50581 4180 79 0.0949 1 1.20E+06 3.14E+06 2.620931 11 HCMP Group 1 GL50803 7715 GL50581 727 67 0.1821 0.99 ND 2.54E+06 ∞ 12 Uncharacterized GL50803 16522 GL50581 352 76 0.1591 NP 1.15E+06 2.21E+06 1.928833 13 HCMP GL50803 12063 GL50581 2622 83 0.246 1 3.62E+05 1.94E+06 5.354665 14 Cathepsin B GL50803 17516 GL50581 2318 72.8 0.2056 1 ND 7.81E+05 ∞ 15 The secretome of human infective Giardia trophozoites of assemblage A and B have a conserved repertoire of abundant secreted factors identified by both Orbitrap MS and Q-Exactive MS. Fifteen proteins were identified as most likely to be secreted by both GS and WB isolates. Thirteen are annotated proteins, and 2 are h ypothetical proteins. Proteins are ranked according to GS Q-Exactive supernatant (SP) protein abundance, from most to least abundant. Of the 12 annotated proteins, 5 are tenascins and 3 are related high-cysteine membrane proteins or VSP, and 3 are cathepsin Bs. The other annotated abundant secreted proteins are an extracellular nuclease and PNPO. Protein ranking represents the proteins’ rank within this table, from most to least abundant. Detailed breakdowns of the secretome for each assemblage by each method are provided in Supplemental Tables S1–S4. dN/dS in bold indicate that protein shows evidence of positive selective pressure during divergence from a common ancestor. Probability of N-terminal signal peptide using SignalP. Not predicted. Not detected. increase coverage. Only proteins identified by both techniques and 24 in the supernatant only, with 1127 WB proteins in pellet within the 3 replicate datasets were included in the analysis to only. increase the robustness of the data. The protein quantification was performed using a label-free method: intensity-based abso- Giardia secretome lute quantification (iBAQ), which calculates the sum of parent ion intensities of identified peptides per protein [ 16]. The aver- To evaluate supernatant enrichment, proteins identified in the age normalized abundance was divided by the iBAQ values, giv- supernatant (SP) datasets were gathered and compared with ing the “Abundance-iBAQ.” The quantitative datasets from both their concentration in the pellet (P) to provide a ratio using the SP abundance−iB AQ MS techniques and for each independent replicate were shown following formula: . These proteins were then Pabundance−iB AQ to be strongly correlated by a Spearman correlation test (data ranked from highest to lowest by ratiometric value, and an ar- not shown) and therefore exploitable for proteomic analysis. bitrary cutoff was invoked such that the top 50 were considered The Q-Exactive MS identified almost all of the proteins iden- the most likely to be secreted. Proteins identified only in SP were tified by use of the Orbitrap MS, and in total the 2 techniques also included in the analysis as most likely to be secreted. All the identified 1587 GS proteins and 1690 WB proteins (Additional File proteins selected as “of interest” were ranked according to their 1: Fig. S2). This represents more than a quarter of the open read- SP expression from most to least abundant to obtain a quantita- ing frames predicted by the respective genomes in this single tive enrichment profile for each isolate, and this was performed life cycle stage under this steady state set of in vitro culture con- for each platform. Orbitrap and Q-Exactive enrichment pro- ditions and compares favourably with other recent proteomic files were compared, and proteins were considered most likely analyses of Giardia [17, 18]. Lists of proteins detected in only 1 to be enriched in the supernatant when identified as such by of the 2 assemblages are provided (Additional file 2: Tables S1 Q-Exactive MS and confirmed by Orbitrap MS. The different en- and S2). Protein from 2 of the 8 predicted assemblage-specific richment profiles were then also compared between assem- genes previously identified by comparative genomics was de- blages. tected [19]. The results yielded a set of 15 orthologous proteins that Overall, both assemblages gave comparable and consistent were identified in both isolates by both techniques (Table 1). results using both platforms, with the sensitivity of detection Eleven of these were predicted to possess an N-terminal sig- being greater for Q-Exactive MS, which provided a range of de- nal sequence. Just 2 of these were of unknown function, and tection spanning 5 logs. In total, Q-Exactive MS identified 1542 2 groups dominated the annotated genes encoding the rest of GS proteins and 1641 WB proteins (Fig. S3). Of these, 946 GS pro- these proteins; 5 were annotated as tenascins and 3 as cathepsin teins were present in both pellet and supernatant, 27 in the su- B cysteine proteases. The most abundant enriched protein was pernatant only, and 569 GS proteins in pellet only. By compari- found to be pyridoxamine 5’-phosphate oxidase (PNPO), a flavin son, 490 WB proteins were identified in supernatant and pellet mononucleotide–dependent enzyme capable of fixing molecular Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Giardia secretome oxygen that lacks a signal peptide and which was also recently termine whether Giardia-secreted virulence factors could induce identified as a secreted Giardia trophozoite protein upregulated changes in the behaviour of the intestinal epithelium, short- during interaction with epithelial cells [15]. An extracellular nu- circuit current (Isc) was continuously measured across polar- clease was also present, along with a high-cysteine membrane ized CaCo-2 epithelial cells that had either been cultured with- protein, as well as a protein product of a gene misannotated as out any additions, co-cultured with Giardia trophozoites, or co- a variant surface protein (VSP; as it was well conserved between cultured with diluted (1:1000) Giardia supernatants (Fig. 2B). Fur- assemblages). ther experiments demonstrated that either after 24-hour co- We considered that where proteins were shown to be en- culture with Giardia (Fig. 2C) or 24-hour co-culture with diluted riched in the supernatant by both platforms and in both assem- Giardia supernatants (Fig. 2D), both experimental conditions dra- blages and possessed an N-terminal signal sequence, they were matically inhibit both the cAMP-stimulated Isc (basolateral ap- truly secreted proteins. Secreted proteins involved in adapting plication of 10 μM of forskolin) and the calcium-activated Isc Giardia to the host environment of the human gut might be ex- (basolateral application of 100 μM of UTP). In order to identify pected to be engaged in Red Queen evolution and have dN/dS which ion channels were being affected, the CFTR chloride ion indicative of positive selection. While amino acid divergence be- channel inhibitor, GlyH101 (50 μM), and the calcium-activated tween orthologs of secreted proteins varied considerably from chloride ion channel inhibitor, DIDS (100 μM), were added to the 67% for the high-cysteine membrane protein (HCMP) to 83% for, apical side of the Ussing chamber. The cAMP-stimulated Isc is e.g., the extracellular nuclease, only 3 proteins showed evidence predominantly due to activation of CFTR chloride channels as it of positive selections, 2 tenascins and 1 of the cathepsins. One is inhibited by GlyH101 (Fig. 2B–D). The calcium-activated Isc is cathepsin and 1 tenascin in particular showed evidence of evo- predominantly due to activation of calcium-activated chloride lution under a very high degree of selective pressure (Table 1). channels as it is inhibited by DIDS (Fig. 2B–D). Interestingly, some cathepsins and some tenascins with similar levels of amino acid identity between the assemblages to those under high selective pressure showed little or no evidence of Discussion positive selection. We considered whether lineage-specific soluble mediators In this study, we have identified proteins secreted by tropho- might also be present and identified by this method, compar- zoites of both human-infecting assemblages. Contaminating ing those proteins identified by both methods as having the host serum proteins (mainly bovine albumin) in the supernatant highest relative expression in the supernatant (Tables S3 and samples were a concern, as previously described by others [20]. S4). The 5 most abundant conserved secreted proteins from Ta- Such serum proteins bind to the parasite’s surface and are con- ble 1 were also present in the top 10 secreted proteins from tinuously released, which interferes with the characterization each assemblage amongst other VSPs, tenascins, and cathep- of Giardia secretome. To overcome this issue, parasites were sin B, and this regardless of the MS technique or the isolate. cleansed from the serum proteins and incubated in serum-free Unsurprisingly, VSPs were the primary proteins enriched in su- DMEM before collecting supernatants and pellets. To increase pernatants that were lineage-specific. Amongst the multigene the coverage and robustness of the analysis, 2 mass spectrome- families, however, there were also differences in the cathep- ters (Orbitrap and Q-Exactive MS) were used on the same repli- sin B and tenascins/HCMP repertoires. No other proteins with cates, and proteins identified by both MS were included in the N-terminal peptides were encoded in either assemblage except analysis. for 1 CxC-rich protein. Interestingly, none of the 8 proteins en- Previous studies have focused on protein secretion during coded by assemblage-specific genes and identified by compara- Giardia trophozoite encystation; or protein secretion upon inter- tive genomics was found to be enriched in the supernatants. action with (or attachment to) host cells. Here instead, we chose When comparing secretion profiles between the 2 assem- to provide a detailed baseline from cultured Giardia trophozoites blages, 7 proteins were over-represented in the supernatants secreting proteins under a steady state in vitro. Nevertheless, our by only 1 assemblage or only identified by Q-Exactive MS or results are strongly supportive of a recent proteomic study look- present at very low abundance in 1 of the 2 (Table 2). Only 2 ing at the effect of host attachment on the profile of Giardia- proteins, sentrin and A-type flavoprotein lateral transfer candi- secreted proteins [15]. Prior to that study, several metabolic en- date, were present in the top 50 proteins of assemblage B (GS zymes had been proposed to be released by Giardia trophozoites strain) trophozoites secretome, whereas the other 5, 1 elonga- upon interaction with intestinal epithelial cells (IECs) [13], such tion factor 1-α (EF-1α), 1 ATP-binding cassette protein 5, 1 CxC- as arginine deiminase (ADI), enolase, and ornithine carbamoyl- rich protein, 1 translation initiation inhibitor, and a peptide me- transferase (OCT), which we were also able to identify from the thionine sulfoxide reductase MsrB, were present in the top 55 culture supernatants of both assemblages. proteins of assemblage A (WB strain) trophozoites secretomes. Our study does confirm the previously observed enrichment Interestingly, A-type flavoprotein lateral transfer candidate was of EF-1α in assemblage A culture supernatants (Table 2;Table also present in the top 50 supernatant proteins by assemblage S4) [20]. EF-1α is a key enzyme in the protein synthesis process A trophozoites; however, its low supernatant enrichment ratio in eukaryotic cells [21], but many organisms have been shown (<0.2) suggests that this protein is unlikely to be secreted by as- to express EF-1α in excess, which suggests that this protein may semblage A trophozoites. have some other functions [21]. In the context of pathogenic- ity and virulence, the secreted Leishamnia EF-1α has been shown to downregulate host inflammatory cell signalling [ 22]. In Giar- Giardia soluble mediators disrupt intestinal cell dia, EF-1α has been shown to be an immunoreactive protein rec- functions ognized by antibodies from patients who have previously had Soluble and diffusible agents, able to disrupt gut function, giardiasis [20]. Yet its role as putatively secreted virulence fac- could potentially mediate more diffuse and profound pathol- tor in Giardia pathogenesis remains elusive. That this protein ogy for giardiasis than close range interactions between the is only released by assemblage A trophozoites raises the pos- trophozoites and the gastrointestinal epithelium alone. To de- sibility of associating its function with observable differences in Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 5 Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Table 2: Assemblage specific component proteins of the Giardia secretome. Protein abundance Abundance ranking in Assemblage A Assemblage B secretome GI number GI number Signal Pellet (P) Supernatant Pellet (P) Supernatant Assemblage Assemblage Protein description assemblage A assemblage B peptide iBAQ (SP) iBAQ SP/P ratio iBAQ (SP) iBAQ SP/P ratio A B A-type flavoprotein lateral GL50803 10358 GL50581 1626 NP 2.11E+07 3.70E+06 0.17546 4.35E+06 8.70E+06 2.001741 47 10 candidate c c c c Sentrin GL50803 7760 GL50581 3210 NP 1.44E+05 ND ND ND 3.67E+04 ∞ – 31 c c EF-1α GL50803 112312 – NP ND 2.22E+06 ∞ ––– 17 – ATP-binding cassette GL50803 8227 GL50581 3399 NP 2.93E+06 2.89E+06 0.98546 2.01E+05 1.43E+04 0.071052 15 897 protein 5 CxCrichprotein GL50803 17476 GL50581 4509 1 4.80E+05 2.83E+05 0.58945 4.35E+04 3.59E+04 0.823376 43 819 Peptide methionine GL50803 5180 GL50581 3084 NP 1.24E+05 8.80E+04 0.70952 1.09E+06 1.30E+06 1.19568 53 331 sulfoxide reducast MsrB Translation initiation GL50803 480 GL50581 4017 NP 9.47E+06 4.08E+06 0.43038 1.06E+07 9.76E+06 0.920148 15 95 inhibitor Human infective Giardia trophozoites of assemblage A and B secrete a small set of different proteins. Seven proteins were identified as the most likely to be secreted by either G S or WB isolates. Two are the most likely to be secreted by GS isolate (shown in italics), and 5 are the most likely to be secreted by WB isolate (shown in bold). One GS isolate secreted and 2 WB isolates secreted were identified only via Q-Exactive MS in the other assemblage’s dataset (shown in underline). The abundance ranking represents the protein ranking within the secretome of both assemblages according to their abundances in the supernatant. Probability of N-terminal signal peptide using SignalP. Not predicted. Not detected. 