Orthologues of Streptococcus pyogenes nuclease A (SpnA) and Streptococcal 5′-nucleotidase A (S5nA) found in Streptococcus iniae

Orthologues of Streptococcus pyogenes nuclease A (SpnA) and Streptococcal 5′-nucleotidase A... Abstract Streptococcus pyogenes nuclease A (SpnA) and streptococcal 5′ nucleosidase A (S5nA) are two recently described virulence factors from the human pathogen S. pyogenes. In vitro studies have shown that SpnA is a nuclease that cleaves ssDNA and dsDNA, including the DNA backbone of neutrophil extracellular traps. S5nA was shown to hydrolyse AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. S5nA also generates the macrophage-toxic deoxyadenosine from dAMP. However, detailed in vivo studies of the two enzymes have been hampered by difficulties with using current animal models for this exclusive human pathogen. Here we report the identification of two novel enzymes from the fish pathogen Streptococcus iniae that show similarities to SpnA and S5nA in amino acid sequence, protein domain structure and biochemical properties. We propose that SpnAi and S5nAi are orthologues of the S. pyogenes enzymes, providing a rationale to analyse the in vivo function of the two enzymes using a S. iniae-zebrafish infection model. cell surface-anchored enzyme, enzyme synergy, group A Streptococcus, LPXTG motif, Streptococcus iniae, Streptococcus pyogenes nuclease A (SpnA), Streptococcal 5′-nucleotidase A (S5nA), zebrafish infection Streptococcus pyogenes also known as Group A Streptococcus (GAS) is an exclusively human pathogen that can cause a wide range of diseases ranging from non-invasive pharyngitis and impetigo to more severe invasive diseases, such as toxic shock syndrome, necrotizing fasciitis and acute rheumatic fever (ARF) (1–3). The number of deaths due to severe GAS disease are more than half a million per year with the major burden due to ARF and rheumatic heart disease (RHD), followed by invasive disease. A 2005 report showed that the prevalence of severe GAS disease is at least 18.1 million cases, with 1.78 million new cases each year (4). GAS produces a plethora of virulence factors that facilitate colonization, bacterial spreading and immune evasion (5–7). We have recently characterized two novel cell wall-anchored immune evasion factors of GAS, the Streptococcus pyogenes nuclease A (SpnA) and Streptococcal 5′-nucleotidase A (S5nA) (8, 9). Both are expressed as precursor proteins with an N-terminal signal peptide sequence for secretion and a C-terminal cell wall-anchor (CWA) domain including a conserved sortase A recognition motif (LPXTG). SpnA is a Ca2+/Mg2+-dependent nuclease that cleaves, double stranded (ds) linear DNA, chromosomal DNA and the DNA backbone of neutrophil extracellular traps (NETs) (8). NETs are innate immune structures that are released from neutrophils after activation. They consist of a DNA scaffold with several bactericidal proteins, such as neutrophil elastase, myeloperoxidase and histones, that aid in bacterial clearance (10, 11). SpnA is able to enhance the survival of GAS in human blood and in neutrophil killing assays and was shown to facilitate virulence in a murine infection model (8, 12). More recently, it was shown in a Galleria mellonella (wax moth) infection model that nuclease activity is not solely responsible for SpnA mediated virulence and that SpnA has another, yet unknown virulence function (13). S5nA is a recently discovered nucleotidase that cleaves AMP, dAMP and ADP to generate the immunomodulatory molecules adenosine and deoxyadenosine (9). Adenosine antagonises the effect of ATP by stimulation of adenosine receptors suppressing the pro-inflammatory response (14–17). As a result, adenosine decreases the phagocytic activity of macrophages by suppressing the generation of nitric oxide (18), superoxide (19, 20) and pro-inflammatory cytokines (21). Adenosine also inhibits neutrophil degranulation (22). Despite a wealth of knowledge on the in vitro function of GAS virulence factors, information on in vivo function is limited due to difficulties with using current animal models for this exclusively human pathogen. Streptococcus iniae is a major fish pathogen that shares many virulence traits with GAS, and can also cause infections in humans who handle and prepare infected fish (23–25). S. iniae is a beta-hemolytic Gram-positive coccus that was first isolated from a subcutaneous abscess of a captive freshwater dolphin (26). S. iniae infections range from skin infections to major invasive diseases in at least 27 species of saltwater and fresh water fish (27, 28). Phylogenetic analysis based on 16S rRNA analysis revealed a close genetic relationship with other pathogenic streptococci, including S. pyogenes (29, 30). A zebrafish infection model has been established for S. iniae that allows the investigation of streptococcal virulence factors in a natural host organism (28, 31, 32). We have identified two genes on the S. iniae genome that are similar to the GAS genes encoding SpnA and S5nA. In this study, we show that the corresponding S. iniae proteins, termed SpnAi and S5nAi, are also functionally similar to their GAS counterparts making them true orthologues. These results provide the basis for further studies to determine the in vivo function of these proteins using a S. iniae-zebrafish infection model. Materials and Methods Bioinformatic analysis SpnAi and S5nAi were identified by a BLAST search of the S. iniae 9117 genome at https://blast.ncbi.nlm.nih.gov/Blast.cgi using the amino acid sequences of SpnA (AAK33693) and S5nA (NP 269071). The Signal P 4.0 server at http://www.cbs.dtu.dk/services/SignalP/ was used to predict the presence and length of the N-terminal signal peptide. For domain structure predictions, we searched the InterProScan software at EMBL-EBI. BLAST searches were performed using the tblastn program. Sequence alignments and the phylogenetic tree were generated using the ClustalW server. The Genbank accession numbers for SpnAi and S5nAi are EKB52944 and EKB52830, repectively. Bacterial strains and DNA manipulations Escherichia coli BL21 cells were grown in Luria Bertani (LB, BD Biosciences) at 37°C with aeration. When appropriate, 50 µg/ml ampicillin or 30 µg/ml chloramphenicol was added to the culture. S. iniae strain 9117, a human clinical isolate from a patient with cellulitis (kindly provided by Dr. Sarah Highlander, JCVI, La Jolla, CA), was grown in Todd Hewitt broth medium supplemented with 0.2% yeast extract (THY, BD Biosciences). The SpnAi ORF without the regions encoding the N-terminal signal peptide sequence and the C-terminal cell wall-anchor domain (nucleotide positions 103–2721) was amplified from genomic DNA of S. iniae strain 9117. The DNA was amplified with primers SpnAi.fw (5′-GAAGGATCCGAAGAAATCATAGGACCC-3′) and SpnAi.rev (5′-GCCAAGCTTTTATACTTTTCCTTTTTTTTTGAC-3′) by 25 cycles of PCR using iProofTM high-fidelity DNA polymerase (Bio-Rad) at an annealing temperature of 56°C. A stop codon (TAA) was introduced with the reverse primer at the 3′ end of the gene (nucleotide position 2722–2724). The S5nAi ORF (nucleotide position 82–1926) was amplified under the same conditions using the primers S5nAi.fw (5′-CGGATCCGATCAGGTTGATGTTCAAATTC-3′) and S5nAi.rev (5′- GGCGAATTCTTATTCTTGTTTCTTAGCCATTG-3′). The SpnAi and S5nAi PCR products were cloned separately into the BamHI/HindIII and BamHI/EcoRI cloning sites of the pPROEX-Htb expression vector (Life Technologies), respectively, followed by transformation into E. coli BL21. The cloned DNA sequences were analysed by the dideoxy chain termination method using the DNA sequencing facility at the School of Biological Science, University of Auckland. Expression and purification of recombinant proteins Recombinant SpnAi and S5nAi were expressed in E. coli BL21. Cultures were grown at 37°C until OD600 of 0.6 and protein expression was induced for 4 h after adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma). The recombinant His6-tagged proteins were purified using Ni2+-iminodiacetic acid (IDA) sepharose (Sigma) according to the manufacturer’s instruction. The eluates containing the recombinant proteins were collected and analysed on 10% SDS-polyacrylamide gels according to the procedure of Laemmli. Enzyme activity assays for rSpnAi Varying concentrations of rSpnAi were incubated with lambda DNA (GE Healthcare) in nuclease reaction buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM CaCl2 and incubated for 1 h at 37°C. For pH titration at pH 4.0–6.5, Tris–HCl was replaced with 50 mM acetate buffer. For Mg2+ and Ca2+ titrations, MgCl2 or CaCl2 were omitted from the reaction buffer and added separately at indicated concentrations. After incubation, all enzyme reactions were stopped by adding EDTA to a final concentration of 20 mM. Samples were then loaded onto 1% agarose gels. Lambda DNA without the addition of rSpnAi was loaded as the control for each set of experiment. DNA bands were visualized after SYBR® safe staining (Invitrogen) using a Gel DocTM EZ system (Bio-Rad) and quantified using Image LabTM Software V5.2.1 (Bio-Rad). The percentage of DNA digestion was calculated as [1 - (band intensity with rSpnAi/band intensity without rSpnAi)] × 100%. All samples were analysed in triplicates for each of the three independent experiments. Enzyme activity assays for rS5nAi Enzymatic reactions with rS5nAi were carried out in 50 mM Tris–HCl pH 7, 10 mM MgCl2, 1 mM substrate and 0.1 µM of rS5nAi in a total volume of 20 µl. The reaction mixture was incubated for 20 min at 37°C. Substrates included AMP, ADP, ATP, dAMP, GMP, CMP and TMP (Sigma Aldrich). For pH titration at pH 5.0–6.5, Tris-HCl was replaced with 50 mM acetate buffer. Dependence on metal cations was analysed for Mg2+, Ca2+, Mn2+ and Zn2+ at three different concentrations (0.1, 1 and 10 mM) at pH 7 and 37°C. The enzyme kinetics were analysed by incubation of a fixed concentration (0.1 µM) of rS5nAi with increasing amounts of AMP in a total volume of 50 µl at pH 7 and 37°C. The enzymatic reactions were stopped by adding EDTA to a final concentration of 50 mM. The release of inorganic phosphate (Pi) was quantified using a malachite green phosphate colorimetic assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. Release of Pi was measured at Abs650nm and the amount of Pi was calculated based on a standard Pi curve. Michaelis-Menten curve fitting using non-linear regression was performed using GraphPad Prism version 7.03 software. To test for synergy with SpnAi, a 200 μl reaction mix containing 50 μg/ml of UltraPureTM salmon sperm DNA (Invitrogen), 10 μg/ml rSpnAi, 50 mM Tris–HCl (pH 7.0), 150 mM NaCl, 2 mM MgCl2 and 2 mM CaCl2 was incubated at 37°C. After 1 h, 0.1 μM S5nAi and 10 mM MgCl2 were added and the reaction was incubated for another 1 h. Generation of Pi was analysed as described above. All samples were analysed in triplicate for each of the three independent experiments. Results and Discussion Identification of SpnAi and S5nAi To investigate if recently discovered GAS virulence factor genes spnA and s5nA would also be present on the S. iniae genome, protein sequences of SpnA and S5nA derived from GAS strain SF370 (serotype M1) were used to search the S. iniae strain 9117 using the tblastn option at the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Two hypothetical open reading frames (ORFs) were identified that shared amino acid sequence identities of 63% and 62% with SpnA and S5nA, respectively, and were named SpnAi and S5nAi. These sequences were then used to search the entire nucleotide collection at NCBI. ORFs with significant sequence similarities were used to generate a phylogenetic tree (Fig. 1A). Notably, ORFs with significant sequence similarities to the putative S. iniae nuclease SpnAi (62–70%) were only found in certain streptococcal species (S. dysgalactiae, S. suis and S. equi) and not in any other genera. In contrast, and as previously reported, S5nA related nucleotidases are found in a wide range of organisms, including Gram-positive and Gram-negative bacteria, as well as mammals (9). The putative S. iniae nucleotidase S5nAi is most closely related to the UshA protein of S. dysgalactiae and to an uncharacterized ORF (SEQ1278) in S. equi, both sharing 68% sequence identity (Fig. 1A). Fig. 1 View largeDownload slide Bioinformatic analysis and purification of SpnAi and S5nAi. (A) Rooted phylogenetic tree UPGMA, (unweighted pair group method with arithmetic mean) of cell wall-anchored nucleases (left) and cell wall-anchored 5′-nucleotidases (right). The trees were generated with ClustalW using complete protein sequences of SpnAi (S. iniae), WP_003050123 (S. dysgalactiae), WP_024395691 (S. suis), WP_012679382 (S. equi), S5nAi (S. iniae), S5nA (S. pyogenes, AAK33792), AdsA (Staphylococcus aureus, ESR29110), ecto-5′-nucleotidase A (Nt5e) (S. sanguinis, AFK32764), Ssads (S. suis, YP001197640), NudP (S. agalactiae, CDN66659), UshA (S. dysgalactiae, ADX24386), SEQ1278 (S. equi, CAW94038), 5′-nucleotidase (5′NT) (E. coli, AJM76137) and CD73 (Homo sapiens, AAH65937). The number in parentheses show amino acid sequence identities with SpnAi and S5nAi. (B) Schematic presentation of SpnAi (top) and S5nAi (below) domain structure based on the prediction using InterProScan software at EMBL-EBI. The numbers on top represent amino acid positions. SP, signal peptide sequence; CWA, cell wall anchor domain. The arrows above indicate the regions that were generated as recombinant proteins in E. coli. (C) Recombinant form of SpnAi (left) and S5nAi (right) were expressed and purified from E. coli by immobilized metal chelate chromatography. The purity of the proteins was ≥95% as estimated from a 10% SDS-polyacrylamide gel. M, BenchMarkTM molecular weight marker. Fig. 1 View largeDownload slide Bioinformatic analysis and purification of SpnAi and S5nAi. (A) Rooted phylogenetic tree UPGMA, (unweighted pair group method with arithmetic mean) of cell wall-anchored nucleases (left) and cell wall-anchored 5′-nucleotidases (right). The trees were generated with ClustalW using complete protein sequences of SpnAi (S. iniae), WP_003050123 (S. dysgalactiae), WP_024395691 (S. suis), WP_012679382 (S. equi), S5nAi (S. iniae), S5nA (S. pyogenes, AAK33792), AdsA (Staphylococcus aureus, ESR29110), ecto-5′-nucleotidase A (Nt5e) (S. sanguinis, AFK32764), Ssads (S. suis, YP001197640), NudP (S. agalactiae, CDN66659), UshA (S. dysgalactiae, ADX24386), SEQ1278 (S. equi, CAW94038), 5′-nucleotidase (5′NT) (E. coli, AJM76137) and CD73 (Homo sapiens, AAH65937). The number in parentheses show amino acid sequence identities with SpnAi and S5nAi. (B) Schematic presentation of SpnAi (top) and S5nAi (below) domain structure based on the prediction using InterProScan software at EMBL-EBI. The numbers on top represent amino acid positions. SP, signal peptide sequence; CWA, cell wall anchor domain. The arrows above indicate the regions that were generated as recombinant proteins in E. coli. (C) Recombinant form of SpnAi (left) and S5nAi (right) were expressed and purified from E. coli by immobilized metal chelate chromatography. The purity of the proteins was ≥95% as estimated from a 10% SDS-polyacrylamide gel. M, BenchMarkTM molecular weight marker. The 2,823 bp nuclease gene spnAi from S. iniae strain 9117 encodes a 940 amino acid precursor protein with a predicted signal peptide sequence (position 1–34) and a C-terminal CWA domain (907–940). The s5nAi gene is 2,031 bp in length and encodes a 676 amino acid gene product with a predicted signal peptide sequence (position 1–27) and a C-terminal CWA domain (642–676) (Fig. 1B). In order to determine if SpnAi and S5nAi are functionally similar to the GAS proteins SpnA and S5nA, soluble recombinant proteins (rSpnAi and rS5nAi) that lack the N-terminal signal peptide sequence and the C-terminal CWA domain (Fig. 1B) were expressed with an N-terminal (His)6-tag in E. coli. The proteins were purified by Ni2+-affinity chromatography to a purity of approximately 95%, as estimated from an SDS-polyacrylamide gel (Fig. 1C). rSpnA and rS5nAi migrated at ∼100 and ∼70 kDa, respectively, which is in agreement with their calculated molecular weight of 96280.61 and 66935.19 Da, respectively. Biochemical analysis of recombinant SpnAi SpnA has a very strong dependence on Mg2+ and Ca2+ and is completely inactive if one of the cations is absent (8). To determine if SpnAi displays similar cation-dependence, purified rSpnAi was incubated with double-stranded linear lambda DNA in the presence of Ca2+ and Mg2+. Complete DNA digestion by rSpnAi was observed after 1 h incubation at 37°C. However, in the absence of either Ca2+/Mg2+ or both, the DNase activity of rSpnAi was completely undetectable (Fig. 2A) indicating a similar cation requirement as SpnA. The Mg2+ and Ca2+ dependence was further analysed at different concentrations of MgCl2 and CaCl2 (Fig. 2B and C). rSpnAi was active between 0.19 and 25 mM Mg2+ with maximum activity at 0.78 and 3.125 mM Mg2+, but was completely inactive at 50 mM Mg2+. This was similar to the results previously reported for rSpnA, which showed maximum activity at 1.56–3.125 mM and no activity at 50 mM (8). Although, the optimum Ca2+ concentration was similar between SpnAi (0.78-6.25 mM) and SpnA (0.78 mM), the S. iniae enzyme was still active at high concentrations of Ca2+ (∼70% activity at 200 mM), whereas the GAS enzyme was completely inactive at ⩾25 mM Ca2+(8). Fig. 2 View largeDownload slide Biochemical analysis of rSpnAi. (A) SpnAi activity depends on the presence of Ca2+ and Mg2+. Lambda DNA was digested in the presence or absence of each of the cations and analysed on a 1% agarose gel. C, control: reaction mixture without rSpnAi. Lambda DNA was digested with rSpnAi in the presence of varying concentrations of MgCl2 (B), varying concentrations of CaCl2 (C), at different pH (D) and at different temperature (E). Digested DNA was run on a 1% agarose gel and DNA bands were visualized after SYBR® safe staining and quantified using Image LabTM Software V5.2.1. (F) The minimum enzyme required for complete cleavage of 1 µg of lambda DNA was analysed at optimum reaction conditions (1 mM CaCl2, 3 mM MgCl2, pH 7, 37°C). The error bars show the standard deviation of three independent experiments performed in triplicates. Fig. 2 View largeDownload slide Biochemical analysis of rSpnAi. (A) SpnAi activity depends on the presence of Ca2+ and Mg2+. Lambda DNA was digested in the presence or absence of each of the cations and analysed on a 1% agarose gel. C, control: reaction mixture without rSpnAi. Lambda DNA was digested with rSpnAi in the presence of varying concentrations of MgCl2 (B), varying concentrations of CaCl2 (C), at different pH (D) and at different temperature (E). Digested DNA was run on a 1% agarose gel and DNA bands were visualized after SYBR® safe staining and quantified using Image LabTM Software V5.2.1. (F) The minimum enzyme required for complete cleavage of 1 µg of lambda DNA was analysed at optimum reaction conditions (1 mM CaCl2, 3 mM MgCl2, pH 7, 37°C). The error bars show the standard deviation of three independent experiments performed in triplicates. Next, we examined the effect of pH on rSpnAi activity using the constant optimized Mg2+ concentration of 3 mM. Greater than 50% activity was observed between pH 5 and 8.5, with maximum activity seen between pH 6.5 and 8. Reduced activity (∼25%) was also measurable at pH 4, while the enzyme was almost inactive at pH 9 (∼8% activity) (Fig. 2D). The pH range was slightly shifted into the alkaline range compared to rSpnA, which showed optimal activity between pH 5.5 and 7 (8). This might be due to adaptation to a different host, as most fish species have a normal blood pH in the range of 7.7–8.0 (33, 34), whereas human blood has a pH of 7.3–7.4. Recombinant SpnAi activity was determined for a range of different temperatures (12–47°C) at pH 7 and 3 mM Mg2+ (Fig. 2E). Maximum rSpnAi activity was found between 32–37°C, while activity was reduced to ∼80% at 27°C, ∼60% at 22°C, ∼40% at 17°C and 42°C and ∼25% at 47°C. Only marginal activity was observed at 12°C (<10%). This is only slightly lower than the optimal temperature range for rSpnA which was found to between 32 and 42°C (8). The enzymatic activity of rSpnAi under optimal conditions (1 mM CaCl2, 3 mM MgCl2, pH 7, 37°C) was determined by serial dilution of rSpnAi starting from 35 pM. As shown in Fig. 2F, 20 pmol of rSpnAi is the minimum amount of enzyme required to completely digest 1 µg of lambda DNA in 1 h. This is similar to rSpnA, for which 26 pmol was required for the same activity (8). All biochemical properties for rSpnAand rSpnAi are summarized in Table I. Table I. Summary of biochemical properties of rSpnAi and rSpnA Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  #without signal peptide and cell wall anchor regions. Table I. Summary of biochemical properties of rSpnAi and rSpnA Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  #without signal peptide and cell wall anchor regions. Biochemical analysis of recombinant S5nAi We have previously shown that S5nA from S. pyogenes hydrolyses AMP and ADP, but not ATP, to produce the immunomodulatory product adenosine (9). We have tested rS5nAi for activity against the same components and found a similar substrate preference (Fig. 3A). The highest activity was observed against AMP, while ATP was not hydrolysed. In contrast to rS5nA, rS5nAi showed higher activity for ADP (9). There were also small differences in the activity for other nucleoside monophosphates. rS5nAi hydrolysed