Functions of CaPhm7 in the regulation of ion homeostasis, drug tolerance, filamentation and virulence in Candida albicans

Functions of CaPhm7 in the regulation of ion homeostasis, drug tolerance, filamentation and... Background: Calcium-permeable transient receptor potential (TRP) channels exist in eukaryotic cells from yeasts to animals and plants. and they act as sensors for various stresses. Arabidopsis thaliana calcium permeable stress-gated cation channel 1 (AtCSC1) was the first plant calcium-permeable TRP to be described and can be activated by hyperosmotic shock. Candida albicans CaPHM7 is one of the sequence homologs of AtCSC1, but its function remains unknown. Results: We show here that CaPhm7 is localized to the plasma membrane in both the yeast and hyphal cells of C. albicans. C. albicans cells lacking CaPHM7 are sensitive to SDS and ketoconazole but tolerant to rapamycin and zinc. In addition, deletion of CaPHM7 leads to a filamentation defect, reduced colony growth and attenuated virulence in the mouse model of systemic infection. Conclusions: CaPhm7 is involved in the regulation of ion homeostasis, drug tolerance, filamentation and virulence in this important human fungal pathogen. CaPhm7 could be a potential target of antifungal drugs. Keywords: Anoctamin, TMEM16, Calcium permeable stress-gated cation channel, Phm7, CSC1, Ion Background stress-gated cation channel 1 (CSC1) [8]. AtCSC1 was also Calcium homeostasis and calcium signaling are highly con- independently isolated through a genetic approach and 2+ served during evolution in eukaryotic cells [1, 2]. The cyto- named as reduced hyperosmolarity-induced [Ca ] in- solic calcium concentration is maintained by calcium crease 1 (OSCA1) [7]. AtCSC1 and its Saccharomyces cere- channels, pumps, exchangers and other regulators in the visiae homolog ScCSC1 also belong to the anoctamin/ plasma membrane and intracellular organelle membranes TMEM16 family [8]. of eukaryotic cells [2–4]. Elevation of the cytosolic calcium The anoctamin/TMEM16 family members function in concentration is one of the earliest responses to stress in many physiological processes, including ion homeostasis eukaryotic cells [5, 6]. Calcium-permeable transient recep- and phospholipid scrambling [14–16]. TMEM16A (also tor potential (TRP) channels exist in animals, plants and known as ANO1) and TMEM16B (ANO2) form the 2+ − yeasts, and act as sensors for various stresses, including Ca -activated Cl channel, which is important for temperature, pH, osmolarity and nutrient availability [7– transepithelial ion transport, olfaction, phototransduc- 13]. The first calcium-permeable TRP, initially isolated tion, smooth muscle contraction, nociception, cell from Arabidopsis thaliana, can be activated by hyperosmo- proliferation and control of neuronal excitability [14]. + − tic shock and, therefore, is named as calcium permeable The Ca2 -activated Cl channel formed by CaCC and TMEM16F regulates phospholipid scramblase [15]. TMEM16F itself also functions as a phospholipid * Correspondence: linghuojiang@sdut.edu.cn scramblase [16]. However, functions of other family Laboratory for Yeast Molecular and Cell Biology, the Research Center of members remain to be elucidated. Mutations in four Fermentation Technology, School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo 255000, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jiang and Pan BMC Microbiology (2018) 18:49 Page 2 of 8 anoctamin genes (ANO3, ANO5, ANO6, and ANO10) Table 2 Plasmids used in this study cause various genetic diseases. For example, mutations Plasmid Description Source in the plasma membrane protein TMEM16F impair CIp10 C. albicans integration vector [30] 2+ with URA3 marker Ca -dependent externalization of phosphatidylserine in activated platelets, erythrocytes and lymphocytes, which CIp10-CaPHM7 Full-length CaPHM7 gene in This study CIp10 leads to Scott syndrome, a bleeding disorder [15, 16]. In addition, a few other anoctamin-related diseases have pSFS2 The SAT1 flipper cassette [23] been identified, such as ataxia and dystonia, persistent pGFP-URA3 C-terminal GFP integration [24] with URA3 marker borrelia and mycobacteria infection, skeletal syndromes, gnathodiaphyseal dysplasia as well as cancers [17]. TMEM16C (ANO3) is associated with childhood mumps DNA manipulation and rubella vaccination-related febrile seizures [18]. There is one PstI site (CTGCAG), which contains a codon Candida albicans is the most common opportunistic (bolded), encoding the A499 residue of CaPhm7, in both human fungal pathogen, which causes life-threatening sys- alleles A and B of the CaPHM7 gene. However, due to the temic infections in immunocompromised patients [19, presence of a synonymous coding single nucleotide poly- 20]. To explore the biological functions of C. albicans ho- morphism (GCA→GCT) (cSNP) between alleles A and B mologs of CSC1 and other anoctamin/TMEM16 family of the CaPHM7 gene encoding the A705 residue of members, using the amino acid sequence of AtCsc1 as a CaPhm7, there is an additional PstI site (CTGCAG) over- query, we blasted the C. albicans genome and revealed lapping with this cSNP site in the allele A of CaPHM7. five AtCsc1 homologous proteins, CaPhm7, CaRsn1, Therefore, using the primer pair pRS316-CaPHM7-F/ CaSpo75, orf19.1823 (C1_06270W) and orf19.5932 CaPHM7 TY-UP-R, we first amplified the DNA fragment (C3_04720C). CaPhm7 is a protein of 927 amino acids, containing the 739-bp promoter and the upstream which shares 21% (42%), 36% (59%), 26% (44%), 20% 1498-bp ORF region of CaPHM7, cut it with HindIII and (41%) and 21% (42%) identities (similarities) with AtCsc1, PstI, and cloned it into the CIp10 vector, yielding CaRsn1, CaSpo75, C1_06270Wp and C3_04720Cp, CIp10-CaPHM7UP. Second, using the primer pair respectively, in amino acid sequences (Additional file 1: CaPHM7 TY-DOWN-F/CaPHM7 TY-DOWN-R, from Figure S1). In this study, we have characterized the bio- thealleleBof CaPHM7 we amplified a 1.58-kb DNA frag- logical functions of CaPHM7 in C. albicans. ment containing the downstream 1307-bp ORF region and the 321-bp terminator region of CaPHM7, cut with Methods PstI, and clone the 1.58-kb DNA fragment into the PstI Strains, media, plasmids and primers site of CIp10-CaPHM7UP, generating CIp10-CaPHM7. C. albicans strains and plasmids used in this study are These inserts were sequenced to ensure no mutation and listed in Tables 1 and 2, respectively. Primers used in this correct orientation. study