6 Giardia secretome pathogenesis or host range between the 2 human infective as- cysteine membrane proteins, variant surface proteins, and semblages. tenascins. Our study shows some other differences in secretions be- The cathepsin B family of Giardia are confirmed virulence tween assemblage A and B trophozoites (Table 2). A-type flavo- factors involved in many of the parasite’s processes such as protein lateral transfer candidate and sentrin were present in as- encystation and excystation [6]; secreted GCATBs degrade host semblage B (GS strain) trophozoites secretome; ATP-binding cas- IL-8 and inhibit neutrophil chemotaxis [3]. GCATB contains se- sette (ABC) protein 5, CxC-rich protein, translation initiation in- creted and nonsecreted trophozoite-expressed proteins; the or- hibitor, and peptide methionine sulfoxide reductase (MsrB) were thologues of which are predominantly common to GS (B) and present in assemblage A (WB strain) secretome. WB (A) assemblages (Fig. 1). Expression of 16 GCATBs was pro- A-type flavoprotein lateral transfer candidate has a high oxy- teomically confirmed, of which 11 were shown by our proteomic gen reductase activity during Giardia infection, suggesting an O analysis to be secreted. These 11 fell into 6 orthologous groups, scavenging function upon release in the host intestinal environ- and for 3 of these groups, all group members were shown to ment [23], thus potentially affording increased resilience to Gi- be secreted. Secreted GCTAB GL50803 15564 (WB) and its or- ardia trophozoites in the small intestine and manipulating the tholog GL50581 2036 (GS) show dN/dS values of >26, indicative parasites’ immediate microenvironment. Whether assemblage of strong positive selective pressure. Interestingly, when GS was B trophozoites require A-type flavoprotein lateral transfer can- resequenced, GL50803 15564 was found to comprise 3 recently didate throughout the infection or just in its early stage remains diverged orthologs (GSB 153537, GSB 155477, GSB 150353), and unclear. Sentrin is involved in the ubiquitination of proteins to it may be that the positive selection pressure observed has been render them resistant to degradation [24]. Sentrin is evolution- generated as a result of recent gene duplications in the assem- arily conserved and has been identified in prokaryotic and eu- blage B strain. GL50803 16779, an assemblage A (WB) GCATB, karyotic organisms such as S. cerevisiae, A. thaliana,and Homo has previously been shown to be upregulated and involved in sapiens, which suggests a conserved specialized function in cell trophozoite motility in the early pathogenesis of Giardia [15]. In metabolism [24]. With its ubiquitination function, sentrin was this study, this protein was found to be in WB’s top 5 secreted expected to be only present in Giardia proteome but not in its se- proteins (Table S4); its GS ortholog (GL50581 78) was also present cretome. Why this protein would be secreted or released by Gia- but at a considerably lower level, suggesting that for this GCATB rdia trophozoites remains unclear and raises the question of the may play a more significant role in assemblage A than assem- advantages, for the parasite, of releasing sentrin into the host blage B. environment upon infection. HCMPs are an enigmatic group of proteins with few associ- ABC proteins are a large and diverse canonical group of mem- ated functional studies. They may protect trophozoites against brane proteins typically resident in the plasma membrane and proteolysis [30, 31] and oxidative damage [32]. In Giardia,itap- associated, in eukaryotes, with the ATP-dependent egress of pears that 1 lineage of HCMPs has given rise to the VSPs, while metabolites and toxins; they can be determinants of virulence another has given rise to a group with high homology to mam- and drug resistance [25]. Here 1 Giardia ABC protein shows en- malian tenascins. Tenascin, VSPs, and HCMPs are then related richment in the supernatant of WB but not of GS, and it will be multigene families that together form the largest group of pro- interesting to see if a functional correlation can be found. The teins enriched in the Giardia supernatants. Interestingly, when CxC-rich protein belongs to the HCMP superfamily, which also aligned and analysed phylogenetically, the secreted tenascins includes VSPs, tenascins, and HCMPs. The presence of orthologs segregate into a monophyletic group (Fig. S4). Both WB and GS in both strains is consistent with it not being a VSP protein. As orthologs of 5 tenascin gene products were secreted, and in WB, with several other HCMPs, this CxC-rich protein had a very high 2 other secreted tenascins were also detected that were not de- signal and only 1 TM domain suggesting that it may be a labile tected for the GS strain (Fig. 1B). surface protein in WB, but its specific role and why it is much VSPs are well-characterized surface glycoproteins with more abundant in the WB supernatant than the GS supernatant transmembrane domains, which are expressed one at a time is not clear. Translation initiation inhibitors are proteins inhibit- by Giardia trophozoites through an RNAi-regulated mechanism. ing the initiation of the translation of messenger RNA (mRNA) They are quintessential virulence factors, responsible for anti- into proteins and are mainly located in the cell cytosol [26]. Yet, genic variation. VSPs are hypervariable by nature, and thus it is 1 translation initiation inhibitor is over-represented in the as- to be expected that they do not form orthologous pairs. This was semblage A trophozoite secretome (top 20 secreted proteins), the case for most we observed; intriguingly though, a few pro- probably due to its high solubility and stability. Peptide methio- teins annotated as VSPs were conserved between isolates, sug- nine sulfoxide reductase (MsrB) catalyzes the reduction of free- gesting that they are not actually VSPs and would not be subject and protein-bound methionine sulfoxides to corresponding me- to “one at a time” controlled expression—but are actually mis- thionines, which constitutes a mechanism for the scavenging of annotated HCMPs, which may have a conserved function in both reactive oxygen species (ROS) responsible for a fundamental in- GS and WB isolates. This study was not able to resolve whether nate defence against pathogens in various host organisms [27]. the enrichment of such proteins in the supernatant observed is MsrB is an antioxidant protein protecting organisms from the due to clipping or shedding from the parasite surface or whether cytotoxic effects of ROS and therefore from cell death. This pro- the proteins are also secreted. tein is crucial for the virulence of S. typhimurium and the im- Tenascins are characterized by the presence of EGF repeats mune evasion of Schistosoma mansoni [28, 29]. Whether msrB has and are able to act as ligands for EGF receptors. Mammalian a similar role in Giardia assemblage A pathogenicity remains tenascins are extracellular matrix proteins, which modulate unclear. cell adhesion and migration [33]. They appear to have evolved The difference in secretion between the 2 human infective from a group of proteins specific to vertebrates, presumably assemblages observed in this study may also go some way to co-evolving with the EGF receptor, and so the presence of homol- explaining the differences in pathogenesis, symptoms, and host ogous proteins in Giardia evolving independently from HCMPs range previously observed between assemblage A and B. is a clear example of the kind of convergent evolution best de- The most abundant proteins, in both human isolates, scribed as molecular mimicry. Interestingly, a Giardia tenascin primarily belong to 4 families of proteins: GCATB, high- (WB-GL50803 8687/GS-GL50581 4316), secreted by both strains, Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 7 Figure 1: Neighbour joining tree showing clustering of (A) cathepsin B and (B) tenascin gene families. Genes were retrieved by gene name search on GiardiaDB. Gene sequences were downloaded and aligned using ClustalW generated with the MEGA 6 software package. Maximum composite likelihood method was used, with 2000 bootstrap replicates. Bootstrap values greater than 50% are shown above the branches. Proteins confirmed to be secreted using our proteomic analysis. and Giardia tenascin (WB-GL50803 14573/GS-GL50581 1475), interaction with the host and luminal environment is sup- secreted only by the WB strain (Table 1; Table S4), were found ported by the very high dN/dS values of some family mem- to be induced by host soluble factors and implicated in the bers. Correlation of variation within assemblages at these loci regulation of trophozoite attachment [15], supporting the case with strain virulence is the essential next step for their use in for secreted tenascins acting as virulence factors in Giardia the diagnosis of virulent strains, risk assessment, and disease pathogenesis. prognosis. Most published studies concerning host cell–Giardia inter- Our results indicate that Giardia secretions are sufficient to actions have focused on the effects on the host intestinal ep- disable normal function in enteric epithelial cells, making them ithelia upon attachment of the trophozoites to the cells. In this less able to extract fluids from the lumen. In particular, they im- study, we have shown that diluted supernatant obtained from plicate PNPO, an extracellular nuclease, GCATBs, and tenascins. thesteadygrowthof Giardia trophozoites in vitro has an effect The fact that both extracellular nuclease and GCATBs can be on the intestinal cell function. The effect observed on chloride involved in the degradation of the intestinal mucus layer and secretion by Giardia supernatants indicates that Giardia secretes that both GCATBs and tenascins can be associated with intesti- a soluble factor, which is likely affecting secretion across the in- nal intracellular junction disruption suggests collaboration be- testinal epithelial cells. Physiologically, cultured intestinal cells tween these proteins. Therefore, we propose a pathogenic mech- show sensitivity to Giardia proteins released by the parasite even anism (Fig. 3) whereby PNPO produces a reducing environment at high dilution. Fig. 2D demonstrates that intestinal epithelial favouring growth of trophozoites and the extracellular nucle- cells when acutely exposed to such Giardia proteins lose the abil- ase degrades the outer layer of the intestinal mucus, improving ity to stimulate CFTR and calcium-activated chloride channels, access for GCATBs for further degradation of the protective mu- the clear implication being that virulence determinants released cous barrier and subsequent disruption of intestinal intracellu- from Giardia trophozoites interact with epithelial cell receptors lar junctions. Lastly, tenascins are involved in maintaining in- and ion channels. testinal cell separation by ligation of EGF receptors present at the In this analysis, we have identified the proteins that are se- surface of intestinal cells and exacerbation of epithelial damage creted by human infective Giardia trophozoites. Just 2 groups via increased levels of apoptosis amongst these more detached form the majority of these proteins, GCATBs and the HCMP su- cells. Once the intestinal barrier is breached by the actions of perfamily, encoding known virulence factors in addition to an Giardia-secreted virulence factors, the sites of damage become abundant extracellular nuclease and an oxygen-fixing enzyme. prone to secondary infection by other opportunist microbes res- The elucidation of this group of proteins dramatically increases ident in the intestinal lumen and sensitive to irritation by aller- our understanding of the pathogenic mechanisms underlying gens in foodstuffs, leading to further inflammation and to the giardiasis at a molecular level. The genes encoding GCATBs characteristic symptoms of the disease. Further investigations and HCMP superfamily proteins are among the most heteroge- are necessary to verify this proposed mechanism of the patho- neous of all genes between assemblages. Their probable role in genesis of giardiasis. Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8 Giardia secretome Figure 2: The effect of co-culture with Giardia or Giardia supernatants on the electrophysiological properties of CaCo-2 monolayers. (A) Transepithelial electrical resistance in CaCo-2 monolayers following seeding on permeable supports. Data show an increase in TEER as the monolayer develops. Confluence occurre d around day 6. Giardia were added on day 6 after the confluent monolayer formed and co-cultured with the Caco-2 monolayer for 24 hours. TEER was measured after 24 hours and compared with TEER in monolayers that had not been exposed to Giardia (n = 6). (B) A representative short circuit current (Isc) against time recording from single monolayers of CaCo-2 cells in an Ussing chamber. The trace shows the activation of CFTR chloride channels (basolateral application of 10 μM of forskolin) and calcium-activated chloride channels (basolateral application of 100 μM of UTP). Specificity of activation is confirmed by inhibition of Isc by the specific CFTR channel blocker, GlyH101; and specific calcium-activated chloride channel blocker, DIDS. The effect on Isc of 24-hour co-incubation of CaCo-2 monol ayers with Giardia or with Giardia supernatant (1:1000 dilution) is also shown. (C) Effect of 24-hour co-incubation of CaCo-2 monolayers with different strains of Giardia (WB, GS, and patient samples) on forskolin-stimulated and UTP-stimulated Isc (n = 3). (D) Effect of supernatant co-incubation from different strains of Giardia (WB, GS, and patient samples) on forskolin-stimulated and UTP-stimulated Isc (n = 3) from Caco-2 monolayers. The results were analysed by the Student t test and expressed as mean ∗ ∗∗ values ± standard error mean (SEM). Significant difference expressed as P < 0.05, P < 0.01 compared with control. Global Health at the University of Liverpool for mass spectrom- Methods etry analysis (Fig. S3) [41]. Proteomic analysis Protein samples were dispensed into low protein-binding mi- Sample preparation crocentrifuge tubes (Sarstedt, Leicester, UK) and made up to 160 μl by addition of 25 mM of Ambic. The proteins were de- Giardia trophozoites from the genome reference strains WB (as- TM semblage A, ATCC 50803) and GS (assemblage B, ATCC 50581) natured using 10 μl of 1% (w/v) RapiGest (Waters MS Tech- nologies, Manchester, UK) in 25 mM of Ambic, followed by 3 were cultured in TYI-S-33 under standard conditions (5% CO ) [34] and harvested during the mid–log phase of their in vitro cycles of freeze-thaw and 2 cycles of 10 minutes’ sonication in water bath. The sample was then incubated at 80 Cfor 10 growth curves. The total trophozoites (adhered and nonadhered) were washed ×3 in phosphate buffer saline (PBS) and then incu- minutes and reduced (addition of 10 μl of 60-mM DTT and incubation at 65 C for 10 minutes) and alkylated (addition of bated in nonsupplemented DMEM, with antibiotics to conserve an axenic milieu, for 45 minutes at 37 C(Fig. S5A) [38]. After 10 μl of 180-mM iodoacetamide and incubation at room tem- perature for 30 minutes in the dark). Trypsin (Sigma-Aldrich, incubation, an aliquot was analysed by flow cytometry to eval- Dorset, UK) was reconstituted in 50 mM of acetic acid to a con- uate the viability of the Giardia samples. Trophozoites and su- pernatant