GMP, CMP and TMP with almost equal efficiency, but lower than for AMP, whereas rS5nA showed a high preference towards CMP (9). Both enzymes hydrolysed dAMP with similar efficiency as AMP, which is different from the NudP nucleosidase produced by Streptococcus agalactiae (Group B Streptococcus), which is unable to hydrolyse dAMP (35). Fig. 3 View largeDownload slide Biochemical analysis of rS5nAi. (A) Reaction mixtures containing 0.1 µM rS5nAi were incubated with 1 mM of various substrates for 20 min in the presence of 10 mM MgCl2 at 37°C. Hydrolysis of 1 mM AMP by 0.1 µM rS5nAi over 20 min was analysed in the presence of different divalent metal cations at varying concentrations (B), at different pH at 37°C (C) and at different temperature at pH 7 (D). (E) The time to reaction equilibrium was determined with 1 mM AMP and 0.1 µM rS5nAi in the presence of 10 mM MgCl2 at pH 7 and 37°C. (F) Enzyme kinetics of rS5nAi for AMP hydrolysis were determined by velocity measurements with various substrate concentrations in the presence of 10 mM MgCl2 at pH 7 and 37°C using GraphPad Prism V6.03 software. The Pi released in each experiment was quantified using a malachite green phosphate colorimetric assay kit. The error bars show the standard deviation of three independent experiments performed in triplicates. Fig. 3 View largeDownload slide Biochemical analysis of rS5nAi. (A) Reaction mixtures containing 0.1 µM rS5nAi were incubated with 1 mM of various substrates for 20 min in the presence of 10 mM MgCl2 at 37°C. Hydrolysis of 1 mM AMP by 0.1 µM rS5nAi over 20 min was analysed in the presence of different divalent metal cations at varying concentrations (B), at different pH at 37°C (C) and at different temperature at pH 7 (D). (E) The time to reaction equilibrium was determined with 1 mM AMP and 0.1 µM rS5nAi in the presence of 10 mM MgCl2 at pH 7 and 37°C. (F) Enzyme kinetics of rS5nAi for AMP hydrolysis were determined by velocity measurements with various substrate concentrations in the presence of 10 mM MgCl2 at pH 7 and 37°C using GraphPad Prism V6.03 software. The Pi released in each experiment was quantified using a malachite green phosphate colorimetric assay kit. The error bars show the standard deviation of three independent experiments performed in triplicates. Divalent cations are important as co-factors for nucleosidase activity. We analysed the activity of rS5nAi for AMP with either Mg2+, Mn2+, Ca2+ or Zn2+ at 37°C and pH 7 (Fig. 3B). The activity of rS5nAi was slightly higher in the presence of Mg2+ compared to Ca2+ in a dose-dependent manner with highest activity at 10 mM. Highest activity was also achieved with lower concentrations of Mn2+ (0.1 and 1 mM), whereas a higher concentration of 10 mM had an inhibitory effect. In contrast, rS5nAi hydrolysis of AMP was strongly decreased with Zn2+, in particular at 10 mM. These results are very similar to those observed for rS5nA (9). As observed for the nuclease enzyme, the optimal pH range was higher for S5nAi (pH 5–7.5) compared to S5nA (pH 5–6.6) (Fig. 3C), which might reflect an adaptation to the higher pH of fish blood compared to human blood (33, 34). The temperature requirements for rS5nA and S5nAi are very similar (9). Like rS5nA, rS5nAi shows maximum activity at 42°C, but is active over a wide range of temperatures (Fig. 3D). Even at 21 and 52°C, the enzymatic activity was found to be over 25% suggesting strong thermostability. Finally we tested the enzymatic activity against AMP at 37°C, pH 7 and 10 mM MgCl2. A time course showed that AMP hydrolysis with 0.1 μM rS5nAi reached equilibrium after 30 min (Fig. 3E), which is comparable to 25 min for rS5nA (9). The reaction kinetics followed the Michaelis–Menten model for a single substrate with a Km of 121.3 ± 1.683 μM and a Vmax of 7,808 ± 78.3 nmol of released Pi/mg enzyme/min (Fig. 3F). This corresponds well with the kinetics previously reported for rS5nA (Km of 168.3 ± 38 μM and a Vmax of 7,550 ± 326 nmol of released Pi/mg enzyme/min) (9). All biochemical properties for rS5nA and rS5nAi are summarized in Table II. Table II. Summary of biochemical properties of rS5nAi and rS5nA Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  aWithout signal peptide and cell wall anchor regions. bAMP hydrolysis with 0.1 μM rS5nAi at 37°C, pH 7 and 10 mM MgCl2. Table II. Summary of biochemical properties of rS5nAi and rS5nA Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  aWithout signal peptide and cell wall anchor regions. bAMP hydrolysis with 0.1 μM rS5nAi at 37°C, pH 7 and 10 mM MgCl2. Synergy between SpnAi and S5nAi Based on work conducted with Staphylococus aureus, it has previously been suggested that nucleases and nucleotidases might work in synergy to evade the host immune system. It was proposed that the nuclease would hydrolyze DNA, including NETs, to generate the nucleotidase substrate dAMP, which would then be hydrolyzed to produce deoxyadenosine (dAdo) to trigger the caspase-3-mediated death of macrophages and monocytes restricting macrophage influx into abscesses (36, 37). To test the possibility that such a synergy also exists between SpnAi and S5nAi, we tested both enzymes together using salmon sperm DNA as substrate. A strong production of inorganic phosphate (Pi) was observed when both enzymes were mixed together. In contrast, addition of either SpnAi or S5nAi alone (or no enzyme) did not generate detectable Pi (Fig. 4). Fig. 4 View largeDownload slide Synergy between rSpnAi and rS5nAi. Incubation of salmon sperm DNA with both enzymes, but not with individual enzymes, results in the generation of Pi which was quantified using a malachite green phosphate colorimetric assay kit. This suggests that SpnAi hydrolyses dsDNA to produce deoxynucleotide monophosphates including dAMP, which can then be used as a substrate by S5nAi to generate deoxyadenosine (dAdo) and Pi. The error bars show the standard deviation of three independent experiments performed in triplicates. Fig. 4 View largeDownload slide Synergy between rSpnAi and rS5nAi. Incubation of salmon sperm DNA with both enzymes, but not with individual enzymes, results in the generation of Pi which was quantified using a malachite green phosphate colorimetric assay kit. This suggests that SpnAi hydrolyses dsDNA to produce deoxynucleotide monophosphates including dAMP, which can then be used as a substrate by S5nAi to generate deoxyadenosine (dAdo) and Pi. The error bars show the standard deviation of three independent experiments performed in triplicates. Conclusion The biochemical properties of the S. iniae enzymes SpnAi and S5nAi are very similar to those reported for S. pyogenes SpnA and S5nA, respectively. Together with similar protein lengths, domain structures and amino acid similarities, it is highly likely that these proteins are true orthologues and act together as immune evasion factors. Future work will include the generation of spnAi and s5nAi deletion mutants in S. iniae and their use in a zebrafish infection model (28, 31, 32). Investigation into bacterial dissemination in the host, NET degradation, and migration of neutrophils and macrophages to the site of infection will provide new insights into the function of the two enzymes. This might allow us to draw conclusions on similar virulence mechanisms used by S. pyogenes in the human host. Acknowledgements We thank Dr. Sarah Highlander, JCVI, La Jolla, C.A. for sending us the S. iniae 9117 strain. Funding This study was financially supported by a Graduate Student fund from the University of Auckland. K.Y.S. was supported by an University of Auckland International Doctoral Scholarship. J.M.S.L. is the recipient of a New Zealand National Heart Foundation Research Fellowship. Conflict of Interest None declared. References 1 Stevens D.L., Bryant A.E. ( 2016) Severe group A streptococcal infections in Streptococcus pyogenes: Basic Biology to Clinical Manifestations  ( Ferretti J.J., Stevens D.L., Fischetti V.A., eds.), University of Oklahoma Health Sciences Center, Oklahoma City (OK) 2 Cunningham M.W. ( 2012) Streptococcus and rheumatic fever. Curr. Opin. Rheumatol.  