are listed in Additional file 1: Table S1. C. albicans cells were maintained in YPD medium (2% glucose, 2% Disruption of two alleles for CaPHM7 peptone, 1% yeast extract) or SD medium (0.67% yeast ni- To delete the first allele of CaPHM7 in the CAI4 back- trogen base without amino acids, 2% glucose, and auxo- ground, we PCR amplified the SAT1 flipper cassette with trophic amino acids as needed) at 30 °C. Antifungal drugs primers CaPHM7-NAT-UP and CaPHM7-NAT-DOWN were obtained from Sigma. from the plasmid pSFS2 [21–23], and transformed it into the CAI4 strain. The resulting PHBCA57 strain (PHM7/ Table 1 Strains used in this study phm7::natR) was grown in YPD medium containing − 1 Strain Genotype Source 25 μgml nourseothricin for FLP-mediated excision of the SAT1 cassette, which generated PHBCA58 strain CAI4 ura3::λimm434/ura3::λimm434 [38] (PHM7/phm7::frt). Similarly, we disrupted the second al- PHBCA57 CAI4 PHM7/phm7::natR This study lele, which generated the PHBCA62 strain (phm7::frt/ PHBCA58 CAI4 PHM7/phm7::frt This study phm7::natR) (Additional file 1: Figure S2). PHBCA62 CAI4 phm7::frt/phm7::natR This study XDCA06 CAI4/CIp10 [32] Chromosomal tagging of CaPhm7 with green fluorescent PHBCA100 CAI4 PHM7/phm7::frt/CIp10 This study protein (GFP) To generate a C-terminal GFP-tagged CaPHM7-GFP fu- PHBCA102 CAI4 phm7::frt/phm7::frt/CIp10 This study sion protein, from the plasmid pGFP-URA3 [24], we used PHBCA105 CAI4 phm7::frt/phm7::frt/CIp10-CaPHM7 This study the primers CaPHM7-GFP-UP and CaPHM7-GFP-DOWN PHBCA107 CAI4 PHM7/PHM7::GFP-URA3 This study to amplify the GFP-URA3 cassette flanked with 100-bp PHBCA126 CAI4 PHM7::GFP-URA3/phm7::natR This study CaPHM7 DNA fragments. This cassette was transformed Jiang and Pan BMC Microbiology (2018) 18:49 Page 3 of 8 into both the wild type CAI4 and the heterozygous mutant accordance with the guidelines established by the Ethics PHBCA57. Genotypes of the resulted CaPHM7-GFP Committee of Nantong University, China. strains PHBCA107 and PHBCA126 were verified by PCR with primer pairs CaPHM7-GFP-F/CaPHM7-GFP-R and Results GFP-R/CaURA3-F, respectively (Data not shown). Deletion of CaPHM7 causes C. albicans cells sensitive to SDS and ketoconazole and tolerant to rapamycin and zinc ion Phenotype tests Unlike AtCsc1 with nine TM domains, CaPhm7 has 11 Test strains were grown overnight in liquid medium at TM domains (Additional file 1: Figure S1). However, 30 °C, serially diluted by 10-fold and spotted sequentially CaPhm7 is highly conserved in ascomycetes including onto plates as described previously [25, 26]. Plates were other human pathogenic Candida species, and shares 94% photographed after they were incubated for 2–3daysat (98%), 42% (64%), 82% (91%), 43% (64%) and 46% (67%) 30 °C. Filamentation was assessed by inoculating cells with identity (similarity), respectively, with C. dubliniensis an appropriate dilution into various filamentation-induction CdPhm7, C. glabrata CgPhm7, C. tropicalis CtPhm7, liquid media, and cells were grown with shaking at 37 °C. S. cerevisiae ScPhm7 and Schizosaccharomyces pombe For colony morphology observation 20 cells from each SpPhm7 (Additional file 1: Figure S3). To understand strain was plated onto YPD 10% serum, Lee’sand Spider the biological functions of CaPhm7 in C. albicans, medium [27, 28], and incubated for 5–7 days at 37 °C. we disrupted the two alleles of CaPHM7 (Table 1; These experiments were repeated at least three times. Additional file 1:FigureS2). Ascomparedtothe wild type, the homozygous mutant was sensitive to SDS and Growth curve assay tolerant to rapamycin (Fig. 1a). In contrast, both the het- Cells of the wild type CAI4 + CIp10, the heterozygous mu- erozygous mutant and the homozygous mutant were sen- tant PHM7/phm7 + CIp10, the homozygous mutant sitive to ketoconazole (KCZ) and tolerant to ZnCl phm7/phm7 + CIp10 and the revertant phm7/phm7 + (Fig. 1b). However, no alternation was observed in the CIp10-CaPHM7 were cultured overnight in SD-URA sensitivity of these mutants to other agents such as cal- medium at 30 °C, serially diluted for 10 times in fresh cium (0.6 M), manganese (1 mM), magnesium (10 mM), YPD medium and grown further for 10 h. Culture samples sodium (1.5 M), potassium (1.5 M), lithium and cadmium of each strain were taken every 2 h for OD measure- (100 μM) ions (Data not shown). The SDS-sensitive, 600nm ment. Data were the average of three independent experi- rapamycin-tolerant and zinc-tolerant phenotypes of the ments and analyzed by GraphPad Prism software. P values homozygous mutant could be reversed by introducing one of < 0.05 were considered to be significant. allele of the CaPHM7 gene (Fig. 1). However, it should be noted that reintroduction of CaPHM7 only partially re- Virulence assays stored growth of the homozygous mutant on ketocona- Strains tested for virulence were integrated with the zole. On zinc, the revertant (one copy of CaPHM7) plasmid CIp10, so that URA3 was expressed from the restored the wild type phenotype, but the heterozygous neutral RPS1 locus [29, 30]. Phenotypes of all mutant grew at levels similar to the homozygous mutant CIp10-integrated mutants were confirmed (Data not (Fig. 1). Furthermore, the colonies of both the homozy- shown). Male BALB/c mice of 8 weeks old and 25–30 g gous mutant and the revertant were slightly smaller than (Geruisiwei Inc., Suzhou, China) were maintained in in- those of the wild type on solid YPD plates (Fig. 1). How- dividually ventilated cages. Cell suspension at a concen- ever, the growth rates of both the homozygous mutant 7 − 1 tration of 1 × 10 cells ml in 0.9% (w/v) NaCl solution and the revertant were not significantly different from the was prepared for each strain. Twelve mice for each wild type (Additional file 1:FigureS4). strain were inoculated with 0.1 ml of cell suspension per mouse via a lateral tail vein. Mouse survival rates were CaPhm7 is localized to the plasma membrane monitored daily. If mice appeared to be curled up, hair To examine the subcellular localization of CaPhm7 pro- fluffy, dull and not responsive to outside stimuli, they teins, we integrated the GFP tag at the C-terminus of the were sacrificed through cervical vertebra. Survival curves CaPHM7 allele in the wild type CAI4 and the heterozygous were generated according to the Kaplan–Meier method mutant, which generated PHBCA107 and PHBCA126 using the PRISM program (GraphPad Software) and strains, respectively. Both showed similar phenotypes to the compared