were separated by centrifugation, and both tropho- centration of 0.2 μg/μl. Digestion was performed by the ad- dition of 10 μl of trypsin to the sample, followed by incuba- zoite pellet and supernatant were harvested. Proteins con- ◦ TM tained in supernatant were concentrated in Vivaspin columns tion at 37 C overnight. The RapiGest was removed from the sample by acidification (1 μl of trifluoroacetic acid and incu- (3000 MWCO) with 25 mM of ammonium bicarbonate (Ambic) (Fig. S5 B) [39]. Supernatants were analysed by SDS PAGE [40] bation at 37 C for 45 minutes) and centrifugation (15 000 × g for 15 minutes) [41]. After protein digestion, 1 μg of digest was and were tested on cultured epithelial cells (Caco-2) to ensure the presence of proteins and biological activity (see below). Su- injected into both the Orbitrap Velos and the Q-Exactive MS for all samples. pernatants and pellets were sent to the Institute of Infection and Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 9 Figure 3: Proposed novel mechanism of pathogenicity for Giardia involving PNPO, extracellular nuclease, GCATB, tenascin. PNPO ( ) renders the intestinal environment more favourable to trophozoite’s growth. Once a new Giardia colony is established, trophozoites release extracellular nuclease ( ), GCATB ( ), and tenascin ( ). Extracellular nuclease may contribute to reducing the viscosity of the intestinal outer mucus layer, while GCATB may degrade mucins and disrupt intracellular junction. Finally, tenascins may maintain intestinal cells apart by attaching to the EGF receptors present at the surface of intestinal cells that could, over time, lead to the apoptosis of these isolated intestinal cells. Orbitrap Velos ature of 35 C, and the LC system coupled to a Q-Exactive mass Peptide mixtures were analysed by online nanoflow liquid chro- spectrometer (Thermo Fisher Scientific). The Q-Exactive was op- matography using the nanoACQUITY-nLC system (Waters MS erated in data-dependent mode with survey scans acquired at a technologies, Manchester, UK) coupled to an LTQ-Orbitrap Velos resolution of 70 000 at m/z 200. Up to the top 10 most abundant (ThermoFisher Scientific, Bremen, Germany) mass spectrome- isotope patterns with charge states +2, +3, and/or +4fromthe ter equipped with the manufacturer’s nanospray ion source. The survey scan were selected with an isolation window of 2.0 Th TM analytical column (nanoACQUITY UPLC BEH130 C18 15 cm × and fragmented by higher-energy collisional dissociation with 75 μm, 1.7-μm capillary column) was maintained at 35 Cand a normalized collision energies of 30. The maximum ion injection flow rate of 300 nl/min. The gradient consisted of 3%–40% ace- times for the survey scan and the MS/MS scans were 250 and 100 tonitrile in 0.1% formic acid for 90 minutes, then a ramp of 40%– ms, respectively, and the ion target value was set to 1E6 for sur- 85% acetonitrile in 0.1% formic acid for 3 minutes. Full scan MS vey scans and 1E4 for the MS/MS scans. Repetitive sequencing spectra (m/z range 300–2000) were acquired by the Orbitrap at a of peptides was minimized through dynamic exclusion of the resolution of 30 000. Analysis was performed in data-dependant sequenced peptides for 20 seconds. mode. The top 20 most intense ions from the MS1 scan (full MS) were selected for tandem MS by collision-induced dissociation Data analysis (CID), and all product spectra were acquired in the LTQ ion trap. Thermo RAW files were imported into Progenesis LC–MS Ion trap and orbitrap maximal injection times were set to 50 ms (version 4.1, Nonlinear Dynamics, Newcastle upon Tyne, UK). and 500 ms, respectively. Replicate runs were time-aligned using default settings and an auto-selected run as a reference. Peaks were picked by the soft- Q-Exactive MS ware using default settings and filtered to include only peaks Digests (2 μl) were analysed on a 50-cm Easy-Spray column with with a charge state of between +2and +6. Peptide intensities of an internal diameter of 75 μm, packed with 2-μm C18 particles, replicates were normalized against the reference run by Proge- fused to a silica nano-electrospray emitter (Thermo Fisher Sci- nesis LC-MS. Spectral data were transformed to .mgf files with entific). Reversed phase liquid chromatography was performed Progenesis LC-MS (Liquid Chromatography-Mass Spectrome- using the Ultimate 3000 nano system with a binary buffer sys- try) and exported for peptide identification using the PEAKS tem consisting of 0.1% formic acid (buffer A) and 80% acetonitrile Studio 7 (Bioinformatics Solutions Inc., Waterloo, Canada) in 0.1% formic acid (buffer B). The peptides were separated by a search engine. A multiple–search engine platform provided linear gradient of 5%–40% buffer B over 110 minutes at a flow rate by PEAKS Studio named inChorus was used, which combines of 300 nl/min. The column was operated at a constant temper- searching results from PEAKS DB (Bioinformatics Solutions Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 10 Giardia secretome Inc.), Mascot (Matrix Science, London, UK), OMSSA (National experiments with Giardia parasites, or for culture with Giardia Center for Biotechnology Information, Bethesda, USA), and X! supernatants [42]. Tandem (Global Proteome Machine Organization). Tandem MS data were searched against a custom database that con- CaCo-2 co-culture experiments with Giardia or Giardia supernatant tained the common contamination and internal standards Confluent CaCo-2 monolayers were taken, and the CaCo-2 GiardiaDB-3.1 GintestinalisAssemblageA AnnotatedProteins cell media was removed and replenished with a combination and GiardiaDB-3.1 GintestinalisAssemblageB AnnotatedProteins. of 90% complete DMEM/10% Giardia medium plus or minus The search parameters for Orbitrap-Velos were as follows; pre- Giardia trophozoites (100 000 total parasites per insert). Control cursor mass tolerance was set to 10 ppm, and fragment mass cultures were maintained in a separate plate to prevent parasite tolerance was set to 0.5 Da. One missed tryptic cleavage was contamination. Control inserts were inspected under the micro- permitted. Carbamidomethylation was set as a fixed modifi- scope to ensure there was no Giardia cross-contamination. The cation, and oxidation (M) set as a variable modification. The co-cultures were incubated at 37 C and 5% CO for 24 hours, af- search parameters for Q Exactive were as follows; precursor ter which the Giardia parasites were removed [42]. mass tolerance was set to 10 ppm, and fragment mass tolerance Confluent Caco-2 monolayers were also cultured with diluted was set to 0.01 Da. One missed tryptic cleavage was permitted. (1:1000) Giardia supernatants for 24 hours. Briefly, the culture Carbamidomethylation was set as a fixed modification, and media was removed from the insert, and Caco-2 cell media was oxidation (M) set as a variable modification. The false discovery replaced with a combination of 99.9% complete DMEM/0.1% Gi- rates (FDRs) were set at 1%, and at least 2 unique peptides ardia medium plus or minus Giardia supernatant [42]. were required for reporting protein identifications. Protein abundance (iBAQ) was calculated as the sum of all the peak Transepithelial electrical resistance assay intensities (from Progenesis output) divided by the number Monolayers of CaCo-2 cells were grown on 6-well Transwell fil- of theoretically observable tryptic peptides [16]. Protein abun- ters (0.4-μm pore size) for 7–15 days until confluent. The devel- dance was normalized by dividing the protein iBAQ value by the opment of the polarized monolayer was assessed by measuring summed iBAQ values for that sample. The reported abundance the transepithelial electrical resistance (TEER) over a 7–15-day is the mean of the biological replicates. period. Once confluent, Giardia were added to the apical side of The mass spectrometry proteomics data have been de- the Transwell filter and incubated for 24 hours. The integrity of posited to the ProteomeXchange Consortium via the PRIDE part- the confluent polarized monolayer was assessed by measuring ner repository [24] with the dataset identifier PXD004398 and the TEER before and/or after apical infection by Giardia [42]. 10.6019/PXD004398. Electrophysiology assay Monolayers of CaCo-2 cells on Transwell filters were mounted Electrophysiology into a Physiological Instruments EM-CSYS-2 Ussing chamber Giardia trophozoites culture setup after establishment of a confluent monolayer, and the Giardia lamblia WB and GS strains as well as the patients’ strains short circuit current (I ) across the monolayer was continuously SC (obtained from 3 patients with giardiasis from the NNUH) were measured [42]. grown in filter-sterilized, modified TYI-S-33 medium with 10% Both sides of the epithelium were bathed in 5 ml of Krebs adult bovine serum and 0.05% bovine bile [28]at37 Cinmi- Henseleit solution, which was continuously circulated through croaerophilic conditions and subcultured when confluent. To the half chambers, maintained at 37 C, and continuously bub- collect parasites for experiments, the medium was removed bled with 95% O /5% CO . The composition of the Krebs Hense- 2 2 from the culture to eliminate unattached or dead parasites. The leit bath solution used was similar to that used by Cuthbert tube was refilled with cold, sterile medium, and trophozoites de- [35] and had the following composition (in mM): NaCl 118, KCl tached by chilling on ice for 15 minutes. 4.7, CaCl 2.5, MgCl 1.2, NaHCO 25, KH PO 1.2, and glucose 2 2 3 2 4 Parasites were collected by centrifugation (1500 × g for 5 11.1 (pH 7.4). The permeable supports were left for 30 minutes minutes at 4 C) and washed once with the plating medium of to equilibrate before experiments were started. All filters were 90% complete DMEM/10% Giardia medium. Parasites were then treated with 10 μM of amiloride apically to eliminate electro- counted using a haemocytometer and diluted to the appropriate genic sodium absorption through epithelial sodium channels number. [42]. To collect Giardia supernatant for experiments, the Giardia culture bottle was placed on ice for 15 minutes. The bottle then Data analysis underwent centrifugation (1500 × g for 5 minutes at 4 C). The su- I was continuously monitored across the monolayers by a SC pernatant was then collected and filtered 3 times using 15-mm Physiological Instruments Multichannel Voltage/Current Clamp diameter syringe filters (0.2- μm pore size). Subsequently, the (VCC MC6) through 3M KCl/agar, Ag/AgCl cartridge electrodes postfiltered Giardia supernatant was diluted 1:1000 and saved in (Physiologic Instruments), and the raw data for I , transepithe- sc a–20 C freezer until required. lial resistance, and transepithelial voltage were recorded using Acquire and Analyse version 1.3 software (Physiological Instru- Mammalian cell line (CaCo-2) preparation ments). Data were exported to Microsoft Excel initially and then CaCo-2 cells (passages 20–25) were grown in DMEM supple- into the GraphPad Prism version 5.0 for Windows package for mented with nonessential amino acids, penicillin (12 IU/ml), data representation and statistical analysis. streptomycin (12 μg/ml), gentamycin (47 μg/ml), and 20% (vol/vol) heat-inactivated fetal calf serum (all from AMIMED, Chemicals and Inhibitors Bioconcept). The cells were seeded at a density of 6 × 10 Forskolin (10 μM), UTP (100 μM), Amiloride (10 μM), and DIDS cells/cm in 6-well Transwell filters (0.4- μm pore size) and cul- (100 μM) were obtained from Sigma Aldrich, and GlyH-101 tured for 7–15 days until confluent. Confluent monolayers were (50 μM) was obtained from Merck Chemicals. Stock solutions then used for electrophysiological experiments, for co-culture of Amiloride (10 mM) and GlyH-101 (50 mM) were made by Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 11 dissolving in DMSO. Final concentrations of drugs are as indi- teins are ranked according to Q-Exactive supernatant (S) abun- cated in the text or figures and where produced by adding the dance from most to least abundant. appropriate volume of stock concentration to 5 ml of either the Table S5: List of the 1553 proteins identified in the pellet of basolateral or apical bathing solution. Giardia GS strain trophozoites via Q-Exactive and Orbitrap MS. Hypothetical and annotated proteins are shown in blue and black, respectively. Proteins are ranked according to their Q- Phylogeny Exactive protein abundance–iBAQ values from least to most abundant. To look for sequence similarities between proteins of interest Tables S6: List of the 996 proteins identified in the super- from the same protein family, the coding sequences of these pro- natant of Giardia GS strain trophozoites via Q-Exactive and Orbi- teins were retrieved from GiardiaDB (v 3.1, 4.0, and 5.0), aligned, trap MS. Hypothetical and annotated proteins are shown in blue and compared using ClustalW. and black, respectively. Proteins are ranked according to their Phylogenetic trees were built for these proteins via the max- Q-Exactive protein abundance–iBAQ values, from least to most imum likelihood approach using MEGA software (v. 6.06). abundant. Table S7: List of the 1657 proteins identified in the pellet of Giardia WB strain trophozoites via Q-Exactive and Orbitrap MS. Availability of supporting data Hypothetical and annotated proteins are shown in blue and All proteomic datasets are held by and can be accessed for free at black, respectively. Proteins are ranked according to their Q- the European Bioinformatics PRoteomics IDEntifications (PRIDE) Exactive protein abundance–iBAQ values from least to most database (accession number PXD004398). Free integrated func- abundant. tionality with other Giardia large datasets is hosted at EupathDB Table S8: List of the 558 proteins identified in the super- [36]. Supporting data, including raw data in .csv format, align- natant of Giardia WB strain trophozoites via Q-Exactive and Orbi- ments, and phylogenetic analyses, are also available via the trap MS. Hypothetical and annotated proteins are shown in blue GigaScience repository, GigaDB [37]. All protocols used in this and black, respectively. Proteins are ranked according to their study are available and can be accessed at the protocols.io Q-Exactive protein abundance–iBAQ values from least to most database [38–43]. abundant. Figure S1: Giardia trophozoites are viable after incubation in nonsupplemented DMEM. Parasites were chilled on ice for 15 Additional files minutes, washed 3 times in prewarmed PBS, centrifuged 10 min- Table S1: List of the Giardia assemblage A (WB strain) lineage- utes at 3000 rotations per minute (rpm) between each wash; and specific proteins identified via Orbitrap and Q-Exactive MS. Pro- then incubated in prewarmed nonsupplemented DMEM for 45 tein sequences were compared with their coding sequence and minutes at 37 C. After 45 minutes’ incubation, parasites were matched