24, 408 Google Scholar CrossRef Search ADS PubMed  3 Martin W.J., Steer A.C., Smeesters P.R., Keeble J., Inouye M., Carapetis J., Wicks I.P. ( 2015) Post-infectious group A streptococcal autoimmune syndromes and the heart. Autoimmun. Rev . 14, 710– 725 Google Scholar CrossRef Search ADS PubMed  4 Carapetis J.R., Steer A.C., Mulholland E.K., Weber M. ( 2005) The global burden of group A streptococcal diseases. Lancet Infect. Dis.  5, 685– 694 Google Scholar CrossRef Search ADS PubMed  5 Bisno A.L., Brito M.O., Collins C. ( 2003) Molecular basis of group A streptococcal virulence. Lancet Infect. Dis . 3, 191– 200 Google Scholar CrossRef Search ADS PubMed  6 Cole J.N., Barnett T.C., Nizet V., Walker M.J. ( 2011) Molecular insight into invasive group A streptococcal disease. Nat. Rev. Micro.  9, 724– 736 Google Scholar CrossRef Search ADS   7 Courtney H.S., Hasty D.L., Dale J.B. ( 2002) Molecular mechanisms of adhesion, colonization, and invasion of group A streptococci. Ann. Med.  34, 77– 87 Google Scholar CrossRef Search ADS PubMed  8 Chang A., Khemlani A., Kang H., Proft T. ( 2011) Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor. Mol. Microbiol . 79, 1629– 1642 Google Scholar CrossRef Search ADS PubMed  9 Zheng L., Khemlani A., Lorenz N., Loh J.M., Langley R.J., Proft T. ( 2015) Streptococcal 5′-nucleotidase A (S5nA), a novel Streptococcus pyogenes virulence factor that facilitates immune evasion. J. Biol. Chem.  290, 31126– 31137 Google Scholar CrossRef Search ADS PubMed  10 Papayannopoulos V., Zychlinsky A. ( 2009) NETs: a new strategy for using old weapons. Trends Immunol.  30, 513– 521 Google Scholar CrossRef Search ADS PubMed  11 Brinkmann V., Reichard U., Goosmann C., Fauler B., Uhlemann Y., Weiss D.S., Weinrauch Y., Zychlinsky A. ( 2004) Neutrophil extracellular traps kill bacteria. Science  303, 1532– 1535 Google Scholar CrossRef Search ADS PubMed  12 Hasegawa T., Minami M., Okamoto A., Tatsuno I., Isaka M., Ohta M. ( 2010) Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes. Microbiology  156, 184– 190 Google Scholar CrossRef Search ADS PubMed  13 Chalmers C., Khemlani A.H.J., Sohn C.R., Loh J.M.S., Tsai C.J., Proft T. ( 2017) Streptococcus pyogenes nuclease A (SpnA) mediated virulence does not exclusively depend on nuclease activity. J. Microbiol. Immunol. Infect . pii: S1684-1182(17)30236-0; doi: 10.1016/j.jmii.2017.09.006 [Epub ahead of print]. 14 Gorini S., Gatta L., Pontecorvo L., Vitiello L., la Sala A. ( 2013) Regulation of innate immunity by extracellular nucleotides. Am. J. Blood Res.  3, 14– 28 Google Scholar PubMed  15 Hasko G., Cronstein B.N. ( 2004) Adenosine: an endogenous regulator of innate immunity. Trends Immunol.  25, 33– 39 Google Scholar CrossRef Search ADS PubMed  16 Vitiello L., Gorini S., Rosano G., la Sala A. ( 2012) Immunoregulation through extracellular nucleotides. Blood  120, 511– 518 Google Scholar CrossRef Search ADS PubMed  17 Idzko M., Ferrari D., Eltzschig H.K. ( 2014) Nucleotide signalling during inflammation. Nature  509, 310– 317 Google Scholar CrossRef Search ADS PubMed  18 Xaus J., Mirabet M., Lloberas J., Soler C., Lluis C., Franco R., Celada A. ( 1999) IFN-gamma up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation. J. Immunol . 162, 3607– 3614 Google Scholar PubMed  19 Cronstein B.N., Kramer S.B., Weissmann G., Hirschhorn R. ( 1983) Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J. Exp. Med . 158, 1160– 1177 Google Scholar CrossRef Search ADS PubMed  20 Edwards C.K.3rd, Watts L.M., Parmely M.J., Linnik M.D., Long R.E., Borcherding D.R. ( 1994) Effect of the carbocyclic nucleoside analogue MDL 201, 112 on inhibition of interferon-gamma-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression. J. Leukoc. Biol.  56, 133– 144 Google Scholar CrossRef Search ADS PubMed  21 Hasko G., Szabo C., Nemeth Z.H., Kvetan V., Pastores S.M., Vizi E.S. ( 1996) Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J. Immunol . 157, 4634– 4640 Google Scholar PubMed  22 Bouma M.G., Jeunhomme T.M., Boyle D.L., Dentener M.A., Voitenok N.N., van den Wildenberg F.A., Buurman W.A. ( 1997) Adenosine inhibits neutrophil degranulation in activated human whole blood: involvement of adenosine A2 and A3 receptors. J. Immunol . 158, 5400– 5408 Google Scholar PubMed  23 Weinstein M.R., Litt M., Kertesz D.A., Wyper P., Rose D., Coulter M., McGeer A., Facklam R., Ostach C., Willey B.M., Borczyk A., Low D.E. ( 1997) Invasive infections due to a fish pathogen, Streptococcus iniae. N Engl. J. Med.  337, 589– 594 Google Scholar CrossRef Search ADS PubMed  24 Lau S.K., Woo P.C., Luk W.-K., Fung A.M., Hui W.-T., Fong A.H., Chow C.-W., Wong S.S., Yuen K.-Y. ( 2006) Clinical isolates of Streptococcus iniae from Asia are more mucoid and β-hemolytic than those from North America. Diagn. Microbiol. Infect. Dis . 54, 177– 181 Google Scholar CrossRef Search ADS PubMed  25 Baiano J.C., Barnes A.C. ( 2009) Towards control of Streptococcus iniae. Emerg. Infect. Dis.  15, 1891 Google Scholar CrossRef Search ADS PubMed  26 Pier G.B., Madin S.H. ( 1976) Streptococcus iniae sp-nov, a beta-hemolytic streptococcus isolated from an amazon freshwater dolphin, Inia Geoffrensis. Int. J. Syst. Bacteriol . 26, 545– 553 Google Scholar CrossRef Search ADS   27 Miller J.D., Neely M.N. ( 2005) Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae. Infect. Immun . 73, 921– 934 Google Scholar CrossRef Search ADS PubMed  28 Rowe H.M., Withey J.H., Neely M.N. ( 2014) Zebrafish as a model for zoonotic aquatic pathogens. Dev. Comp. Immunol.  46, 96– 107 Google Scholar CrossRef Search ADS PubMed  29 Facklam R., Elliott J., Shewmaker L., Reingold A. ( 2005) Identification and characterization of sporadic isolates of Streptococcus iniae isolated from humans. J. Clin. Microbiol.  43, 933– 937 Google Scholar CrossRef Search ADS PubMed  30 Zlotkin A., Hershko H., Eldar A. ( 1998) Possible transmission of Streptococcus iniae from wild fish to cultured marine fish. Appl. Environ. Microbiol . 64, 4065– 4067 Google Scholar PubMed  31 Neely M.N., Pfeifer J.D., Caparon M. ( 2002) Streptococcus-zebrafish model of bacterial pathogenesis. Infect. Immunity  70, 3904– 3914 Google Scholar CrossRef Search ADS   32 Saralahti A., Ramet M. ( 2015) Zebrafish and streptococcal infections. Scand. J. Immunol.  