using the log-rank test. wild type CAI4 on plates containing SDS, ketoconazole, After 48 h of inoculation, two mice were randomly exe- rapamycin or ZnCl2 (Fig. 2a), indicating the CaPHM7-GFP cuted, and the colony forming unit (CFU) in their kidneys allele is functional. Western blot analysis indicated that the was analyzed on SD-URA plates as described [31, 32]. CaPHM7-GFP expressed as a protein of 126 kDa, as Stained sections of kidney tissues were examined for fun- expected, in cells of both PHBCA107 and PHBCA126 gal infiltration. Mouse studies were carried out in (Additional file 1:FigureS5).ThisCaPHM7-GFPprotein Jiang and Pan BMC Microbiology (2018) 18:49 Page 4 of 8 Fig. 1 C. albicans cells lacking CaPHM7 are sensitive to SDS and ketoconazole (KCZ) (a) and tolerant to rapamycin and zinc (b). The wild type, the heterozygous mutant and the homozygous mutant, integrated with the CIp10 vector or CIp10-CaPHM7, were grown overnight in SD-URA medium, serially diluted by 10 times and spotted onto YPD plates indicated. Plates were incubated at 30 °C for 48 h (a)or72h(b) before photos were taken. KCZ, ketoconazole; Rapa, rapamycin was localized to the plasma membrane in both the cells of C. albicans was observed in the kidney tissues of log-phase yeast-form cells and the filamentous cells of the mice inoculated with saline as a control (Fig. 4b). Com- PHBCA107 strain induced in YPD + 10% FBS, Spider or pared to kidney tissues of mice infected with the wild Lee’smedia (Fig. 2b). Taken together, these data indicate type and the complemented strain, those infected with that CaPhm7 is a plasma membrane protein. the homozygous mutant were less infiltrated by hyphal filaments (Fig. 4b). Taken together, these results suggest Deletion of CaPHM7 leads to a defect in filamentation, that deletion of CaPHM7 attenuates the virulence of C. reduced colony growth and attenuated virulence albicans cells in this systemic infection model. Hyphal development is related to the virulence of C. albi- cans cells [20, 33]. To examine the function of CaPHM7 in Discussion hyphal development, we performed filamentous growth as- In this study, we have shown that the C. albicans homolog says. In liquid YPD plus 10% FBS, Lee’sorSpidermedia, of AtCSC1, CaPhm7, is localized in the plasma membrane the homozygous mutant showed a defect in filamentation of both yeast-form and filamentous cells. C. albicans cells (Fig. 3a). On these solid media, comparing to the wide type lacking CaPHM7 are sensitive to membrane-disturbing and the heterozygous mutant, the homozygous mutant agents, SDS and ketoconazole. AtCSC1 and ScCSC1 ion 2+ showed reduced colony sizes on solid YPD plus 10% FBS, channels are permeable to various cations, including Ca , + + Lee’sand Spider media (Fig. 3b). These defects of the K and Na and can be activated by hyperosmotic shock 2+ homozygous mutant could be complemented by reintro- [8]. However, lack of CaPHM7 leads to a tolerance to Zn , duction of a single copy of the CaPHM7 allele (Fig. 3). but does not affect the sensitivity of C. albicans cells to 2+ + + 2+ 2+ To determine the role of CaPHM7 in C. albicans viru- other ions tested, including Ca ,K ,Na ,Mg ,Mn , 6 2+ + lence, we infected mice intravenously with 1 × 10 cells Cd ,and Li . C. albicans cells lacking CaPHM7 are also of the wild-type parental control strain and the homozy- not sensitive to osmotic stresses such as 1 M mannitol and gous mutant. Two mice were randomly selected from 4 M KCl. Therefore, whether CaPhm7 is a calcium perme- each group and sacrificed after 48 h to make sure that able stress-gated cation channel in the plasma membrane mice had been infected, with the CFUs/g reaching remains to be determined. It is also interesting to note that 1.02 × 10 (n = 2) in wet kidney tissue of the homozygous haploinsufficiency occurs to the deletion of one of the mutant, 1.56 × 10 (n = 2) in that of the wild-type and CaPHM7 alleles in the response to ketoconazole and zinc 1.38 × 10 (n = 2) in that of the complemented strain. By ion, but not to SDS and rapamycin (Fig. 1b). Among anoc- Day 6, none of the mice injected with the wild type sur- tamins, ANO10 shows the highest degree of homology to vived, while 30% and 80% of the mice inoculated with ScIst2, with a 36% similarity and a 17% identity in their the complemented strain and the homozygous mutant, amino acid sequences [17]. CaPhm7 shares 18% (34%) and respectively, survived. By Day 8, none of the mice inocu- 14% (30%) identity (similarity) with HsAno10 and ScPhm7, lated with the complemented strain survived, while the respectively, in their amino acid sequences (Additional file 1: mice injected with the homozygous mutant survived Figure S6). This suggests that CaPhm7 might be a member until Day 20 (Fig. 4a). As a control, no hyphal or yeast of the anoctamin/TMEM16 family member. Jiang and Pan BMC Microbiology (2018) 18:49 Page 5 of 8 Fig. 2 Subcellular localization and mouse virulence assay for CaPhm7. a Functional assay of the CaPHM7-GFP allele. The wild type CAI4 (XDCA06), the heterozygous mutant PHBCA100, the homozygous mutant PHBCA 102, as well as PHBCA107 and PHBCA126, carrying the CaPHM7-GFP allele, were grown overnight in SD-URA medium, serially diluted by 10 times and spotted onto YPD plates indicated. b Visualization of GFP signals within PHBCA107 cells of log-phase yeast-form and filamentous form induced in YPD + 10% FBS (top left), Spider (bottom left) or Lee’s (bottom right) media at 1000 × magnification on a Nikon ECLIPSE 80i microscope. Visualization of the wild type CAI4 cells (top right) as a control for hyphal morphology and green auto-fluorescence. DIC, differential interference contrast. The scale bars represent 5 μM In addition, we have shown that C. albicans cells lacking 7 and, together with other 21 genes, is regulated by the CaPHM7 are tolerant to rapamycin, the specific inhibitor PHO regulatory pathway in S. cerevisiae [35]. However, the of the target of rapamycin (TOR) kinase, which indicates function of ScPHM7 in phosphate metabolism is not de- that the TOR kinase activity might be inhibited in C. albi- fined up to now. ScPhm7 shares a 43% identity and 64% cans cells lacking CaPHM7.Thisisconsistent with our similarity, respectively, with CaPhm7 in amino acid se- observations that deletion of CaPHM7 leads to a reduced quence (Additional file 1: Figure S1). Our results on colony growth, which might be related to a reduced activ- CaPHM7 suggests that ScPhm7 might be a plasma mem- ity