to their orthologs in assemblage B (GS strain) using chilled on ice for 5 minutes and centrifuged for 10 minutes at the Giardia database: GiardiaDB.org. Annotated proteins are high- 3000 rpm. Pellets were collected and respuspended in PBS (A2 lighted in red, and hypothetical proteins in blue. Proteins were and B2). Trophozoites collected from culture and respuspended ranked according to Q-Exactive S supernatant (S) abundance, in either PBS (A3 and B3) or 2% trigene (detergent; A3 and B3) from most to least abundant. were used as life and death controls, respectively. Proportion of living/dead trophozoites by flow cytometry; 5 μl of propidium io- Table S2: List of the Giardia assemblage B (GS strain) lineage- specific proteins identified via Orbitrap and Q-Exactive MS. Pro- dide (PI) was added in each sample to stain DNA liberated in the tein sequences were compared with their coding sequence and milieu after cell death. Flow cytometry was performed using the TM matched to their orthologs in assemblage A (WB strain) using BD Accuri C6 flow cytometer, with a blue laser ( λ = 488 nm) GiardiaDB.org. Annotated proteins are highlighted in red, and hy- and an optical filter 585/40. Gates P2 and P3 represent living and pothetical proteins in blue. Proteins were ranked according to dead trophozoites, respectively. (A) Flow cytometry analysis for Q-Exactive abundance (S), from most to least abundant. GS isolate B. Flow cytometry analysis for WB isolate. Data were TableS3: Listof the86proteinsmostlikelytobesecretedby analysed using BD Accuri C-flow software (version 1.0.227.4). Giardia GS strain trophozoites. Thirty-one proteins were iden- Figure S2: Protein expression profile for Giardia assemblage A tified via Orbitrap and Q-Exactive MS (in bold), and 55 proteins and B obtained with both MS platforms. Both GS and WB pellets were identified via Q-Exactive MS only (in italics). Fifty-nine pro- (P) and supernatant (S) replicates were analysed via Orbitrap and Q-Exactive MS. Supernatant protein expression profiles are sim- teins are annotated (shown in red), and 27 are hypothetical pro- teins (shown in blue). Ten proteins are lineage-specific. The 15 ilar to each other within each assemblage; so are pellet protein expression profiles (graphs). A total of 1690 and 1587 proteins proteins identified as conserved between the 2 isolates are high- lighted in grey. Only proteins identified via both techniques were were identified for assemblage B and A, respectively (Venn dia- considered secreted and were included in the final analysis. Pro- grams), via both MS techniques. For assemblage A (WB isolate), teins are ranked according to Q-Exactive SP abundance from 1170 proteins were present in both datasets, and 49 and 471 pro- most to least abundant. teins were identified only in the Orbitrap MS and Q-Exactive MS TableS4: Listof the61proteinsmostlikelytobesecretedby datasets, respectively. For assemblage B (GS isolate), 1106 pro- Giardia WB strain trophozoites. Forty-4 proteins were identified teins were present in both datasets, and 42 and 439 were iden- via Orbitrap and Q-Exactive MS (in bold), and 16 proteins were tified only via Orbitrap Ms and Q-Exactive, respectively, for as- identified via Q-Exactive MS only (in italics). Fifty-three proteins semblage B. are annotated (shown in red), and 8 are hypothetical proteins Figure S3: Giardia proteins identified by Orbitrap and Q Ex- (shown in blue). Twelve proteins are lineage-specific. The 15 pro- active MS for assemblage A (WB isolate) and B (GS isolate). The Orbitrap MS analysis showed 639 and 426 proteins identified in teins identified as conserved between the 2 isolates are high- lighted in grey. Only proteins identified via both techniques were both supernatant and pellet for assemblage B (GS isolate) and assemblage A (WB isolate), respectively, but also 51 (GS isolate) considered secreted and were included in the final analysis. Pro- Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 12 Giardia secretome and 35 (WB isolate) in supernatant only and 461 (GS isolate) and 2; DIDS: 4,4-disothiocyanatostibene-2,2-sulfonic acid; DMEM: 758 proteins (WB isolate) in pellet only, respectively. The Q Ex- Dulbecco’s Modified Eagle Medium; EF-1 α: elongation factor active MS showed 946 and 490 proteins identified in both super- 1-alpha; EGF: epidermal growth factor; FDR: false discovery natant and pellet for assemblage B (GS isolate) and assemblage A rate; GCATB: Giardia cathepsin B; GlyH101; HCMP: high-cysteine (WB isolate), respectively, but also 27 (GS isolate) and 24 (WB iso- membrane protein; iBAQ: intensity-based absolute quantifica- late) in supernatant only and 569 (GS isolate) and 1227 proteins tion; IEC: intestinal epithelial cells; IL: interleukine; Isc: short- (WB isolate) in pellet only, respectively. Proteins are ranked ac- circuit current; mRNA: messenger RNA; msrB: peptide methion- cording to assemblage B Q-Exactive supernatant (SP) expression ine sulfoxide reductase B; OCT: ornithine carbamoyltransferase; from most to least abundant. P: pellet; PNPO: ryridoxamine 5-phosphate oxidase; PRIDE: PRo- Figure S4: Neighbour joining tree showing clustering of teomics IDEntifications; rcf: relative centrifugal force; RNA: ri- tenascins in the superfamily of High Cysteine Membrane Pro- bonucleic acid; ROS: reductive oxygen species; rpm: rotations teins (HCMP). Tenascin genes are highlighted in yellow. Genes per minute; SP: supernatant; TEER: transepithelial electrical re- were retrieved by gene name search on GiardiaDB. Gene se- sistance; VSP: variant surface protein. quences were downloaded and aligned using ClustalW gener- ated with the MEGA 6 software package. The maximum com- posite likelihood method was used, with 2000 bootstrap repli- Conflicts of interest cates. Bootstrap values greater than 50% are shown.  indicates The authors declare that they have no competing interests. secreted proteins, as confirmed by our proteomic analysis. Pro- teins are ranked according to assemblage A Q-Exactive super- natant (SP) abundance from most to least abundant. Authors’ contributions Figure S5: Protocol to harvest Giardia pellet and supernatant for proteomic analysis. (A) Preparation of Giardia supernatant K.T., J.M.W., J.P.W., and P.H. conceived and designed the studies. K.T. and A.D. coordinated the experiments. A.D. and S.A.N. per- and pellet samples for proteomic assay. Parasites were chilled for 20 minutes, transferred into 15-ml falcon tubes, and cen- formed the electrophysiology with J.P.W. A.D. performed the flow trifuged at 3000 rpm for 10 minutes. Supernatants were dis- cytometry with D.S. A.D. prepared the proteomic samples. D.X. carded, and pellets were washed 3 times in warmed 1xPBS performed the proteomic experiments. A.D. and M.B. performed (4 ml, 2 ml, and 1 ml, respectively). Pellets were then incubated, the phylogenetic analysis. All authors contributed to the anal- under standard growth conditions and in filtered glass tubes, ysis of the datasets obtained and preparation of figures and ta- for 45 minutes at 37 C either in (1) nonsupplemented DMEM bles. The manuscript was drafted by A.D. and K.T. and improved containing phenol red or (2) nonsupplemented phenol red-free and approved prior to submission by all co-authors. DMEM. After 45 minutes’ incubation, parasites were chilled for 5 minutes, transferred in 15-ml falcon tubes, and centrifuged at Acknowledgements 3000 rpm for 10 minutes. Pellets were stored at –20 C. Proteins present in supernatant samples were concentrated prior to per- The research leading to these results was primarily forming the proteomic assay. (B) Protocol to concentrate proteins funded from the European Union Seventh Framework Programme (FP7/2007–2013; FP7/2007–2011) under grant contained in Giardia supernatant samples prior to proteomic as- say. Supernatants were transferred in Vivaspin columns with a agreement No. 311846. P.R.H. is supported by the National 3000 MWCO and centrifuged at 12 000 relative centrifugal force Institute for Health Research Health Protection Research Unit (rcf) for 30 minutes. Proteins were washed up to 3 times with 25 (NIHR HPRU) in Gastrointestinal Infections at the University of mM of Ambic (depending on the presence of phenol red within Liverpool, in partnership with Public Health England (PHE), and DMEM) and centrifuged at 12 000 rcf for 30 minutes. Then, 50 in collaboration with University of East Anglia, University of μl of 25 mM Ambic was added, and samples were left at room Oxford, and the Institute of Food Research. Professor Hunter is temperature for 1 hour; there was a final spin at 3000 rcf for 2 based at the University of East Anglia. The views expressed are minutes to recover proteins using BCA. those of the authors and not necessarily those of the National Figure S6: Giardia supernatant protein profile and protein Health Service, the National Institute for Health Research, the concentration after incubation in nonsupplemented DMEM. Par- Department of Health, or Public Health England. We thank asites were chilled on ice for 15 minutes, washed 3 times in Susanne Warrenfeltz and the EuPathDB team for invaluable prewarmed PBS, centrifuged for 10 minutes at 3000 rpm be- assistance in making the integrated datasets available. tween each wash, and then incubated in prewarmed nonsupple- mented DMEM for 45 minutes at 37 C. After 45 minutes’ incuba- References tion, parasites were chilled on ice for 5 minutes and centrifuged for 10 minutes at 3000 rpm. Supernatants were collected, and 1. 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Giardia secretome highlights secreted tenascins as a key component of pathogenesis

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Abstract

Background: Giardia is a protozoan parasite of public health relevance that causes gastroenteritis in a wide range of hosts. Two genetically distinct lineages (assemblages A and B) are responsible for the human disease. Although it is clear that differences in virulence occur, the pathogenesis and virulence of Giardia remain poorly understood. Results: The genome of Giardia is believed to contain open reading frames that could encode as many as 6000 proteins. By successfully applying quantitative proteomic analyses to the whole parasite and to the supernatants derived from parasite culture of assemblages A and B, we confirm expression of ∼1600 proteins from each assemblage, the vast majority of which are common to both lineages. To look for signature enrichment of secreted proteins, we considered the ratio of proteins in the supernatant compared with the pellet, which defined a small group of enriched proteins, putatively secreted at a steady state by cultured growing trophozoites of both assemblages. This secretome is enriched with proteins annotated to have N-terminal signal peptide. The most abundant secreted proteins include known virulence factors such as cathepsin B cysteine proteases and members of a Giardia superfamily of cysteine-rich proteins that comprise variant surface proteins, high-cysteine membrane proteins, and a new class of virulence factors, the Giardia tenascins. We demonstrate that physiological function of human enteric epithelial cells is disrupted by such soluble factors even in the absence of the trophozoites. Conclusions: We are able to propose a straightforward model of Giardia pathogenesis incorporating key roles for the major Giardia-derived soluble mediators. Keywords: Giardia; secretion; proteomics; quantitative proteomics; tenascin; cysteine protease; enteric pathogen Received: 12 June 2017; Revised: 23 October 2017; Accepted: 17 January 2018 The Author(s) 2018. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Giardia secretome of the protective mucous afforded by the action of a secreted Background nuclease and GCATB, and damage to cellular junctions by With some 280 million symptomatic cases, giardiasis causes GCATB. Tenascins act by means of epidermal growth factor more bouts of human illness than any other parasitic disease (EGF) receptor ligation, to prevent repair to those damaged [1]. The mechanism and mediators of pathogenesis by Giardia, junctions. however, remain largely unknown. Thanks to human volunteer studies, the association of Giardia infection itself, and the sig- nificance of the virulence of the infecting Giardia strain, is ex- Data Description perimentally unambiguous [2]. The molecular definition asso- Soluble and cytosolic fractions from in vitro grown assemblage ciated with strain virulence, though, is largely unexplored. It is A and B trophozoites, the aetiologic agents of human giardiasis, clear that the majority of Giardia infections are asymptomatic. were extracted in order to establish which proteins are secreted It is also clear that infection is primarily localized to the duode- in the steady state by healthy, growing trophozoite populations. num and that some localized damage, close to the sites of col- We reasoned that secreted proteins would be overrepresented in onization, causes villus atrophy and apoptosis of surrounding the medium in which parasites were incubated compared with cells. However, this localized damage cannot be the sole cause the trophozoites that produced them. This ostensibly straight- of the profound diarrhoea that is often characteristic of the dis- forward assessment relied on the sensitive, specific, and quanti- ease and that appears to affect absorption over a much wider tative detection of the proteins expressed by Giardia trophozoites area of the digestive tract than the site of infection alone. in whole cells and in the medium in which the trophozoites were One of the secreted mediators of damage to the duodenum incubated. is believed to be cathepsin B protease [3]. Cathepsin B-like pro- The WB (assemblage A: ATCC 50803) and GS (assemblage B: teases compose one of the superfamilies belonging to the CA ATCC 50581) reference strains were utilized to facilitate ease clan of cysteine peptidases [4]. Compared with other cathepsins, of comparison between genetically divergent human infective cathepsin B proteases possess an additional 20 amino acid inser- isolates with the available reference genomes. For each ex- tions, named the occluding loop, that enable their function as an periment, trophozoites were harvested from mid–log growth endo- or exopeptidase [5]. Although 27 genes encoding cathep- and incubated in nonsupplemented Dulbecco’s Modified Eagle sin proteases have been identified in Giardia, for the majority medium (DMEM) for 45 minutes at 37 C before supernatants and of these proteases, function remains elusive [6]. While some pellets were collected for proteomic and other analyses includ- parasites may secrete cathepsin B proteases to either evade or ing validation of their viability by flow cytometry (Additional modulate their host’s immune responses [7], a recent study has file 1: Fig. S1). Proteomic analyses were