82, 174– 183 Google Scholar CrossRef Search ADS PubMed  33 Borvinskaya E., Gurkov A., Shchapova E., Baduev B., Shatilina Z., Sadovoy A., Meglinski I., Timofeyev M. ( 2017) Parallel in vivo monitoring of pH in gill capillaries and muscles of fishes using microencapsulated biomarkers. Biol. Open  6, 673– 677 Google Scholar CrossRef Search ADS PubMed  34 Evans D., Claiborne J. ( 1997) The Physiology of Fishes , CRC Press, Boca Raton 35 Firon A., Dinis M., Raynal B., Poyart C., Trieu-Cuot P., Kaminski P.A. ( 2014) Extracellular nucleotide catabolism by the Group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood. J. Biol. Chem.  289, 5479– 5489 Google Scholar CrossRef Search ADS PubMed  36 Thammavongsa V., Missiakas D.M., Schneewind O. ( 2013) Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science  342, 863– 866 Google Scholar CrossRef Search ADS PubMed  37 Thammavongsa V., Schneewind O., Missiakas D.M. ( 2011) Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA). BMC Biochem.  12, 56 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations S5nA Streptococcal 5′-nucleotidase A SpnA Streptococcus pyogenes nuclease A © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Biochemistry Oxford University Press

Orthologues of Streptococcus pyogenes nuclease A (SpnA) and Streptococcal 5′-nucleotidase A (S5nA) found in Streptococcus iniae

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Oxford University Press
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© The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved
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0021-924X
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1756-2651
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10.1093/jb/mvy039
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Abstract

Abstract Streptococcus pyogenes nuclease A (SpnA) and streptococcal 5′ nucleosidase A (S5nA) are two recently described virulence factors from the human pathogen S. pyogenes. In vitro studies have shown that SpnA is a nuclease that cleaves ssDNA and dsDNA, including the DNA backbone of neutrophil extracellular traps. S5nA was shown to hydrolyse AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. S5nA also generates the macrophage-toxic deoxyadenosine from dAMP. However, detailed in vivo studies of the two enzymes have been hampered by difficulties with using current animal models for this exclusive human pathogen. Here we report the identification of two novel enzymes from the fish pathogen Streptococcus iniae that show similarities to SpnA and S5nA in amino acid sequence, protein domain structure and biochemical properties. We propose that SpnAi and S5nAi are orthologues of the S. pyogenes enzymes, providing a rationale to analyse the in vivo function of the two enzymes using a S. iniae-zebrafish infection model. cell surface-anchored enzyme, enzyme synergy, group A Streptococcus, LPXTG motif, Streptococcus iniae, Streptococcus pyogenes nuclease A (SpnA), Streptococcal 5′-nucleotidase A (S5nA), zebrafish infection Streptococcus pyogenes also known as Group A Streptococcus (GAS) is an exclusively human pathogen that can cause a wide range of diseases ranging from non-invasive pharyngitis and impetigo to more severe invasive diseases, such as toxic shock syndrome, necrotizing fasciitis and acute rheumatic fever (ARF) (1–3). The number of deaths due to severe GAS disease are more than half a million per year with the major burden due to ARF and rheumatic heart disease (RHD), followed by invasive disease. A 2005 report showed that the prevalence of severe GAS disease is at least 18.1 million cases, with 1.78 million new cases each year (4). GAS produces a plethora of virulence factors that facilitate colonization, bacterial spreading and immune evasion (5–7). We have recently characterized two novel cell wall-anchored immune evasion factors of GAS, the Streptococcus pyogenes nuclease A (SpnA) and Streptococcal 5′-nucleotidase A (S5nA) (8, 9). Both are expressed as precursor proteins with an N-terminal signal peptide sequence for secretion and a C-terminal cell wall-anchor (CWA) domain including a conserved sortase A recognition motif (LPXTG). SpnA is a Ca2+/Mg2+-dependent nuclease that cleaves, double stranded (ds) linear DNA, chromosomal DNA and the DNA backbone of neutrophil extracellular traps (NETs) (8). NETs are innate immune structures that are released from neutrophils after activation. They consist of a DNA scaffold with several bactericidal proteins, such as neutrophil elastase, myeloperoxidase and histones, that aid in bacterial clearance (10, 11). SpnA is able to enhance the survival of GAS in human blood and in neutrophil killing assays and was shown to facilitate virulence in a murine infection model (8, 12). More recently, it was shown in a Galleria mellonella (wax moth) infection model that nuclease activity is not solely responsible for SpnA mediated virulence and that SpnA has another, yet unknown virulence function (13). S5nA is a recently discovered nucleotidase that cleaves AMP, dAMP and ADP to generate the immunomodulatory molecules adenosine and deoxyadenosine (9). Adenosine antagonises the effect of ATP by stimulation of adenosine receptors suppressing the pro-inflammatory response (14–17). As a result, adenosine decreases the phagocytic activity of macrophages by suppressing the generation of nitric oxide (18), superoxide (19, 20) and pro-inflammatory cytokines (21). Adenosine also inhibits neutrophil degranulation (22). Despite a wealth of knowledge on the in vitro function of GAS virulence factors, information on in vivo function is limited due to difficulties with using current animal models for this exclusively human pathogen. Streptococcus iniae is a major fish pathogen that shares many virulence traits with GAS, and can also cause infections in humans who handle and prepare infected fish (23–25). S. iniae is a beta-hemolytic Gram-positive coccus that was first isolated from a subcutaneous abscess of a captive freshwater dolphin (26). S. iniae infections range from skin infections to major invasive diseases in at least 27 species of saltwater and fresh water fish (27, 28). Phylogenetic analysis based on 16S rRNA analysis revealed a close genetic relationship with other pathogenic streptococci, including S. pyogenes (29, 30). A zebrafish infection model has been established for S. iniae that allows the investigation of streptococcal virulence factors in a natural host organism (28, 31, 32). We have identified two genes on the S. iniae genome that are similar to the GAS genes encoding SpnA and S5nA. In this study, we show that the corresponding S. iniae proteins, termed SpnAi and S5nAi, are also functionally similar to their GAS counterparts making them true orthologues. These results provide the basis for further studies to determine the in vivo function of these proteins using a S. iniae-zebrafish infection model. Materials and Methods Bioinformatic analysis SpnAi and S5nAi were identified by a BLAST search of the S. iniae 9117 genome at https://blast.ncbi.nlm.nih.gov/Blast.cgi using the amino acid sequences of SpnA (AAK33693) and S5nA (NP 269071). The Signal P 4.0 server at