for the central controller of cell growth, TOR [34]. brane CSC ion channel that links the extracellular phos- The S. cerevisiae homolog of CaPHM7, ScPHM7 has ini- phate availability to the intracellular calcium signaling tially been identified to be the phosphate metabolism gene pathway to regulate phosphate metabolism. Recent studies Jiang and Pan BMC Microbiology (2018) 18:49 Page 6 of 8 Fig. 3 Functions of CaPHM7 in filamentation and colony formation. The wild type CAI4, the heterozygous mutant and the homozygous mutant, carrying the CIp10 vector or CIp10-CaPHM7, were grown overnight in SD-URA medium, and inoculated to liquid YPD containing 10% fetal bovine serum (FBS), Lee’s or Spider media for filamentation assay (a) or onto their solid plates for colony morphology oberservation (b). Incubation was carried out at 37 °C. The scale bar in (a) represents 10 μM, while the scale bar in (b) represents 5 mm Jiang and Pan BMC Microbiology (2018) 18:49 Page 7 of 8 infection. Similar to the functions of other members of the anoctamin/TMEM16 family as sensors for various stresses including nutrient availability [7–13], the plasma membrane protein CaPhm7 might function to regulate zinc availability for C. albicans cells in media or within the host environment and thereby affect their filamenta- tion and virulence. Conclusion In this study, we have demonstrated that CaPhm7 is lo- calized to the plasma membrane in both yeast-form and filamentous cells of C. albicans. CaPhm7 regulates ion homeostasis, drug tolerance, filamentation and virulence in this important human fungal pathogen. C. albicans cells lacking CaPHM7 have a defect in filamentation and attenuated virulence in the systemic mouse model. Therefore, CaPhm7 is a potential target for antifungal development. Additional file Additional file 1: Figure S1. Amino acid sequence comparison. Transmembrane domain (TM) regions of AtCsc1 and CaPhm7 are underlined, and TM names of AtCsc1 (blue) and CaPhm7 (red) are indicated. TMs were predicted with SMART (http://smart.embl- heidelberg.de/). Identical amino acid residues are indicated in yellow in color, and similar amino acid residues are indicated in light blue and Fig. 4 Virulence assay. a survival rates of mice (n = 10) infected with green. Figure S2. Disruption of CaPHM7. (A) Disruption strategy of the the wide type CAI4, the homozygous phm7/phm7 mutant and its two alleles. PCR confirmation of genotypes of the homozygous mutant complemented strain. Mice were checked every day for morbidity, and PHBCA62 with primer pairs CaPHM7-ORF-UP/DOWN (B), CaPHM7-DF/DR (C) as well as CaPHM7-DF/NAT-R (middle lane) and CaPHM7-DR/NAT-F survival was monitored for 30 days. The p value (0.009) of the log-rank (last lane) (D). Figure S3. TMs of AtCsc1, ScPhm7 and CaPhm7 as well as test indicates that the survival curves between the wild-type CAI4 and their amino acid sequence comparison. Identical amino acid residues are the phm7/phm7 mutant is significantly different. b histopathological indicated in yellow in color, and similar amino acid residues are indicated examination of kidney tissues of moribund mice infected with the in light blue and green. Figure S4. Growth assay. Cells were cultured in wild-type, the homozygous mutant and its complemented strain. SD-URA medium at 30 °C overnight, diluted for 10 times in fresh YPD Saline buffer was injected as negative control (CK). Infected kidney medium and grown further for indicated hours. Data were the average of tissues from mice were stained with periodic acid-Schiff’s reagent. three independent experiments. Growth rates of the heterozygous mu- Representatives of five kidney cross-sections from two mice per strain tant, the homozygous mutant and the revertant were not significantly were photographed with 40× lenses. The hyphal cells are highlighted different from the wild type. Figure S5. Western blot analysis. The CAI4 control, PHBCA107 and PHBCA126 cells were grown to log-phase and by arrows. The scale bars represent 20 μM collected for total protein extraction. Western blot analysis was carried out with the monoclonal anti-GFP antibody. The CaPHM7-GFP protein of 126 kDa was indicated. Figure S6. Amino acid sequence comparison. have indeed linked the calcium signaling pathway to phos- CaPhm7 shares 18% (34%) and 14% (30%) identity (similarity) with hu- phate metabolism. Both the PHO4 gene, encoding the man HsAno10 and S. cerevisiae ScIst2, respectively. Identical amino acid residues are indicated in yellow in color, and similar amino acid residues transcription factor of the PHO pathway, and the PHO89 are indicated in light blue and green. Table S1. Primers used in this gene, encoding the high-affinity inorganic phosphate (Pi) study. (PDF 5687 kb) transporter, are transcriptionally regulated by the calcium/ calcineurin signaling in S. cerevisiae [36]. Calcineurin is Abbreviations also involved in the regulation of the PHO pathway in As- CSC: Calcium permeable stress-gated cation channel; TRP: Calcium- permeable transient receptor potential pergillus fumigatus [37]. Therefore, like AtScs1 as an osmolarity sensor in the plant A. thaliana [7, 8], ScPhm7 Acknowledgements might function as a sensor for extracellular phosphate We are grateful to Jianqing Cheng for his technical support. levels in S. cerevisiae. In this study, we have observed that the homozygous Funding This work is supported by the National Natural Science Foundation of China mutant for CaPHM7 shows a filamentation defect, to LJ (No. 81571966 and No. 81371784). The funding body plays no role in which might contribute to its reduced colony growth the design of the study and collection, analysis, and interpretation of data and attenuated virulence in the mouse model of systemic and in writing the manuscript. Jiang and Pan BMC Microbiology (2018) 18:49 Page 8 of 8 2+ + Availability of data and materials 22. Wang Y, et al. Genetic interactions between the Golgi Ca /H exchanger The datasets used and/or analyzed during the current study are available Gdt1 and the plasma membrane calcium channel Cch1/Mid1 in the from the corresponding author on reasonable request. regulation of calcium homeostasis, stress response and virulence in Candida albicans. FEMS Yeast Res. 2015;15(7). 23. Reuss O, Vik A, Kolter R, Morschhauser J. 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Functions of CaPhm7 in the regulation of ion homeostasis, drug tolerance, filamentation and virulence in Candida albicans