based on samples from demonstrated that Giardia trophozoites secrete cathepsin B–like 3 distinct biological replicates. Each sample was analysed us- proteases, degrading intestinal IL-8 and thereby reducing the in- ing 2 quantitative proteomic platforms, the Orbitrap MS and the flammation reaction by the host [ 3]. Secreted Giardia cathepsin B Q-Exactive MS. Thus, in total, the results from 24 (2 × 2 × 2 × 3) protease (GCATB) may also contribute to degradation of intesti- proteomic analyses are reported. nal mucin and facilitate trophozoite attachment to intestinal ep- The identification of abundant, secreted, Giardia virulence ithelia [8, 9]. factors led us to consider whether the secretions from Gia- Most of the proteomic studies so far reported for rdia alone could affect changes in the behaviour of enteric Giardia were undertaken in trophozoites undergoing encystation epithelia—even in the absence of the trophozoites themselves. [10–12]. Only a few studies have focused on proteins secreted In order to determine the effect of Giardia trophozoite–secreted by Giardia and their role in the host-pathogen interaction [3, factors on the intestinal epithelia, chopstick-type electrodes 13–15]. These studies were focused on parasite interaction connected to a voltmeter were used to measure the trans- with intestinal cell lines. No studies have yet attempted to epithelial electrical resistance (TEER) of polarized CaCo-2 epithe- quantify proteins that are the product of steady state secretion lial cells grown on permeable supports. CaCo-2 cells were cul- by healthy, growing Giardia trophozoites, which we hypothesize tured over 6 days until confluent. TEER across the developing as the primary mediators of giardiasis pathology. In this study, CaCo-2 monolayer was measured on a daily basis, as shown in we have identified, to the limit of existing technology, the Fig. 2A. Once confluence was established, Giardia trophozoites proteins expressed by populations of healthy, growing human were added to the apical side of the confluent epithelium, and infective Giardia trophozoites. We have provided quantitation after 24 hours’ incubation, the trophozoites were washed from of the relative abundance of retained and released trophozoite the apical surface. In order to determine whether co-cultures of proteins from 2 human infective assemblages, affording cal- Giardia trophozoites or diluted Giardia supernatants affected the culation of the specific enrichment of released proteins and ion channels responsible for secretory movement across the ep- thereby the description of which proteins are most likely to be ithelium, an Ussing chamber system was utilized with different secreted by trophozoites of each assemblage. Thereafter, we chloride secretion inhibitors and activators. compared the profile of enrichment between the 2 assemblages Further details about sample collection, secretome analysis, in order to identify conserved as well as assemblage-specific and electrophysiology can be found in the Methods section and secreted proteins. We provide electrophysiological analysis that protocols provided. confirms that trophozoite-secreted molecules adversely affect the homeostasis of enteric epithelia, and our analysis of the heterogeneity of encoding genes between lineages demon- Analyses strates the direct selective pressure on these virulence factors Protein expression in Giardia trophozoites and affords their use in discriminating clinically important strains and outbreaks. Finally, the discovery of tenascins as a To describe definitive Giardia secretomes under a standard set highly represented and variable group of proteins secreted by of conditions with high confidence and based on a robust trophozoites strongly implicates this new class of virulence dataset and to reduce the potential for technical artefact, the factors in a novel model for the mechanism of Giardia patho- 2 MS techniques, Q-Exactive and Orbitrap MS, were used with genesis. We propose that tenascin action follows degradation similar settings on the same 3 independent replicates to Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 3 Table 1: The proteins which comprise the Giardia secretome ranked by abundance. Protein abundance A:B Protein GI number GI number iden- Signal Pellet Supernatant Abundance description assemblage A assemblage B tity dN/dS peptide (P) iBAQ (SP) iBAQ SP/P ratio ranking PNPO GL50803 5810 GL50581 4133 99.2 0.038 NP 5.71E+07 1.18E+08 2.063091 1 Tenascin GL50803 95162 GL50581 1982 76.2 1.597 0.99 2.97E+07 4.77E+07 1.607024 2 Tenascin GL50803 10330 GL50581 4057 73.5 0.347 0.99 4.66E+06 2.02E+07 4.342293 3 Cathepsin B GL50803 16468 GL50581 438 83.6 0.1072 0.78 9.63E+06 1.79E+07 1.861309 4 Tenascin GL50803 8687 GL50581 4316 77.6 44.176 0.98 6.22E+06 1.10E+07 1.770526 5 Uncharacterized GL50803 5258 GL50581 2767 91.2 0.029 NP 3.93E+06 1.09E+07 2.780918 6 Extracellular GL50803 8742 GL50581 3607 83.1 0.234 1 1.03E+06 4.57E+06 4.436193 7 nuclease Tenascin-37 GL50803 16477 GL50581 3575 79.8 0.1256 0.99 8.35E+05 4.03E+06 4.830956 8 Cathepsin B GL50803 15564 GL50581 2036 79.1 1 1.10E+06 3.95E+06 3.589608 9 26.5782 CKS1 GL50803 2661 GL50581 3484 100 0.001 NP 1.14E+06 3.20E+06 2.803062 10 Tenascin GL50803 113038 GL50581 4180 79 0.0949 1 1.20E+06 3.14E+06 2.620931 11 HCMP Group 1 GL50803 7715 GL50581 727 67 0.1821 0.99 ND 2.54E+06 ∞ 12 Uncharacterized GL50803 16522 GL50581 352 76 0.1591 NP 1.15E+06 2.21E+06 1.928833 13 HCMP GL50803 12063 GL50581 2622 83 0.246 1 3.62E+05 1.94E+06 5.354665 14 Cathepsin B GL50803 17516 GL50581 2318 72.8 0.2056 1 ND 7.81E+05 ∞ 15 The secretome of human infective Giardia trophozoites of assemblage A and B have a conserved repertoire of abundant secreted factors identified by both Orbitrap MS and Q-Exactive MS. Fifteen proteins were identified as most likely to be secreted by both GS and WB isolates. Thirteen are annotated proteins, and 2 are h ypothetical proteins. Proteins are ranked according to GS Q-Exactive supernatant (SP) protein abundance, from most to least abundant. Of the 12 annotated proteins, 5 are tenascins and 3 are related high-cysteine membrane proteins or VSP, and 3 are cathepsin Bs. The other annotated abundant secreted proteins are an extracellular nuclease and PNPO. Protein ranking represents the proteins’ rank within this table, from most to least abundant. Detailed breakdowns of the secretome for each assemblage by each method are provided in Supplemental Tables S1–S4. dN/dS in bold indicate that protein shows evidence of positive selective pressure during divergence from a common ancestor. Probability of N-terminal signal peptide using SignalP. Not predicted. Not detected. increase coverage. Only proteins identified by both techniques and 24 in the supernatant only, with 1127 WB proteins in pellet within the 3 replicate datasets were included in the analysis to only. increase the robustness of the data. The protein quantification was performed using a label-free method: intensity-based abso- Giardia secretome lute quantification (iBAQ), which calculates the sum of parent ion intensities of identified peptides per protein [ 16]. The aver- To evaluate supernatant enrichment, proteins identified in the age normalized abundance was divided by the iBAQ values, giv- supernatant (SP) datasets were gathered and compared with ing the “Abundance-iBAQ.” The quantitative datasets from both their concentration in the pellet (P) to provide a ratio using the SP abundance−iB AQ MS techniques and for each independent replicate were shown following formula: . These proteins were then Pabundance−iB AQ to be strongly correlated by a Spearman correlation test (data ranked from highest to lowest by ratiometric value, and an ar- not shown) and therefore exploitable for proteomic analysis. bitrary cutoff was invoked such that the top 50 were considered The Q-Exactive MS identified almost all of the proteins iden- the most likely to be secreted. Proteins identified only in SP were tified by use of the Orbitrap MS, and in total the 2 techniques also included in the analysis as most likely to be secreted. All the identified 1587 GS proteins and 1690 WB proteins (Additional File proteins selected as “of interest” were ranked according to their 1: Fig. S2). This represents more than a quarter of the open read- SP expression from most to least abundant to obtain a quantita- ing frames predicted by the respective genomes in this single tive enrichment profile for each isolate, and this was performed life cycle stage under this steady state set of in vitro culture con- for each platform. Orbitrap and Q-Exactive enrichment pro- ditions and compares favourably with other recent proteomic files were compared, and proteins were considered most likely analyses of Giardia [17, 18]. Lists of proteins detected in only 1 to be enriched in the supernatant when identified as such by of the 2 assemblages are provided (Additional file 2: Tables S1 Q-Exactive MS and confirmed by Orbitrap MS. The different en- and S2). Protein from 2 of the 8 predicted assemblage-specific richment profiles were then also compared between assem- genes previously identified by comparative genomics was de- blages. tected [19]. The results yielded a set of 15 orthologous proteins that Overall, both assemblages gave comparable and consistent were identified in both isolates by both techniques (Table 1). results using both platforms, with the sensitivity of detection Eleven of these were predicted to possess an N-terminal sig- being greater for Q-Exactive MS, which provided a range of de- nal sequence. Just 2 of these were of unknown function, and tection spanning 5 logs. In total, Q-Exactive MS identified 1542 2 groups dominated the annotated genes encoding the rest of GS proteins and 1641 WB proteins (Fig. S3). Of these, 946 GS pro- these proteins; 5 were annotated as tenascins and 3 as cathepsin teins were present in both pellet and supernatant, 27 in the su- B cysteine proteases. The most abundant enriched protein was pernatant only, and 569 GS proteins in pellet only. By compari- found to be pyridoxamine 5’-phosphate oxidase (PNPO), a flavin son, 490 WB proteins were identified in supernatant and pellet mononucleotide–dependent enzyme capable of fixing molecular Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Giardia secretome oxygen that lacks a signal peptide and which was also recently termine whether Giardia-secreted virulence factors could induce identified as a secreted Giardia trophozoite protein upregulated changes in the behaviour of the intestinal epithelium, short- during interaction with epithelial cells [15]. An extracellular nu- circuit current (Isc) was continuously measured across polar- clease was also present, along with a high-cysteine membrane ized CaCo-2 epithelial cells that had either been cultured with- protein, as well as a protein product of a gene misannotated as out any additions, co-cultured with Giardia trophozoites, or co- a variant surface protein (VSP; as it was well conserved between cultured with diluted (1:1000) Giardia supernatants (Fig. 2B). Fur- assemblages). ther experiments demonstrated that either after 24-hour co- We considered that where proteins were shown to be en- culture with Giardia (Fig. 2C) or 24-hour co-culture with diluted riched in the supernatant by both platforms and in both assem- Giardia supernatants (Fig. 2D), both experimental conditions dra- blages and possessed an N-terminal signal sequence, they were matically inhibit both the cAMP-stimulated Isc (basolateral ap- truly secreted proteins. Secreted proteins involved in adapting plication of 10 μM of forskolin) and the calcium-activated Isc Giardia to the host environment of the human gut might be ex- (basolateral application of 100 μM of UTP). In order to identify pected to be engaged in Red Queen evolution and have dN/dS which ion channels were being affected, the CFTR chloride ion indicative of positive selection. While amino acid divergence be- channel inhibitor, GlyH101 (50 μM), and the calcium-activated tween orthologs of secreted proteins varied considerably from chloride ion channel inhibitor, DIDS (100 μM), were added to the 67% for the high-cysteine membrane protein (HCMP) to 83% for, apical side of the Ussing chamber. The cAMP-stimulated Isc is e.g., the extracellular nuclease, only 3 proteins showed evidence predominantly due to activation of CFTR chloride channels as it of positive selections, 2 tenascins and 1 of the cathepsins. One is inhibited by GlyH101 (Fig. 2B–D). The calcium-activated Isc is cathepsin and 1 tenascin in particular showed evidence of evo- predominantly due to activation of calcium-activated chloride lution under a very high degree of selective pressure (Table 1). channels as it is inhibited by DIDS (Fig. 2B–D). Interestingly, some cathepsins and some tenascins with similar levels of amino acid identity between the assemblages to those under high selective pressure showed little or no evidence of Discussion positive selection. We considered whether lineage-specific soluble mediators In this study, we have identified proteins secreted by tropho- might also be present and identified by this method, compar- zoites of both human-infecting assemblages. Contaminating ing those proteins identified by both methods as having the host serum proteins (mainly bovine albumin) in the supernatant highest relative expression in the supernatant (Tables S3 and samples were a concern, as previously described by others [20]. S4). The 5 most abundant conserved secreted proteins from Ta- Such serum proteins bind to the parasite’s surface and are con- ble 1 were also present in the top 10 secreted proteins from tinuously released, which interferes with the characterization each assemblage amongst other VSPs, tenascins, and cathep- of Giardia secretome. To overcome this issue, parasites were sin B, and this regardless of the MS technique or the isolate. cleansed from the serum proteins and incubated in serum-free Unsurprisingly, VSPs were the primary proteins enriched in su- DMEM before collecting supernatants and pellets. To increase pernatants that were lineage-specific. Amongst the multigene the coverage and robustness of the analysis, 2 mass spectrome- families, however, there were also differences in the cathep- ters (Orbitrap and Q-Exactive MS) were used on the same repli- sin B and tenascins/HCMP repertoires. No other proteins with cates, and proteins identified by both MS were included in the N-terminal peptides were encoded in either assemblage except analysis. for 1 CxC-rich protein. Interestingly, none of the 8 proteins en- Previous studies have focused on protein secretion during coded by assemblage-specific genes and identified by compara- Giardia trophozoite encystation; or protein secretion upon inter- tive genomics was found to be enriched in the supernatants. action with (or attachment to) host cells. Here instead, we