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Life Sciences; Microbiology; Biological Microscopy; Mycology; Parasitology; Virology; Life Sciences, general
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Abstract

Background: Calcium-permeable transient receptor potential (TRP) channels exist in eukaryotic cells from yeasts to animals and plants. and they act as sensors for various stresses. Arabidopsis thaliana calcium permeable stress-gated cation channel 1 (AtCSC1) was the first plant calcium-permeable TRP to be described and can be activated by hyperosmotic shock. Candida albicans CaPHM7 is one of the sequence homologs of AtCSC1, but its function remains unknown. Results: We show here that CaPhm7 is localized to the plasma membrane in both the yeast and hyphal cells of C. albicans. C. albicans cells lacking CaPHM7 are sensitive to SDS and ketoconazole but tolerant to rapamycin and zinc. In addition, deletion of CaPHM7 leads to a filamentation defect, reduced colony growth and attenuated virulence in the mouse model of systemic infection. Conclusions: CaPhm7 is involved in the regulation of ion homeostasis, drug tolerance, filamentation and virulence in this important human fungal pathogen. CaPhm7 could be a potential target of antifungal drugs. Keywords: Anoctamin, TMEM16, Calcium permeable stress-gated cation channel, Phm7, CSC1, Ion Background stress-gated cation channel 1 (CSC1) [8]. AtCSC1 was also Calcium homeostasis and calcium signaling are highly con- independently isolated through a genetic approach and 2+ served during evolution in eukaryotic cells [1, 2]. The cyto- named as reduced hyperosmolarity-induced [Ca ] in- solic calcium concentration is maintained by calcium crease 1 (OSCA1) [7]. AtCSC1 and its Saccharomyces cere- channels, pumps, exchangers and other regulators in the visiae homolog ScCSC1 also belong to the anoctamin/ plasma membrane and intracellular organelle membranes TMEM16 family [8]. of eukaryotic cells [2–4]. Elevation of the cytosolic calcium The anoctamin/TMEM16 family members function in concentration is one of the earliest responses to stress in many physiological processes, including ion homeostasis eukaryotic cells [5, 6]. Calcium-permeable transient recep- and phospholipid scrambling [14–16]. TMEM16A (also tor potential (TRP) channels exist in animals, plants and known as ANO1) and TMEM16B (ANO2) form the 2+ − yeasts, and act as sensors for various stresses, including Ca -activated Cl channel, which is important for temperature, pH, osmolarity and nutrient availability [7– transepithelial ion transport, olfaction, phototransduc- 13]. The first calcium-permeable TRP, initially isolated tion, smooth muscle contraction, nociception, cell from Arabidopsis thaliana, can be activated by hyperosmo- proliferation and control of neuronal excitability [14]. + − tic shock and, therefore, is named as calcium permeable The Ca2 -activated Cl channel formed by CaCC and TMEM16F regulates phospholipid scramblase [15]. TMEM16F itself also functions as a phospholipid * Correspondence: linghuojiang@sdut.edu.cn scramblase [16]. However, functions of other family Laboratory for Yeast Molecular and Cell Biology, the Research Center of members remain to be elucidated. Mutations in four Fermentation Technology, School of Agricultural Engineering and Food Science, Shandong University of Technology, Zibo 255000, China © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Jiang and Pan BMC Microbiology (2018) 18:49 Page 2 of 8 anoctamin genes (ANO3, ANO5, ANO6, and ANO10) Table 2 Plasmids used in this study cause various genetic diseases. For example, mutations Plasmid Description Source in the plasma membrane protein TMEM16F impair CIp10 C. albicans integration vector [30] 2+ with URA3 marker Ca -dependent externalization of phosphatidylserine in activated platelets, erythrocytes and lymphocytes, which CIp10-CaPHM7 Full-length CaPHM7 gene in This study CIp10 leads to Scott syndrome, a bleeding disorder [15, 16]. In addition, a few other anoctamin-related diseases have pSFS2 The SAT1 flipper cassette [23] been identified, such as ataxia and dystonia, persistent pGFP-URA3 C-terminal GFP integration [24] with URA3 marker borrelia and mycobacteria infection, skeletal syndromes, gnathodiaphyseal dysplasia as well as cancers [17]. TMEM16C (ANO3) is associated with childhood mumps DNA manipulation and rubella vaccination-related febrile seizures [18]. There is one PstI site (CTGCAG), which contains a codon Candida albicans is the most common opportunistic (bolded), encoding the A499 residue of CaPhm7, in both human fungal pathogen, which causes life-threatening sys- alleles A and B of the CaPHM7 gene. However, due to the temic infections in immunocompromised patients [19, presence of a synonymous coding single nucleotide poly- 20]. To explore the biological functions of C. albicans ho- morphism (GCA→GCT) (cSNP) between alleles A and B mologs of CSC1 and other anoctamin/TMEM16 family of the CaPHM7 gene encoding the A705 residue of members, using the amino acid sequence of AtCsc1 as a CaPhm7, there is an additional PstI site (CTGCAG) over- query, we blasted the C. albicans genome and revealed lapping with this cSNP site in the allele A of CaPHM7. five AtCsc1 homologous proteins, CaPhm7, CaRsn1, Therefore, using the primer pair pRS316-CaPHM7-F/ CaSpo75, orf19.1823 (C1_06270W) and orf19.5932 CaPHM7 TY-UP-R, we first amplified the DNA fragment (C3_04720C). CaPhm7 is a protein of 927 amino acids, containing the 739-bp promoter and the upstream which shares 21% (42%), 36% (59%), 26% (44%), 20% 1498-bp ORF region of CaPHM7, cut it with HindIII and (41%) and 21% (42%) identities (similarities) with AtCsc1, PstI, and cloned it into the CIp10 vector, yielding CaRsn1, CaSpo75, C1_06270Wp and C3_04720Cp, CIp10-CaPHM7UP. Second, using the primer pair respectively, in amino acid sequences (Additional file 1: CaPHM7 TY-DOWN-F/CaPHM7 TY-DOWN-R, from Figure S1). In this study, we have characterized the bio- thealleleBof CaPHM7 we amplified a 1.58-kb DNA frag- logical functions of CaPHM7 in C. albicans. ment containing the downstream 1307-bp ORF region and the 321-bp terminator region of CaPHM7, cut with Methods PstI, and clone the 1.58-kb DNA fragment into the PstI Strains, media, plasmids and primers site of CIp10-CaPHM7UP, generating CIp10-CaPHM7. C. albicans strains and plasmids used in this study are These inserts were sequenced to ensure no mutation and listed in Tables 1 and 2, respectively. Primers used in this correct orientation. study are listed in Additional file 1: Table S1. C. albicans cells were maintained in YPD medium (2% glucose, 2% Disruption of two alleles for CaPHM7 peptone, 1% yeast extract) or SD medium (0.67% yeast ni- To delete the first allele of CaPHM7 in the CAI4 back- trogen base without amino acids, 2% glucose, and auxo- ground, we PCR amplified the SAT1 flipper cassette with trophic amino acids as needed) at 30 °C. Antifungal drugs primers CaPHM7-NAT-UP and CaPHM7-NAT-DOWN were obtained from Sigma. from the plasmid pSFS2 [21–23], and transformed it into the CAI4 strain. The resulting PHBCA57 strain (PHM7/ Table 1 Strains used in this study phm7::natR) was grown in YPD medium containing − 1 Strain Genotype Source 25 μgml nourseothricin for FLP-mediated excision of the SAT1 cassette, which generated PHBCA58 strain CAI4 ura3::λimm434/ura3::λimm434 [38] (PHM7/phm7::frt). Similarly, we disrupted the second al- PHBCA57 CAI4 PHM7/phm7::natR This study lele, which generated the PHBCA62 strain (phm7::frt/ PHBCA58 CAI4 PHM7/phm7::frt This study phm7::natR) (Additional file 1: Figure S2). PHBCA62 CAI4 phm7::frt/phm7::natR This study XDCA06 CAI4/CIp10 [32] Chromosomal tagging of CaPhm7 with green fluorescent PHBCA100 CAI4 PHM7/phm7::frt/CIp10 This study protein (GFP) To generate a C-terminal GFP-tagged CaPHM7-GFP fu- PHBCA102 CAI4 phm7::frt/phm7::frt/CIp10 This study sion protein, from the plasmid pGFP-URA3 [24], we used PHBCA105 CAI4 phm7::frt/phm7::frt/CIp10-CaPHM7 This study the primers CaPHM7-GFP-UP and CaPHM7-GFP-DOWN PHBCA107 CAI4 PHM7/PHM7::GFP-URA3 This study to amplify the GFP-URA3 cassette flanked with 100-bp PHBCA126 CAI4 PHM7::GFP-URA3/phm7::natR This study CaPHM7 DNA fragments. This cassette was transformed Jiang and Pan BMC Microbiology (2018) 18:49 Page 3 of 8 into both the wild type CAI4 and the heterozygous mutant accordance with the guidelines established by the Ethics PHBCA57. Genotypes of the resulted CaPHM7-GFP Committee of Nantong University, China. strains PHBCA107 and PHBCA126 were verified by PCR with primer pairs CaPHM7-GFP-F/CaPHM7-GFP-R and Results GFP-R/CaURA3-F, respectively (Data not shown). Deletion of CaPHM7 causes C. albicans cells sensitive to SDS and ketoconazole and tolerant to rapamycin and zinc ion Phenotype tests Unlike AtCsc1 with nine TM domains, CaPhm7 has 11 Test strains were grown overnight in liquid medium at TM domains (Additional file 1: Figure S1). However, 30 °C, serially diluted by 10-fold and spotted sequentially CaPhm7 is highly conserved in ascomycetes including onto plates as described previously [25, 26]. Plates were other human pathogenic Candida species, and shares 94% photographed after they were incubated for 2–3daysat (98%), 42% (64%), 82% (91%), 43% (64%) and 46% (67%) 30 °C. Filamentation was assessed by inoculating cells with identity (similarity), respectively, with C. dubliniensis an appropriate dilution into various filamentation-induction CdPhm7, C. glabrata CgPhm7, C. tropicalis CtPhm7, liquid media, and cells were grown with shaking at 37 °C. S. cerevisiae ScPhm7 and Schizosaccharomyces pombe For colony morphology observation 20 cells from each SpPhm7 (Additional file 1: Figure S3). To understand strain was plated onto YPD 10% serum, Lee’sand Spider the biological functions of CaPhm7 in C. albicans, medium [27, 28], and incubated for 5–7 days at 37 °C. we disrupted the two alleles of CaPHM7 (Table 1; These experiments were repeated at least three times. Additional file 1:FigureS2). Ascomparedtothe wild type, the homozygous mutant was sensitive to SDS and Growth curve assay tolerant to rapamycin (Fig. 1a). In contrast, both the het- Cells of the wild type CAI4 + CIp10, the heterozygous mu- erozygous mutant and the homozygous mutant were sen- tant PHM7/phm7 + CIp10, the homozygous mutant sitive to ketoconazole (KCZ) and tolerant to ZnCl phm7/phm7 + CIp10 and the revertant phm7/phm7 + (Fig. 1b). However, no alternation was observed in the CIp10-CaPHM7 were cultured overnight in SD-URA sensitivity of these mutants to other agents such as cal- medium at 30 °C, serially diluted for 10 times in fresh cium (0.6 M), manganese (1 mM), magnesium (10 mM), YPD medium and grown further for 10 h. Culture samples sodium (1.5 M), potassium (1.5 M), lithium and cadmium of each strain were taken every 2 h for OD measure- (100 μM) ions (Data not shown). The SDS-sensitive, 600nm ment. Data were the average of three independent experi- rapamycin-tolerant and zinc-tolerant phenotypes of the ments and analyzed by GraphPad Prism software. P values homozygous mutant could be reversed by introducing one of < 0.05 were considered to be significant. allele of the CaPHM7 gene (Fig. 1). However, it should be noted that reintroduction of CaPHM7 only partially re- Virulence assays stored growth of the homozygous mutant on ketocona- Strains tested for virulence were integrated with the zole. On zinc, the revertant (one copy of CaPHM7) plasmid CIp10, so that URA3 was expressed from the restored the wild type phenotype, but the heterozygous neutral RPS1 locus [29, 30]. Phenotypes of all mutant grew at levels similar to the homozygous mutant CIp10-integrated mutants were confirmed (Data not (Fig. 1). Furthermore, the colonies of both the homozy- shown). Male BALB/c mice of 8 weeks old and 25–30 g gous mutant and the revertant were slightly smaller than (Geruisiwei Inc., Suzhou, China) were maintained in in- those of the wild type on solid YPD plates (Fig. 1). How- dividually ventilated cages. Cell suspension at a concen- ever, the growth rates of both the homozygous mutant 7 − 1 tration of 1 × 10 cells ml in 0.9% (w/v) NaCl solution and the revertant were not significantly different from the was prepared for each strain. Twelve mice for each wild type (Additional file 1:FigureS4). strain were inoculated with 0.1 ml of cell suspension per mouse via a lateral tail vein. Mouse survival rates were CaPhm7 is localized to the plasma membrane monitored daily. If mice appeared to be curled up, hair To examine the subcellular localization of CaPhm7 pro- fluffy, dull and not responsive to outside stimuli, they teins, we integrated the GFP tag at the C-terminus of the were sacrificed through cervical vertebra. Survival curves CaPHM7 allele in the wild type CAI4 and the heterozygous were generated according to the Kaplan–Meier method mutant, which generated PHBCA107 and PHBCA126 using the PRISM program (GraphPad Software) and strains, respectively. Both showed similar