chose When comparing secretion profiles between the 2 assem- to provide a detailed baseline from cultured Giardia trophozoites blages, 7 proteins were over-represented in the supernatants secreting proteins under a steady state in vitro. Nevertheless, our by only 1 assemblage or only identified by Q-Exactive MS or results are strongly supportive of a recent proteomic study look- present at very low abundance in 1 of the 2 (Table 2). Only 2 ing at the effect of host attachment on the profile of Giardia- proteins, sentrin and A-type flavoprotein lateral transfer candi- secreted proteins [15]. Prior to that study, several metabolic en- date, were present in the top 50 proteins of assemblage B (GS zymes had been proposed to be released by Giardia trophozoites strain) trophozoites secretome, whereas the other 5, 1 elonga- upon interaction with intestinal epithelial cells (IECs) [13], such tion factor 1-α (EF-1α), 1 ATP-binding cassette protein 5, 1 CxC- as arginine deiminase (ADI), enolase, and ornithine carbamoyl- rich protein, 1 translation initiation inhibitor, and a peptide me- transferase (OCT), which we were also able to identify from the thionine sulfoxide reductase MsrB, were present in the top 55 culture supernatants of both assemblages. proteins of assemblage A (WB strain) trophozoites secretomes. Our study does confirm the previously observed enrichment Interestingly, A-type flavoprotein lateral transfer candidate was of EF-1α in assemblage A culture supernatants (Table 2;Table also present in the top 50 supernatant proteins by assemblage S4) [20]. EF-1α is a key enzyme in the protein synthesis process A trophozoites; however, its low supernatant enrichment ratio in eukaryotic cells [21], but many organisms have been shown (<0.2) suggests that this protein is unlikely to be secreted by as- to express EF-1α in excess, which suggests that this protein may semblage A trophozoites. have some other functions [21]. In the context of pathogenic- ity and virulence, the secreted Leishamnia EF-1α has been shown to downregulate host inflammatory cell signalling [ 22]. In Giar- Giardia soluble mediators disrupt intestinal cell dia, EF-1α has been shown to be an immunoreactive protein rec- functions ognized by antibodies from patients who have previously had Soluble and diffusible agents, able to disrupt gut function, giardiasis [20]. Yet its role as putatively secreted virulence fac- could potentially mediate more diffuse and profound pathol- tor in Giardia pathogenesis remains elusive. That this protein ogy for giardiasis than close range interactions between the is only released by assemblage A trophozoites raises the pos- trophozoites and the gastrointestinal epithelium alone. To de- sibility of associating its function with observable differences in Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 5 Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Table 2: Assemblage specific component proteins of the Giardia secretome. Protein abundance Abundance ranking in Assemblage A Assemblage B secretome GI number GI number Signal Pellet (P) Supernatant Pellet (P) Supernatant Assemblage Assemblage Protein description assemblage A assemblage B peptide iBAQ (SP) iBAQ SP/P ratio iBAQ (SP) iBAQ SP/P ratio A B A-type flavoprotein lateral GL50803 10358 GL50581 1626 NP 2.11E+07 3.70E+06 0.17546 4.35E+06 8.70E+06 2.001741 47 10 candidate c c c c Sentrin GL50803 7760 GL50581 3210 NP 1.44E+05 ND ND ND 3.67E+04 ∞ – 31 c c EF-1α GL50803 112312 – NP ND 2.22E+06 ∞ ––– 17 – ATP-binding cassette GL50803 8227 GL50581 3399 NP 2.93E+06 2.89E+06 0.98546 2.01E+05 1.43E+04 0.071052 15 897 protein 5 CxCrichprotein GL50803 17476 GL50581 4509 1 4.80E+05 2.83E+05 0.58945 4.35E+04 3.59E+04 0.823376 43 819 Peptide methionine GL50803 5180 GL50581 3084 NP 1.24E+05 8.80E+04 0.70952 1.09E+06 1.30E+06 1.19568 53 331 sulfoxide reducast MsrB Translation initiation GL50803 480 GL50581 4017 NP 9.47E+06 4.08E+06 0.43038 1.06E+07 9.76E+06 0.920148 15 95 inhibitor Human infective Giardia trophozoites of assemblage A and B secrete a small set of different proteins. Seven proteins were identified as the most likely to be secreted by either G S or WB isolates. Two are the most likely to be secreted by GS isolate (shown in italics), and 5 are the most likely to be secreted by WB isolate (shown in bold). One GS isolate secreted and 2 WB isolates secreted were identified only via Q-Exactive MS in the other assemblage’s dataset (shown in underline). The abundance ranking represents the protein ranking within the secretome of both assemblages according to their abundances in the supernatant. Probability of N-terminal signal peptide using SignalP. Not predicted. Not detected. 6 Giardia secretome pathogenesis or host range between the 2 human infective as- cysteine membrane proteins, variant surface proteins, and semblages. tenascins. Our study shows some other differences in secretions be- The cathepsin B family of Giardia are confirmed virulence tween assemblage A and B trophozoites (Table 2). A-type flavo- factors involved in many of the parasite’s processes such as protein lateral transfer candidate and sentrin were present in as- encystation and excystation [6]; secreted GCATBs degrade host semblage B (GS strain) trophozoites secretome; ATP-binding cas- IL-8 and inhibit neutrophil chemotaxis [3]. GCATB contains se- sette (ABC) protein 5, CxC-rich protein, translation initiation in- creted and nonsecreted trophozoite-expressed proteins; the or- hibitor, and peptide methionine sulfoxide reductase (MsrB) were thologues of which are predominantly common to GS (B) and present in assemblage A (WB strain) secretome. WB (A) assemblages (Fig. 1). Expression of 16 GCATBs was pro- A-type flavoprotein lateral transfer candidate has a high oxy- teomically confirmed, of which 11 were shown by our proteomic gen reductase activity during Giardia infection, suggesting an O analysis to be secreted. These 11 fell into 6 orthologous groups, scavenging function upon release in the host intestinal environ- and for 3 of these groups, all group members were shown to ment [23], thus potentially affording increased resilience to Gi- be secreted. Secreted GCTAB GL50803 15564 (WB) and its or- ardia trophozoites in the small intestine and manipulating the tholog GL50581 2036 (GS) show dN/dS values of >26, indicative parasites’ immediate microenvironment. Whether assemblage of strong positive selective pressure. Interestingly, when GS was B trophozoites require A-type flavoprotein lateral transfer can- resequenced, GL50803 15564 was found to comprise 3 recently didate throughout the infection or just in its early stage remains diverged orthologs (GSB 153537, GSB 155477, GSB 150353), and unclear. Sentrin is involved in the ubiquitination of proteins to it may be that the positive selection pressure observed has been render them resistant to degradation [24]. Sentrin is evolution- generated as a result of recent gene duplications in the assem- arily conserved and has been identified in prokaryotic and eu- blage B strain. GL50803 16779, an assemblage A (WB) GCATB, karyotic organisms such as S. cerevisiae, A. thaliana,and Homo has previously been shown to be upregulated and involved in sapiens, which suggests a conserved specialized function in cell trophozoite motility in the early pathogenesis of Giardia [15]. In metabolism [24]. With its ubiquitination function, sentrin was this study, this protein was found to be in WB’s top 5 secreted expected to be only present in Giardia proteome but not in its se- proteins (Table S4); its GS ortholog (GL50581 78) was also present cretome. Why this protein would be secreted or released by Gia- but at a considerably lower level, suggesting that for this GCATB rdia trophozoites remains unclear and raises the question of the may play a more significant role in assemblage A than assem- advantages, for the parasite, of releasing sentrin into the host blage B. environment upon infection. HCMPs are an enigmatic group of proteins with few associ- ABC proteins are a large and diverse canonical group of mem- ated functional studies. They may protect trophozoites against brane proteins typically resident in the plasma membrane and proteolysis [30, 31] and oxidative damage [32]. In Giardia,itap- associated, in eukaryotes, with the ATP-dependent egress of pears that 1 lineage of HCMPs has given rise to the VSPs, while metabolites and toxins; they can be determinants of virulence another has given rise to a group with high homology to mam- and drug resistance [25]. Here 1 Giardia ABC protein shows en- malian tenascins. Tenascin, VSPs, and HCMPs are then related richment in the supernatant of WB but not of GS, and it will be multigene families that together form the largest group of pro- interesting to see if a functional correlation can be found. The teins enriched in the Giardia supernatants. Interestingly, when CxC-rich protein belongs to the HCMP superfamily, which also aligned and analysed phylogenetically, the secreted tenascins includes VSPs, tenascins, and HCMPs. The presence of orthologs segregate into a monophyletic group (Fig. S4). Both WB and GS in both strains is consistent with it not being a VSP protein. As orthologs of 5 tenascin gene products were secreted, and in WB, with several other HCMPs, this CxC-rich protein had a very high 2 other secreted tenascins were also detected that were not de- signal and only 1 TM domain suggesting that it may be a labile tected for the GS strain (Fig. 1B). surface protein in WB, but its specific role and why it is much VSPs are well-characterized surface glycoproteins with more abundant in the WB supernatant than the GS supernatant transmembrane domains, which are expressed one at a time is not clear. Translation initiation inhibitors are proteins inhibit- by Giardia trophozoites through an RNAi-regulated mechanism. ing the initiation of the translation of messenger RNA (mRNA) They are quintessential virulence factors, responsible for anti- into proteins and are mainly located in the cell cytosol [26]. Yet, genic variation. VSPs are hypervariable by nature, and thus it is 1 translation initiation inhibitor is over-represented in the as- to be expected that they do not form orthologous pairs. This was semblage A trophozoite secretome (top 20 secreted proteins), the case for most we observed; intriguingly though, a few pro- probably due to its high solubility and stability. Peptide methio- teins annotated as VSPs were conserved between isolates, sug- nine sulfoxide reductase (MsrB) catalyzes the reduction of free- gesting that they are not actually VSPs and would not be subject and protein-bound methionine sulfoxides to corresponding me- to “one at a time” controlled expression—but are actually mis- thionines, which constitutes a mechanism for the scavenging of annotated HCMPs, which may have a conserved function in both reactive oxygen species (ROS) responsible for a fundamental in- GS and WB isolates. This study was not able to resolve whether nate defence against pathogens in various host organisms [27]. the enrichment of such proteins in the supernatant observed is MsrB is an antioxidant protein protecting organisms from the due to clipping or shedding from the parasite surface or whether cytotoxic effects of ROS and therefore from cell death. This pro- the proteins are also secreted. tein is crucial for the virulence of S. typhimurium and the im- Tenascins are characterized by the presence of EGF repeats mune evasion of Schistosoma mansoni [28, 29]. Whether msrB has and are able to act as ligands for EGF receptors. Mammalian a similar role in Giardia assemblage A pathogenicity remains tenascins are extracellular matrix proteins, which modulate unclear. cell adhesion and migration [33]. They appear to have evolved The difference in secretion between the 2 human infective from a group of proteins specific to vertebrates, presumably assemblages observed in this study may also go some way to co-evolving with the EGF receptor, and so the presence of homol- explaining the differences in pathogenesis, symptoms, and host ogous proteins in Giardia evolving independently from HCMPs range previously observed between assemblage A and B. is a clear example of the kind of convergent evolution best de- The most abundant proteins, in both human isolates, scribed as molecular mimicry. Interestingly, a Giardia tenascin primarily belong to 4 families of proteins: GCATB, high- (WB-GL50803 8687/GS-GL50581 4316), secreted by both strains, Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 7 Figure 1: Neighbour joining tree showing clustering of (A) cathepsin B and (B) tenascin gene families. Genes were retrieved by gene name search on GiardiaDB. Gene sequences were downloaded and aligned using ClustalW generated with the MEGA 6 software package. Maximum composite likelihood method was used, with 2000 bootstrap replicates. Bootstrap values greater than 50% are shown above the branches. Proteins confirmed to be secreted using our proteomic analysis. and Giardia tenascin (WB-GL50803 14573/GS-GL50581 1475), interaction with the host and luminal environment is sup- secreted only by the WB strain (Table 1; Table S4), were found ported by the very high dN/dS values of some family mem- to be induced by host soluble factors and implicated in the bers. Correlation of variation within assemblages at these loci regulation of trophozoite attachment [15], supporting the case with strain virulence is the essential next step for their use in for secreted tenascins acting as virulence factors in Giardia the diagnosis of virulent strains, risk assessment, and disease pathogenesis. prognosis. Most published studies concerning host cell–Giardia inter- Our results indicate that Giardia secretions are sufficient to actions have focused on the effects on the host intestinal ep- disable normal function in enteric epithelial cells, making them ithelia upon attachment of the trophozoites to the cells. In this less able to extract fluids from the lumen. In particular, they im- study, we have shown that diluted supernatant obtained from plicate PNPO, an extracellular nuclease, GCATBs, and tenascins. thesteadygrowthof Giardia trophozoites in vitro has an effect The fact that both extracellular nuclease and GCATBs can be on the intestinal cell function. The effect observed on chloride involved in the degradation of the intestinal mucus layer and secretion by Giardia supernatants indicates that Giardia secretes that both GCATBs and tenascins can be associated with intesti- a soluble factor, which is likely affecting secretion across the in- nal intracellular junction disruption suggests collaboration be- testinal epithelial cells. Physiologically, cultured intestinal cells tween these proteins. Therefore, we propose a pathogenic mech- show sensitivity to Giardia proteins released by the parasite even anism (Fig. 3) whereby PNPO produces a reducing environment at high dilution. Fig. 2D demonstrates that intestinal epithelial favouring growth of trophozoites and the extracellular nucle- cells when acutely exposed to such Giardia proteins lose the abil- ase degrades the outer layer of the intestinal mucus, improving ity to stimulate CFTR and calcium-activated chloride channels, access for GCATBs for further degradation of the protective mu- the clear implication being that virulence determinants released cous barrier and subsequent disruption of intestinal intracellu- from Giardia trophozoites interact with epithelial cell receptors lar junctions. Lastly, tenascins are involved in maintaining in- and ion channels. testinal cell separation by ligation of EGF receptors present at the In this analysis, we have identified the proteins that are se- surface of intestinal cells and exacerbation of epithelial damage creted by human infective Giardia trophozoites. Just 2 groups via increased levels of apoptosis amongst these more detached form the majority of these proteins, GCATBs and the HCMP su- cells. Once the intestinal barrier is breached by the actions of perfamily, encoding known virulence factors in addition to an Giardia-secreted virulence factors, the sites of damage become abundant extracellular nuclease and an oxygen-fixing enzyme. prone to secondary infection by other opportunist microbes res- The elucidation of this group of proteins dramatically increases ident in the intestinal lumen and sensitive to irritation by aller- our understanding of the pathogenic mechanisms underlying gens in foodstuffs, leading to further inflammation and to the giardiasis at a molecular level. The genes encoding GCATBs characteristic symptoms of the disease. Further investigations and HCMP superfamily proteins are among the most heteroge- are necessary to verify this proposed mechanism of the patho- neous of all genes between assemblages. Their probable role in genesis of giardiasis. Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 8 Giardia secretome Figure 2: The effect of co-culture with Giardia or Giardia supernatants on the electrophysiological properties of CaCo-2 monolayers. (A) Transepithelial electrical resistance in CaCo-2 monolayers following seeding on permeable supports. Data show an increase in TEER as the monolayer develops. Confluence occurre d around day 6. Giardia were added on day 6 after the confluent monolayer formed and co-cultured with the Caco-2 monolayer for 24 hours. TEER was measured after 24 hours and compared with TEER in monolayers that had not been exposed to Giardia (n = 6). (B) A representative short circuit current (Isc) against time recording from single monolayers of CaCo-2 cells in an Ussing chamber. The trace shows the activation of CFTR chloride channels (basolateral application of 10 μM of forskolin) and calcium-activated chloride channels (basolateral application of 100 μM of UTP). Specificity of activation is confirmed by inhibition of Isc by the specific CFTR channel blocker, GlyH101; and specific calcium-activated chloride channel blocker, DIDS. The effect on Isc of 24-hour co-incubation of CaCo-2 monol ayers with Giardia or with Giardia supernatant (1:1000 dilution) is also shown. (C) Effect of 24-hour co-incubation of CaCo-2 monolayers with different strains of Giardia (WB, GS, and patient samples) on forskolin-stimulated and UTP-stimulated Isc (n = 3). (D) Effect of supernatant co-incubation from different strains of Giardia (WB, GS, and patient samples) on forskolin-stimulated and UTP-stimulated Isc (n = 3) from Caco-2 monolayers. The results were analysed by the Student t test and expressed as mean ∗ ∗∗ values ± standard error mean (SEM). Significant difference expressed as P < 0.05, P < 0.01 compared with control. Global Health at the University of Liverpool for mass spectrom- Methods etry analysis (Fig. S3) [41]. Proteomic analysis Protein samples were dispensed into low protein-binding mi- Sample preparation crocentrifuge tubes (Sarstedt, Leicester, UK) and made up to 160 μl by addition of 25 mM of Ambic. The proteins were de- Giardia trophozoites from the genome reference strains WB (as- TM semblage A, ATCC 50803) and GS (assemblage B, ATCC 50581) natured using 10 μl of 1% (w/v) RapiGest (Waters MS Tech- nologies, Manchester, UK) in 25 mM of Ambic, followed by 3 were cultured in TYI-S-33 under standard conditions (5% CO ) [34] and harvested during the mid–log phase of their in vitro cycles of freeze-thaw and 2 cycles of 10 minutes’ sonication in water bath. The sample was then incubated at 80 Cfor 10 growth curves. The total trophozoites (adhered and nonadhered) were washed ×3 in phosphate buffer saline (PBS) and then incu- minutes and reduced (addition of 10 μl of 60-mM DTT and incubation at 65 C for 10 minutes) and alkylated (addition of bated in nonsupplemented DMEM, with antibiotics to conserve an axenic milieu, for 45 minutes at 37 C(Fig. S5A) [38]. After 10 μl of 180-mM iodoacetamide and incubation at room tem- perature for 30 minutes in the dark). Trypsin (Sigma-Aldrich, incubation, an aliquot was analysed by flow cytometry to eval- Dorset, UK) was reconstituted in 50 mM of acetic acid to a con- uate the viability of the Giardia samples. Trophozoites and su- pernatant were separated by centrifugation, and both tropho- centration of 0.2 μg/μl. Digestion was performed by the ad- dition of 10 μl of trypsin to the sample, followed by incuba- zoite pellet and supernatant were harvested. Proteins con- ◦ TM tained in supernatant were concentrated in Vivaspin columns tion at 37 C overnight. The RapiGest was removed from the sample by acidification (1 μl of trifluoroacetic acid and incu- (3000 MWCO) with 25 mM of ammonium bicarbonate (Ambic) (Fig. S5 B) [39]. Supernatants were analysed by SDS PAGE [40] bation at 37 C for 45 minutes) and centrifugation (15 000 × g for 15 minutes) [41]. After protein digestion, 1 μg of digest was and were tested on cultured epithelial cells (Caco-2) to ensure the presence of proteins and biological activity (see below). Su- injected into both the Orbitrap Velos and the Q-Exactive MS for all samples. pernatants and pellets were sent to the Institute of Infection and Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 9 Figure 3: Proposed novel mechanism of pathogenicity for Giardia involving PNPO, extracellular nuclease, GCATB, tenascin. PNPO ( ) renders the intestinal environment more favourable to trophozoite’s growth. Once a new Giardia colony is established, trophozoites release extracellular nuclease ( ), GCATB ( ), and tenascin ( ). Extracellular nuclease may contribute to reducing the viscosity of the intestinal outer mucus layer, while GCATB may degrade mucins and disrupt intracellular junction. Finally, tenascins may maintain intestinal cells apart by attaching to the EGF receptors present at the surface of intestinal cells that could, over time, lead to the apoptosis of these isolated intestinal cells. Orbitrap Velos ature of 35 C, and the LC system coupled to a Q-Exactive mass Peptide mixtures were analysed by online nanoflow liquid chro- spectrometer (Thermo Fisher Scientific). The Q-Exactive was op- matography using the nanoACQUITY-nLC system (Waters MS erated in data-dependent mode with survey scans acquired at a technologies, Manchester, UK) coupled to an LTQ-Orbitrap Velos resolution of 70 000 at m/z 200. Up to the top 10 most abundant (ThermoFisher Scientific, Bremen, Germany) mass spectrome- isotope patterns with charge states +2, +3, and/or +4fromthe ter equipped with the manufacturer’s nanospray ion source. The survey scan were selected with an isolation window of 2.0 Th TM analytical column (nanoACQUITY UPLC BEH130 C18 15 cm × and fragmented by higher-energy collisional dissociation with 75 μm, 1.7-μm capillary column) was maintained at 35 Cand a normalized collision energies of 30. The maximum ion injection flow rate of 300 nl/min. The gradient consisted of 3%–40% ace- times for the survey scan and the MS/MS scans were 250 and 100 tonitrile in 0.1% formic acid for 90 minutes, then a ramp of 40%– ms, respectively, and the ion target value was set to 1E6 for sur- 85% acetonitrile in 0.1% formic acid for 3 minutes. Full scan MS vey scans and 1E4 for the MS/MS scans. Repetitive sequencing spectra (m/z range 300–2000) were acquired by the Orbitrap at a of peptides was minimized through dynamic exclusion of the resolution of 30 000. Analysis was performed in data-dependant sequenced peptides for 20 seconds. mode. The top 20 most intense ions from the MS1 scan (full MS) were selected for tandem MS by collision-induced dissociation Data analysis (CID), and all product spectra were acquired in the LTQ ion trap. Thermo RAW files were imported into Progenesis LC–MS Ion trap and orbitrap maximal injection times were set to 50 ms (version 4.1, Nonlinear Dynamics, Newcastle upon Tyne, UK). and 500 ms, respectively. Replicate runs were time-aligned using default settings and an auto-selected run as a reference. Peaks were picked by the soft- Q-Exactive MS ware using default settings and filtered to include only peaks Digests (2 μl) were analysed on a 50-cm Easy-Spray column with with a charge state of between +2and +6. Peptide intensities of an internal diameter of 75 μm, packed with 2-μm C18 particles, replicates were normalized against the reference run by Proge- fused to a silica nano-electrospray emitter (Thermo Fisher Sci- nesis LC-MS. Spectral data were transformed to .mgf files with entific). Reversed phase liquid chromatography was performed Progenesis LC-MS (Liquid Chromatography-Mass Spectrome- using the Ultimate 3000 nano system with a binary buffer sys- try) and exported for peptide identification using the PEAKS tem consisting of 0.1% formic acid (buffer A) and 80% acetonitrile Studio 7 (Bioinformatics Solutions Inc., Waterloo, Canada) in 0.1% formic acid (buffer B). The peptides were separated by a search engine. A multiple–search engine platform provided linear gradient of 5%–40% buffer B over 110 minutes at a flow rate by PEAKS Studio named inChorus was used, which combines of 300 nl/min. The column was operated at a constant temper- searching results from PEAKS DB (Bioinformatics Solutions Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 10 Giardia secretome Inc.), Mascot (Matrix Science, London, UK), OMSSA (National experiments with Giardia parasites, or for culture with Giardia Center for Biotechnology Information, Bethesda, USA), and X! supernatants [42]. Tandem (Global Proteome Machine Organization). Tandem MS data were searched against a custom database that con- CaCo-2 co-culture experiments with Giardia or Giardia supernatant tained the common contamination and internal standards Confluent CaCo-2 monolayers were taken, and the CaCo-2 GiardiaDB-3.1 GintestinalisAssemblageA AnnotatedProteins cell media was removed and replenished with a combination and GiardiaDB-3.1 GintestinalisAssemblageB AnnotatedProteins. of 90% complete DMEM/10% Giardia medium plus or minus The search parameters for Orbitrap-Velos were as follows; pre- Giardia trophozoites (100 000 total parasites per insert). Control cursor mass tolerance was set to 10 ppm, and fragment mass cultures were maintained in a separate plate to prevent parasite tolerance was set to 0.5 Da. One missed tryptic cleavage was contamination. Control inserts were inspected under the micro- permitted. Carbamidomethylation was set as a fixed modifi- scope to ensure there was no Giardia cross-contamination. The cation, and oxidation (M) set as a variable modification. The co-cultures were incubated at 37 C and 5% CO for 24 hours, af- search parameters for Q Exactive were as follows; precursor ter which the Giardia parasites were removed [42]. mass tolerance was set to 10 ppm, and fragment mass tolerance Confluent Caco-2 monolayers were also cultured with diluted was set to 0.01 Da. One missed tryptic cleavage was permitted. (1:1000) Giardia supernatants for 24 hours. Briefly, the culture Carbamidomethylation was set as a fixed modification, and media was removed from the insert, and Caco-2 cell media was oxidation (M) set as a variable modification. The false discovery replaced with a combination of 99.9% complete DMEM/0.1% Gi- rates (FDRs) were set at 1%, and at least 2 unique peptides ardia medium plus or minus Giardia supernatant [42]. were required for reporting protein identifications. Protein abundance (iBAQ) was calculated as the sum of all the peak Transepithelial electrical resistance assay intensities (from Progenesis output) divided by the number Monolayers of CaCo-2 cells were grown on 6-well Transwell fil- of theoretically observable tryptic peptides [16]. Protein abun- ters (0.4-μm pore size) for 7–15 days until confluent. The devel- dance was normalized by dividing the protein iBAQ value by the opment of the polarized monolayer was assessed by measuring summed iBAQ values for that sample. The reported abundance the transepithelial electrical resistance (TEER) over a 7–15-day is the mean of the biological replicates. period. Once confluent, Giardia were added to the apical side of The mass spectrometry proteomics data have been de- the Transwell filter and incubated for 24 hours. The integrity of posited to the ProteomeXchange Consortium via the PRIDE part- the confluent polarized monolayer was assessed by measuring ner repository [24] with the dataset identifier PXD004398 and the TEER before and/or after apical infection by Giardia [42]. 10.6019/PXD004398. Electrophysiology assay Monolayers of CaCo-2 cells on Transwell filters were mounted Electrophysiology into a Physiological Instruments EM-CSYS-2 Ussing chamber Giardia trophozoites culture setup after establishment of a confluent monolayer, and the Giardia lamblia WB and GS strains as well as the patients’ strains short circuit current (I ) across the monolayer was continuously SC (obtained from 3 patients with giardiasis from the NNUH) were measured [42]. grown in filter-sterilized, modified TYI-S-33 medium with 10% Both sides of the epithelium were bathed in 5 ml of Krebs adult bovine serum and 0.05% bovine bile [28]at37 Cinmi- Henseleit solution, which was continuously circulated through croaerophilic conditions and subcultured when confluent. To the half chambers, maintained at 37 C, and continuously bub- collect parasites for experiments, the medium was removed bled with 95% O /5% CO . The composition of the Krebs Hense- 2 2 from the culture to eliminate unattached or dead parasites. The leit bath solution used was similar to that used by Cuthbert tube was refilled with cold, sterile medium, and trophozoites de- [35] and had the following composition (in mM): NaCl 118, KCl tached by chilling on ice for 15 minutes. 