phenotypes to the compared using the log-rank test. wild type CAI4 on plates containing SDS, ketoconazole, After 48 h of inoculation, two mice were randomly exe- rapamycin or ZnCl2 (Fig. 2a), indicating the CaPHM7-GFP cuted, and the colony forming unit (CFU) in their kidneys allele is functional. Western blot analysis indicated that the was analyzed on SD-URA plates as described [31, 32]. CaPHM7-GFP expressed as a protein of 126 kDa, as Stained sections of kidney tissues were examined for fun- expected, in cells of both PHBCA107 and PHBCA126 gal infiltration. Mouse studies were carried out in (Additional file 1:FigureS5).ThisCaPHM7-GFPprotein Jiang and Pan BMC Microbiology (2018) 18:49 Page 4 of 8 Fig. 1 C. albicans cells lacking CaPHM7 are sensitive to SDS and ketoconazole (KCZ) (a) and tolerant to rapamycin and zinc (b). The wild type, the heterozygous mutant and the homozygous mutant, integrated with the CIp10 vector or CIp10-CaPHM7, were grown overnight in SD-URA medium, serially diluted by 10 times and spotted onto YPD plates indicated. Plates were incubated at 30 °C for 48 h (a)or72h(b) before photos were taken. KCZ, ketoconazole; Rapa, rapamycin was localized to the plasma membrane in both the cells of C. albicans was observed in the kidney tissues of log-phase yeast-form cells and the filamentous cells of the mice inoculated with saline as a control (Fig. 4b). Com- PHBCA107 strain induced in YPD + 10% FBS, Spider or pared to kidney tissues of mice infected with the wild Lee’smedia (Fig. 2b). Taken together, these data indicate type and the complemented strain, those infected with that CaPhm7 is a plasma membrane protein. the homozygous mutant were less infiltrated by hyphal filaments (Fig. 4b). Taken together, these results suggest Deletion of CaPHM7 leads to a defect in filamentation, that deletion of CaPHM7 attenuates the virulence of C. reduced colony growth and attenuated virulence albicans cells in this systemic infection model. Hyphal development is related to the virulence of C. albi- cans cells [20, 33]. To examine the function of CaPHM7 in Discussion hyphal development, we performed filamentous growth as- In this study, we have shown that the C. albicans homolog says. In liquid YPD plus 10% FBS, Lee’sorSpidermedia, of AtCSC1, CaPhm7, is localized in the plasma membrane the homozygous mutant showed a defect in filamentation of both yeast-form and filamentous cells. C. albicans cells (Fig. 3a). On these solid media, comparing to the wide type lacking CaPHM7 are sensitive to membrane-disturbing and the heterozygous mutant, the homozygous mutant agents, SDS and ketoconazole. AtCSC1 and ScCSC1 ion 2+ showed reduced colony sizes on solid YPD plus 10% FBS, channels are permeable to various cations, including Ca , + + Lee’sand Spider media (Fig. 3b). These defects of the K and Na and can be activated by hyperosmotic shock 2+ homozygous mutant could be complemented by reintro- [8]. However, lack of CaPHM7 leads to a tolerance to Zn , duction of a single copy of the CaPHM7 allele (Fig. 3). but does not affect the sensitivity of C. albicans cells to 2+ + + 2+ 2+ To determine the role of CaPHM7 in C. albicans viru- other ions tested, including Ca ,K ,Na ,Mg ,Mn , 6 2+ + lence, we infected mice intravenously with 1 × 10 cells Cd ,and Li . C. albicans cells lacking CaPHM7 are also of the wild-type parental control strain and the homozy- not sensitive to osmotic stresses such as 1 M mannitol and gous mutant. Two mice were randomly selected from 4 M KCl. Therefore, whether CaPhm7 is a calcium perme- each group and sacrificed after 48 h to make sure that able stress-gated cation channel in the plasma membrane mice had been infected, with the CFUs/g reaching remains to be determined. It is also interesting to note that 1.02 × 10 (n = 2) in wet kidney tissue of the homozygous haploinsufficiency occurs to the deletion of one of the mutant, 1.56 × 10 (n = 2) in that of the wild-type and CaPHM7 alleles in the response to ketoconazole and zinc 1.38 × 10 (n = 2) in that of the complemented strain. By ion, but not to SDS and rapamycin (Fig. 1b). Among anoc- Day 6, none of the mice injected with the wild type sur- tamins, ANO10 shows the highest degree of homology to vived, while 30% and 80% of the mice inoculated with ScIst2, with a 36% similarity and a 17% identity in their the complemented strain and the homozygous mutant, amino acid sequences [17]. CaPhm7 shares 18% (34%) and respectively, survived. By Day 8, none of the mice inocu- 14% (30%) identity (similarity) with HsAno10 and ScPhm7, lated with the complemented strain survived, while the respectively, in their amino acid sequences (Additional file 1: mice injected with the homozygous mutant survived Figure S6). This suggests that CaPhm7 might be a member until Day 20 (Fig. 4a). As a control, no hyphal or yeast of the anoctamin/TMEM16 family member. Jiang and Pan BMC Microbiology (2018) 18:49 Page 5 of 8 Fig. 2 Subcellular localization and mouse virulence assay for CaPhm7. a Functional assay of the CaPHM7-GFP allele. The wild type CAI4 (XDCA06), the heterozygous mutant PHBCA100, the homozygous mutant PHBCA 102, as well as PHBCA107 and PHBCA126, carrying the CaPHM7-GFP allele, were grown overnight in SD-URA medium, serially diluted by 10 times and spotted onto YPD plates indicated. b Visualization of GFP signals within PHBCA107 cells of log-phase yeast-form and filamentous form induced in YPD + 10% FBS (top left), Spider (bottom left) or Lee’s (bottom right) media at 1000 × magnification on a Nikon ECLIPSE 80i microscope. Visualization of the wild type CAI4 cells (top right) as a control for hyphal morphology and green auto-fluorescence. DIC, differential interference contrast. The scale bars represent 5 μM In addition, we have shown that C. albicans cells lacking 7 and, together with other 21 genes, is regulated by the CaPHM7 are tolerant to rapamycin, the specific inhibitor PHO regulatory pathway in S. cerevisiae [35]. However, the of the target of rapamycin (TOR) kinase, which indicates function of ScPHM7 in phosphate metabolism is not de- that the TOR kinase activity might be inhibited in C. albi- fined up to now. ScPhm7 shares a 43% identity and 64% cans cells lacking CaPHM7.Thisisconsistent with our similarity, respectively, with CaPhm7 in amino acid se- observations that deletion of CaPHM7 leads to a reduced quence (Additional file 1: Figure S1). Our results on colony growth, which might be related to a reduced activ- CaPHM7 suggests that