4.7, CaCl 2.5, MgCl 1.2, NaHCO 25, KH PO 1.2, and glucose 2 2 3 2 4 Parasites were collected by centrifugation (1500 × g for 5 11.1 (pH 7.4). The permeable supports were left for 30 minutes minutes at 4 C) and washed once with the plating medium of to equilibrate before experiments were started. All filters were 90% complete DMEM/10% Giardia medium. Parasites were then treated with 10 μM of amiloride apically to eliminate electro- counted using a haemocytometer and diluted to the appropriate genic sodium absorption through epithelial sodium channels number. [42]. To collect Giardia supernatant for experiments, the Giardia culture bottle was placed on ice for 15 minutes. The bottle then Data analysis underwent centrifugation (1500 × g for 5 minutes at 4 C). The su- I was continuously monitored across the monolayers by a SC pernatant was then collected and filtered 3 times using 15-mm Physiological Instruments Multichannel Voltage/Current Clamp diameter syringe filters (0.2- μm pore size). Subsequently, the (VCC MC6) through 3M KCl/agar, Ag/AgCl cartridge electrodes postfiltered Giardia supernatant was diluted 1:1000 and saved in (Physiologic Instruments), and the raw data for I , transepithe- sc a–20 C freezer until required. lial resistance, and transepithelial voltage were recorded using Acquire and Analyse version 1.3 software (Physiological Instru- Mammalian cell line (CaCo-2) preparation ments). Data were exported to Microsoft Excel initially and then CaCo-2 cells (passages 20–25) were grown in DMEM supple- into the GraphPad Prism version 5.0 for Windows package for mented with nonessential amino acids, penicillin (12 IU/ml), data representation and statistical analysis. streptomycin (12 μg/ml), gentamycin (47 μg/ml), and 20% (vol/vol) heat-inactivated fetal calf serum (all from AMIMED, Chemicals and Inhibitors Bioconcept). The cells were seeded at a density of 6 × 10 Forskolin (10 μM), UTP (100 μM), Amiloride (10 μM), and DIDS cells/cm in 6-well Transwell filters (0.4- μm pore size) and cul- (100 μM) were obtained from Sigma Aldrich, and GlyH-101 tured for 7–15 days until confluent. Confluent monolayers were (50 μM) was obtained from Merck Chemicals. Stock solutions then used for electrophysiological experiments, for co-culture of Amiloride (10 mM) and GlyH-101 (50 mM) were made by Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Dubourg et al. 11 dissolving in DMSO. Final concentrations of drugs are as indi- teins are ranked according to Q-Exactive supernatant (S) abun- cated in the text or figures and where produced by adding the dance from most to least abundant. appropriate volume of stock concentration to 5 ml of either the Table S5: List of the 1553 proteins identified in the pellet of basolateral or apical bathing solution. Giardia GS strain trophozoites via Q-Exactive and Orbitrap MS. Hypothetical and annotated proteins are shown in blue and black, respectively. Proteins are ranked according to their Q- Phylogeny Exactive protein abundance–iBAQ values from least to most abundant. To look for sequence similarities between proteins of interest Tables S6: List of the 996 proteins identified in the super- from the same protein family, the coding sequences of these pro- natant of Giardia GS strain trophozoites via Q-Exactive and Orbi- teins were retrieved from GiardiaDB (v 3.1, 4.0, and 5.0), aligned, trap MS. Hypothetical and annotated proteins are shown in blue and compared using ClustalW. and black, respectively. Proteins are ranked according to their Phylogenetic trees were built for these proteins via the max- Q-Exactive protein abundance–iBAQ values, from least to most imum likelihood approach using MEGA software (v. 6.06). abundant. Table S7: List of the 1657 proteins identified in the pellet of Giardia WB strain trophozoites via Q-Exactive and Orbitrap MS. Availability of supporting data Hypothetical and annotated proteins are shown in blue and All proteomic datasets are held by and can be accessed for free at black, respectively. Proteins are ranked according to their Q- the European Bioinformatics PRoteomics IDEntifications (PRIDE) Exactive protein abundance–iBAQ values from least to most database (accession number PXD004398). Free integrated func- abundant. tionality with other Giardia large datasets is hosted at EupathDB Table S8: List of the 558 proteins identified in the super- [36]. Supporting data, including raw data in .csv format, align- natant of Giardia WB strain trophozoites via Q-Exactive and Orbi- ments, and phylogenetic analyses, are also available via the trap MS. Hypothetical and annotated proteins are shown in blue GigaScience repository, GigaDB [37]. All protocols used in this and black, respectively. Proteins are ranked according to their study are available and can be accessed at the protocols.io Q-Exactive protein abundance–iBAQ values from least to most database [38–43]. abundant. Figure S1: Giardia trophozoites are viable after incubation in nonsupplemented DMEM. Parasites were chilled on ice for 15 Additional files minutes, washed 3 times in prewarmed PBS, centrifuged 10 min- Table S1: List of the Giardia assemblage A (WB strain) lineage- utes at 3000 rotations per minute (rpm) between each wash; and specific proteins identified via Orbitrap and Q-Exactive MS. Pro- then incubated in prewarmed nonsupplemented DMEM for 45 tein sequences were compared with their coding sequence and minutes at 37 C. After 45 minutes’ incubation, parasites were matched to their orthologs in assemblage B (GS strain) using chilled on ice for 5 minutes and centrifuged for 10 minutes at the Giardia database: GiardiaDB.org. Annotated proteins are high- 3000 rpm. Pellets were collected and respuspended in PBS (A2 lighted in red, and hypothetical proteins in blue. Proteins were and B2). Trophozoites collected from culture and respuspended ranked according to Q-Exactive S supernatant (S) abundance, in either PBS (A3 and B3) or 2% trigene (detergent; A3 and B3) from most to least abundant. were used as life and death controls, respectively. Proportion of living/dead trophozoites by flow cytometry; 5 μl of propidium io- Table S2: List of the Giardia assemblage B (GS strain) lineage- specific proteins identified via Orbitrap and Q-Exactive MS. Pro- dide (PI) was added in each sample to stain DNA liberated in the tein sequences were compared with their coding sequence and milieu after cell death. Flow cytometry was performed using the TM matched to their orthologs in assemblage A (WB strain) using BD Accuri C6 flow cytometer, with a blue laser ( λ = 488 nm) GiardiaDB.org. Annotated proteins are highlighted in red, and hy- and an optical filter 585/40. Gates P2 and P3 represent living and pothetical proteins in blue. Proteins were ranked according to dead trophozoites, respectively. (A) Flow cytometry analysis for Q-Exactive abundance (S), from most to least abundant. GS isolate B. Flow cytometry analysis for WB isolate. Data were TableS3: Listof the86proteinsmostlikelytobesecretedby analysed using BD Accuri C-flow software (version 1.0.227.4). Giardia GS strain trophozoites. Thirty-one proteins were iden- Figure S2: Protein expression profile for Giardia assemblage A tified via Orbitrap and Q-Exactive MS (in bold), and 55 proteins and B obtained with both MS platforms. Both GS and WB pellets were identified via Q-Exactive MS only (in italics). Fifty-nine pro- (P) and supernatant (S) replicates were analysed via Orbitrap and Q-Exactive MS. Supernatant protein expression profiles are sim- teins are annotated (shown in red), and 27 are hypothetical pro- teins (shown in blue). Ten proteins are lineage-specific. The 15 ilar to each other within each assemblage; so are pellet protein expression profiles (graphs). A total of 1690 and 1587 proteins proteins identified as conserved between the 2 isolates are high- lighted in grey. Only proteins identified via both techniques were were identified for assemblage B and A, respectively (Venn dia- considered secreted and were included in the final analysis. Pro- grams), via both MS techniques. For assemblage A (WB isolate), teins are ranked according to Q-Exactive SP abundance from 1170 proteins were present in both datasets, and 49 and 471 pro- most to least abundant. teins were identified only in the Orbitrap MS and Q-Exactive MS TableS4: Listof the61proteinsmostlikelytobesecretedby datasets, respectively. For assemblage B (GS isolate), 1106 pro- Giardia WB strain trophozoites. Forty-4 proteins were identified teins were present in both datasets, and 42 and 439 were iden- via Orbitrap and Q-Exactive MS (in bold), and 16 proteins were tified only via Orbitrap Ms and Q-Exactive, respectively, for as- identified via Q-Exactive MS only (in italics). Fifty-three proteins semblage B. are annotated (shown in red), and 8 are hypothetical proteins Figure S3: Giardia proteins identified by Orbitrap and Q Ex- (shown in blue). Twelve proteins are lineage-specific. The 15 pro- active MS for assemblage A (WB isolate) and B (GS isolate). The Orbitrap MS analysis showed 639 and 426 proteins identified in teins identified as conserved between the 2 isolates are high- lighted in grey. Only proteins identified via both techniques were both supernatant and pellet for assemblage B (GS isolate) and assemblage A (WB isolate), respectively, but also 51 (GS isolate) considered secreted and were included in the final analysis. Pro- Downloaded from https://academic.oup.com/gigascience/article-abstract/7/3/1/4818238 by Ed 'DeepDyve' Gillespie user on 16 March 2018 12 Giardia secretome and 35 (WB isolate) in supernatant only and 461 (GS isolate) and 2; DIDS: 4,4-disothiocyanatostibene-2,2-sulfonic acid; DMEM: 758 proteins (WB isolate) in pellet only, respectively. The Q Ex- Dulbecco’s Modified Eagle Medium; EF-1 α: elongation factor active MS showed 946 and 490 proteins identified in both super- 1-alpha; EGF: epidermal growth factor; FDR: false discovery natant and pellet for assemblage B (GS isolate) and assemblage A rate; GCATB: Giardia cathepsin B; GlyH101; HCMP: high-cysteine (WB isolate), respectively, but also 27 (GS isolate) and 24 (WB iso- membrane protein; iBAQ: intensity-based absolute quantifica- late) in supernatant only and 569 (GS isolate) and 1227 proteins tion; IEC: intestinal epithelial cells; IL: interleukine; Isc: short- (WB isolate) in pellet only, respectively. Proteins are ranked ac- circuit current; mRNA: messenger RNA; msrB: peptide methion- cording to assemblage B Q-Exactive supernatant (SP) expression ine sulfoxide reductase B; OCT: ornithine carbamoyltransferase; from most to least abundant. P: pellet; PNPO: ryridoxamine 5-phosphate oxidase; PRIDE: PRo- Figure S4: Neighbour joining tree showing clustering of teomics IDEntifications; rcf: relative centrifugal force; RNA: ri- tenascins in the superfamily of High Cysteine Membrane Pro- bonucleic acid; ROS: reductive oxygen species; rpm: rotations teins (HCMP). Tenascin genes are highlighted in yellow. Genes per minute; SP: supernatant; TEER: transepithelial electrical re- were retrieved by gene name search on GiardiaDB. Gene se- sistance; VSP: variant surface protein. quences were downloaded and aligned using ClustalW gener- ated with the MEGA 6 software package. The maximum com- posite likelihood method was used, with 2000 bootstrap repli- Conflicts of interest cates. Bootstrap values greater than 50% are shown.  indicates The authors declare that they have no competing interests. secreted proteins, as confirmed by our proteomic analysis. Pro- teins are ranked according to assemblage A Q-Exactive super- natant (SP) abundance from most to least abundant. Authors’ contributions Figure S5: Protocol to harvest Giardia pellet and supernatant for proteomic analysis. (A) Preparation of Giardia supernatant K.T., J.M.W., J.P.W., and P.H. conceived and designed the studies. K.T. and A.D. coordinated the experiments. A.D. and S.A.N. per- and pellet samples for proteomic assay. Parasites were chilled for 20 minutes, transferred into 15-ml falcon tubes, and cen- formed the electrophysiology with J.P.W. A.D. performed the flow trifuged at 3000 rpm for 10 minutes. Supernatants were dis- cytometry with D.S. A.D. prepared the proteomic samples. D.X. carded, and pellets were washed 3 times in warmed 1xPBS performed the proteomic experiments. A.D. and M.B. performed (4 ml, 2 ml, and 1 ml, respectively). Pellets were then incubated, the phylogenetic analysis. All authors contributed to the anal- under standard growth conditions and in filtered glass tubes, ysis of the datasets obtained and preparation of figures and ta- for 45 minutes at 37 C either in (1) nonsupplemented DMEM bles. The manuscript was drafted by A.D. and K.T. and improved containing phenol red or (2) nonsupplemented phenol red-free and approved prior to submission by all co-authors. DMEM. After 45 minutes’ incubation, parasites were chilled for 5 minutes, transferred in 15-ml falcon tubes, and centrifuged at Acknowledgements 3000 rpm for 10 minutes. Pellets were stored at –20 C. Proteins present in supernatant samples were concentrated prior to per- The research leading to these results was primarily forming the proteomic assay. (B) Protocol to concentrate proteins funded from the European Union Seventh Framework Programme (FP7/2007–2013; FP7/2007–2011) under grant contained in Giardia supernatant samples prior to proteomic as- say. Supernatants were transferred in Vivaspin columns with a agreement No. 311846. P.R.H. is supported by the National 3000 MWCO and centrifuged at 12 000 relative centrifugal force Institute for Health Research Health Protection Research Unit (rcf) for 30 minutes. Proteins were washed up to 3 times with 25 (NIHR HPRU) in Gastrointestinal Infections at the University of mM of Ambic (depending on the presence of phenol red within Liverpool, in partnership with Public Health England (PHE), and DMEM) and centrifuged at 12 000 rcf for 30 minutes. Then, 50 in collaboration with University of East Anglia, University of μl of 25 mM Ambic was added, and samples were left at room Oxford, and the Institute of Food Research. Professor Hunter is temperature for 1 hour; there was a final spin at 3000 rcf for 2 based at the University of East Anglia. The views expressed are minutes to recover proteins using BCA. those of the authors and not necessarily those of the National Figure S6: Giardia supernatant protein profile and protein Health Service, the National Institute for Health Research, the concentration after incubation in nonsupplemented DMEM. Par- Department of Health, or Public Health England. We thank asites were chilled on ice for 15 minutes, washed 3 times in Susanne Warrenfeltz and the EuPathDB team for invaluable prewarmed PBS, centrifuged for 10 minutes at 3000 rpm be- assistance in making the integrated datasets available. tween each wash, and then incubated in prewarmed nonsupple- mented DMEM for 45 minutes at 37 C. After 45 minutes’ incuba- References tion, parasites were chilled on ice for 5 minutes and centrifuged for 10 minutes at 3000 rpm. Supernatants were collected, and 1. 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