ScPhm7 might be a plasma mem- ity for the central controller of cell growth, TOR [34]. brane CSC ion channel that links the extracellular phos- The S. cerevisiae homolog of CaPHM7, ScPHM7 has ini- phate availability to the intracellular calcium signaling tially been identified to be the phosphate metabolism gene pathway to regulate phosphate metabolism. Recent studies Jiang and Pan BMC Microbiology (2018) 18:49 Page 6 of 8 Fig. 3 Functions of CaPHM7 in filamentation and colony formation. The wild type CAI4, the heterozygous mutant and the homozygous mutant, carrying the CIp10 vector or CIp10-CaPHM7, were grown overnight in SD-URA medium, and inoculated to liquid YPD containing 10% fetal bovine serum (FBS), Lee’s or Spider media for filamentation assay (a) or onto their solid plates for colony morphology oberservation (b). Incubation was carried out at 37 °C. The scale bar in (a) represents 10 μM, while the scale bar in (b) represents 5 mm Jiang and Pan BMC Microbiology (2018) 18:49 Page 7 of 8 infection. Similar to the functions of other members of the anoctamin/TMEM16 family as sensors for various stresses including nutrient availability [7–13], the plasma membrane protein CaPhm7 might function to regulate zinc availability for C. albicans cells in media or within the host environment and thereby affect their filamenta- tion and virulence. Conclusion In this study, we have demonstrated that CaPhm7 is lo- calized to the plasma membrane in both yeast-form and filamentous cells of C. albicans. CaPhm7 regulates ion homeostasis, drug tolerance, filamentation and virulence in this important human fungal pathogen. C. albicans cells lacking CaPHM7 have a defect in filamentation and attenuated virulence in the systemic mouse model. Therefore, CaPhm7 is a potential target for antifungal development. Additional file Additional file 1: Figure S1. Amino acid sequence comparison. Transmembrane domain (TM) regions of AtCsc1 and CaPhm7 are underlined, and TM names of AtCsc1 (blue) and CaPhm7 (red) are indicated. TMs were predicted with SMART (http://smart.embl- heidelberg.de/). Identical amino acid residues are indicated in yellow in color, and similar amino acid residues are indicated in light blue and Fig. 4 Virulence assay. a survival rates of mice (n = 10) infected with green. Figure S2. Disruption of CaPHM7. (A) Disruption strategy of the the wide type CAI4, the homozygous phm7/phm7 mutant and its two alleles. PCR confirmation of genotypes of the homozygous mutant complemented strain. Mice were checked every day for morbidity, and PHBCA62 with primer pairs CaPHM7-ORF-UP/DOWN (B), CaPHM7-DF/DR (C) as well as CaPHM7-DF/NAT-R (middle lane) and CaPHM7-DR/NAT-F survival was monitored for 30 days. The p value (0.009) of the log-rank (last lane) (D). Figure S3. TMs of AtCsc1, ScPhm7 and CaPhm7 as well as test indicates that the survival curves between the wild-type CAI4 and their amino acid sequence comparison. Identical amino acid residues are the phm7/phm7 mutant is significantly different. b histopathological indicated in yellow in color, and similar amino acid residues are indicated examination of kidney tissues of moribund mice infected with the in light blue and green. Figure S4. Growth assay. Cells were cultured in wild-type, the homozygous mutant and its complemented strain. SD-URA medium at 30 °C overnight, diluted for 10 times in fresh YPD Saline buffer was injected as negative control (CK). Infected kidney medium and grown further for indicated hours. Data were the average of tissues from mice were stained with periodic acid-Schiff’s reagent. three independent experiments. Growth rates of the heterozygous mu- Representatives of five kidney cross-sections from two mice per strain tant, the homozygous mutant and the revertant were not significantly were photographed with 40× lenses. The hyphal cells are highlighted different from the wild type. Figure S5. Western blot analysis. The CAI4 control, PHBCA107 and PHBCA126 cells were grown to log-phase and by arrows. The scale bars represent 20 μM collected for total protein extraction. Western blot analysis was carried out with the monoclonal anti-GFP antibody. The CaPHM7-GFP protein of 126 kDa was indicated. Figure S6. Amino acid sequence comparison. have indeed linked the calcium signaling pathway to phos- CaPhm7 shares 18% (34%) and 14% (30%) identity (similarity) with hu- phate metabolism. Both the PHO4 gene, encoding the man HsAno10 and S. cerevisiae ScIst2, respectively. Identical amino acid residues are indicated in yellow in color, and similar amino acid residues transcription factor of the PHO pathway, and the PHO89 are indicated in light blue and green. Table S1. Primers used in this gene, encoding the high-affinity inorganic phosphate (Pi) study. (PDF 5687 kb) transporter, are transcriptionally regulated by the calcium/ calcineurin signaling in S. cerevisiae [36]. Calcineurin is Abbreviations also involved in the regulation of the PHO pathway in As- CSC: Calcium permeable stress-gated cation channel; TRP: Calcium- permeable transient receptor potential pergillus fumigatus [37]. Therefore, like AtScs1 as an osmolarity sensor in the plant A. thaliana [7, 8], ScPhm7 Acknowledgements might function as a sensor for extracellular phosphate We are grateful to Jianqing Cheng for his technical support. levels in S. cerevisiae. In this study, we have observed that the homozygous Funding This work is supported by the National Natural Science Foundation of China mutant for CaPHM7 shows a filamentation defect, to LJ (No. 81571966 and No. 81371784). The funding body plays no role in which might contribute to its reduced colony growth the design of the study and collection, analysis, and interpretation of data and attenuated virulence in the mouse model of systemic and in writing the manuscript. Jiang and Pan BMC Microbiology (2018) 18:49 Page 8 of 8 2+ + Availability of data and materials 22. Wang Y, et al. Genetic interactions between the Golgi Ca /H exchanger The datasets used and/or analyzed during the current study are available Gdt1 and the plasma membrane calcium channel Cch1/Mid1 in the from the corresponding author on reasonable request. regulation of calcium homeostasis, stress response and virulence in Candida albicans. FEMS Yeast Res. 2015;15(7). 23. Reuss O, Vik A, Kolter R, Morschhauser J. 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BMC MicrobiologySpringer Journals

Published: Jun 4, 2018

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