http://www.cbs.dtu.dk/services/SignalP/ was used to predict the presence and length of the N-terminal signal peptide. For domain structure predictions, we searched the InterProScan software at EMBL-EBI. BLAST searches were performed using the tblastn program. Sequence alignments and the phylogenetic tree were generated using the ClustalW server. The Genbank accession numbers for SpnAi and S5nAi are EKB52944 and EKB52830, repectively. Bacterial strains and DNA manipulations Escherichia coli BL21 cells were grown in Luria Bertani (LB, BD Biosciences) at 37°C with aeration. When appropriate, 50 µg/ml ampicillin or 30 µg/ml chloramphenicol was added to the culture. S. iniae strain 9117, a human clinical isolate from a patient with cellulitis (kindly provided by Dr. Sarah Highlander, JCVI, La Jolla, CA), was grown in Todd Hewitt broth medium supplemented with 0.2% yeast extract (THY, BD Biosciences). The SpnAi ORF without the regions encoding the N-terminal signal peptide sequence and the C-terminal cell wall-anchor domain (nucleotide positions 103–2721) was amplified from genomic DNA of S. iniae strain 9117. The DNA was amplified with primers SpnAi.fw (5′-GAAGGATCCGAAGAAATCATAGGACCC-3′) and SpnAi.rev (5′-GCCAAGCTTTTATACTTTTCCTTTTTTTTTGAC-3′) by 25 cycles of PCR using iProofTM high-fidelity DNA polymerase (Bio-Rad) at an annealing temperature of 56°C. A stop codon (TAA) was introduced with the reverse primer at the 3′ end of the gene (nucleotide position 2722–2724). The S5nAi ORF (nucleotide position 82–1926) was amplified under the same conditions using the primers S5nAi.fw (5′-CGGATCCGATCAGGTTGATGTTCAAATTC-3′) and S5nAi.rev (5′- GGCGAATTCTTATTCTTGTTTCTTAGCCATTG-3′). The SpnAi and S5nAi PCR products were cloned separately into the BamHI/HindIII and BamHI/EcoRI cloning sites of the pPROEX-Htb expression vector (Life Technologies), respectively, followed by transformation into E. coli BL21. The cloned DNA sequences were analysed by the dideoxy chain termination method using the DNA sequencing facility at the School of Biological Science, University of Auckland. Expression and purification of recombinant proteins Recombinant SpnAi and S5nAi were expressed in E. coli BL21. Cultures were grown at 37°C until OD600 of 0.6 and protein expression was induced for 4 h after adding 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) (Sigma). The recombinant His6-tagged proteins were purified using Ni2+-iminodiacetic acid (IDA) sepharose (Sigma) according to the manufacturer’s instruction. The eluates containing the recombinant proteins were collected and analysed on 10% SDS-polyacrylamide gels according to the procedure of Laemmli. Enzyme activity assays for rSpnAi Varying concentrations of rSpnAi were incubated with lambda DNA (GE Healthcare) in nuclease reaction buffer containing 50 mM Tris–HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM CaCl2 and incubated for 1 h at 37°C. For pH titration at pH 4.0–6.5, Tris–HCl was replaced with 50 mM acetate buffer. For Mg2+ and Ca2+ titrations, MgCl2 or CaCl2 were omitted from the reaction buffer and added separately at indicated concentrations. After incubation, all enzyme reactions were stopped by adding EDTA to a final concentration of 20 mM. Samples were then loaded onto 1% agarose gels. Lambda DNA without the addition of rSpnAi was loaded as the control for each set of experiment. DNA bands were visualized after SYBR® safe staining (Invitrogen) using a Gel DocTM EZ system (Bio-Rad) and quantified using Image LabTM Software V5.2.1 (Bio-Rad). The percentage of DNA digestion was calculated as [1 - (band intensity with rSpnAi/band intensity without rSpnAi)] × 100%. All samples were analysed in triplicates for each of the three independent experiments. Enzyme activity assays for rS5nAi Enzymatic reactions with rS5nAi were carried out in 50 mM Tris–HCl pH 7, 10 mM MgCl2, 1 mM substrate and 0.1 µM of rS5nAi in a total volume of 20 µl. The reaction mixture was incubated for 20 min at 37°C. Substrates included AMP, ADP, ATP, dAMP, GMP, CMP and TMP (Sigma Aldrich). For pH titration at pH 5.0–6.5, Tris-HCl was replaced with 50 mM acetate buffer. Dependence on metal cations was analysed for Mg2+, Ca2+, Mn2+ and Zn2+ at three different concentrations (0.1, 1 and 10 mM) at pH 7 and 37°C. The enzyme kinetics were analysed by incubation of a fixed concentration (0.1 µM) of rS5nAi with increasing amounts of AMP in a total volume of 50 µl at pH 7 and 37°C. The enzymatic reactions were stopped by adding EDTA to a final concentration of 50 mM. The release of inorganic phosphate (Pi) was quantified using a malachite green phosphate colorimetic assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. Release of Pi was measured at Abs650nm and the amount of Pi was calculated based on a standard Pi curve. Michaelis-Menten curve fitting using non-linear regression was performed using GraphPad Prism version 7.03 software. To test for synergy with SpnAi, a 200 μl reaction mix containing 50 μg/ml of UltraPureTM salmon sperm DNA (Invitrogen), 10 μg/ml rSpnAi, 50 mM Tris–HCl (pH 7.0), 150 mM NaCl, 2 mM MgCl2 and 2 mM CaCl2 was incubated at 37°C. After 1 h, 0.1 μM S5nAi and 10 mM MgCl2 were added and the reaction was incubated for another 1 h. Generation of Pi was analysed as described above. All samples were analysed in triplicate for each of the three independent experiments. Results and Discussion Identification of SpnAi and S5nAi To investigate if recently discovered GAS virulence factor genes spnA and s5nA would also be present on the S. iniae genome, protein sequences of SpnA and S5nA derived from GAS strain SF370 (serotype M1) were used to search the S. iniae strain 9117 using the tblastn option at the NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). Two hypothetical open reading frames (ORFs) were identified that shared amino acid sequence identities of 63% and 62% with SpnA and S5nA, respectively, and were named SpnAi and S5nAi. These sequences were then used to search the entire nucleotide collection at NCBI. ORFs with significant sequence similarities were used to generate a phylogenetic tree (Fig. 1A). Notably, ORFs with significant sequence similarities to the putative S. iniae nuclease SpnAi (62–70%) were only found in certain streptococcal species (S. dysgalactiae, S. suis and S. equi) and not in any other genera. In contrast, and as previously reported, S5nA related nucleotidases are found in a wide range of organisms, including Gram-positive and Gram-negative bacteria, as well as mammals (9). The putative S. iniae nucleotidase S5nAi is most closely related to the UshA protein of S. dysgalactiae and to an uncharacterized ORF (SEQ1278) in S. equi, both sharing 68% sequence identity (Fig. 1A). Fig. 1 View largeDownload slide Bioinformatic analysis and purification of SpnAi and S5nAi. (A) Rooted phylogenetic tree UPGMA, (unweighted pair group method with arithmetic mean) of cell wall-anchored nucleases (left) and cell wall-anchored 5′-nucleotidases (right). The trees were generated with ClustalW using complete protein sequences of SpnAi (S. iniae), WP_003050123 (S. dysgalactiae), WP_024395691 (S. suis), WP_012679382 (S. equi), S5nAi (S. iniae), S5nA (S. pyogenes, AAK33792), AdsA (Staphylococcus aureus, ESR29110), ecto-5′-nucleotidase A (Nt5e) (S. sanguinis, AFK32764), Ssads (S. suis, YP001197640), NudP (S. agalactiae, CDN66659), UshA (S. dysgalactiae, ADX24386), SEQ1278 (S. equi, CAW94038), 5′-nucleotidase (5′NT) (E. coli, AJM76137) and CD73 (Homo sapiens, AAH65937). The number in parentheses show amino acid sequence identities with SpnAi and S5nAi. (B) Schematic presentation of SpnAi (top) and S5nAi (below) domain structure based on the prediction using InterProScan software at EMBL-EBI. The numbers on top represent amino acid positions. SP, signal peptide sequence; CWA, cell wall anchor domain. The arrows above indicate the regions that were generated as recombinant proteins in E. coli. (C) Recombinant form of SpnAi (left) and S5nAi (right) were expressed and purified from E. coli by immobilized metal chelate chromatography. The purity of the proteins was ≥95% as estimated from a 10% SDS-polyacrylamide gel. M, BenchMarkTM molecular weight marker. Fig. 1 View largeDownload slide Bioinformatic analysis and purification of SpnAi and S5nAi. (A) Rooted phylogenetic tree UPGMA, (unweighted pair group method with arithmetic mean) of cell wall-anchored nucleases (left) and cell wall-anchored 5′-nucleotidases (right). The trees were generated with ClustalW using complete protein sequences of SpnAi (S. iniae), WP_003050123 (S. dysgalactiae), WP_024395691 (S. suis), WP_012679382 (S. equi), S5nAi (S. iniae), S5nA (S. pyogenes, AAK33792), AdsA (Staphylococcus aureus, ESR29110), ecto-5′-nucleotidase A (Nt5e) (S. sanguinis, AFK32764), Ssads (S. suis, YP001197640), NudP (S. agalactiae, CDN66659), UshA (S. dysgalactiae, ADX24386), SEQ1278 (S. equi, CAW94038), 5′-nucleotidase (5′NT) (E. coli, AJM76137) and CD73 (Homo sapiens, AAH65937). The number in parentheses show amino acid sequence identities with SpnAi and S5nAi. (B) Schematic presentation of SpnAi (top) and S5nAi (below) domain structure based on the prediction using InterProScan software at EMBL-EBI. The numbers on top represent amino acid positions. SP, signal peptide sequence; CWA, cell wall anchor domain. The arrows above indicate the regions that were generated as recombinant proteins in E. coli. (C) Recombinant form of SpnAi (left) and S5nAi (right) were expressed and purified from E. coli by immobilized metal chelate chromatography. The purity of the proteins was ≥95% as estimated from a 10% SDS-polyacrylamide gel. M, BenchMarkTM molecular weight marker. The 2,823 bp nuclease gene spnAi from S. iniae strain 9117 encodes a 940 amino acid precursor protein with a predicted signal peptide sequence (position 1–34) and a C-terminal CWA domain (907–940). The s5nAi gene is 2,031 bp in length and encodes a 676 amino acid gene product with a predicted signal peptide sequence (position 1–27) and a C-terminal CWA domain (642–676) (Fig. 1B). In order to determine if SpnAi and S5nAi are functionally similar to the GAS proteins SpnA and S5nA, soluble recombinant proteins (rSpnAi and rS5nAi) that lack the N-terminal signal peptide sequence and the C-terminal CWA domain (Fig. 1B) were expressed with an N-terminal (His)6-tag in E. coli. The proteins were purified by Ni2+-affinity chromatography to a purity of approximately 95%, as estimated from an SDS-polyacrylamide gel (Fig. 1C). rSpnA and rS5nAi migrated at ∼100 and ∼70 kDa, respectively, which is in agreement with their calculated molecular weight of 96280.61 and 66935.19 Da, respectively. Biochemical analysis of recombinant SpnAi SpnA has a very strong dependence on Mg2+ and Ca2+ and is completely inactive if one of the cations is absent (8). To determine if SpnAi displays similar cation-dependence, purified rSpnAi was incubated with double-stranded linear lambda DNA in the presence of Ca2+ and Mg2+. Complete DNA digestion by rSpnAi was observed after 1 h incubation at 37°C. However, in the absence of either Ca2+/Mg2+ or both, the DNase activity of rSpnAi was completely undetectable (Fig. 2A) indicating a similar cation requirement as SpnA. The Mg2+ and Ca2+ dependence was further analysed at different concentrations of MgCl2 and CaCl2 (Fig. 2B and C). rSpnAi was active between 0.19 and 25 mM Mg2+ with maximum activity at 0.78 and 3.125 mM Mg2+, but was completely inactive at 50 mM Mg2+. This was similar to the results previously reported for rSpnA, which showed maximum activity at 1.56–3.125 mM and no activity at 50 mM (8). Although, the optimum Ca2+ concentration was similar between SpnAi (0.78-6.25 mM) and SpnA (0.78 mM), the S. iniae enzyme was still active at high concentrations of Ca2+ (∼70% activity at 200 mM), whereas the GAS enzyme was completely inactive at ⩾25 mM Ca2+(8). Fig. 2 View largeDownload slide Biochemical analysis of rSpnAi. (A) SpnAi activity depends on the presence of Ca2+ and Mg2+. Lambda DNA was digested in the presence or absence of each of the cations and analysed on a 1% agarose gel. C, control: reaction mixture without rSpnAi. Lambda DNA was digested with rSpnAi in the presence of varying concentrations of MgCl2 (B), varying concentrations of CaCl2 (C), at different pH (D) and at different temperature (E). Digested DNA was run on a 1% agarose gel and DNA bands were visualized after SYBR® safe staining and quantified using Image LabTM Software V5.2.1. (F) The minimum enzyme required for complete cleavage of 1 µg of lambda DNA was analysed at optimum reaction conditions (1 mM CaCl2, 3 mM MgCl2, pH 7, 37°C). The error bars show the standard deviation of three independent experiments performed in triplicates. Fig. 2 View largeDownload slide Biochemical analysis of rSpnAi. (A) SpnAi activity depends on the presence of Ca2+ and Mg2+. Lambda DNA was digested in the presence or absence of each of the cations and analysed on a 1% agarose gel. C, control: reaction mixture without rSpnAi. Lambda DNA was digested with rSpnAi in the presence of varying concentrations of MgCl2 (B), varying concentrations of CaCl2 (C), at different pH (D) and at different temperature (E). Digested DNA was run on a 1% agarose gel and DNA bands were visualized after SYBR® safe staining and quantified using Image LabTM Software V5.2.1. (F) The minimum enzyme required for complete cleavage of 1 µg of lambda DNA was analysed at optimum reaction conditions (1 mM CaCl2, 3 mM MgCl2, pH 7, 37°C). The error bars show the standard deviation of three independent experiments performed in triplicates. Next, we examined the effect of pH on rSpnAi activity using the constant optimized Mg2+ concentration of 3 mM. Greater than 50% activity was observed between pH 5 and 8.5, with maximum activity seen between pH 6.5 and 8. Reduced activity (∼25%) was also measurable at pH 4, while the enzyme was almost inactive at pH 9 (∼8% activity) (Fig. 2D). The pH range was slightly shifted into the alkaline range compared to rSpnA, which showed optimal activity between pH 5.5 and 7 (8). This might be due to adaptation to a different host, as most fish species have a normal blood pH in the range of 7.7–8.0 (33, 34), whereas human blood has a pH of 7.3–7.4. Recombinant SpnAi activity was determined for a range of different temperatures (12–47°C) at pH 7 and 3 mM Mg2+ (Fig. 2E). Maximum rSpnAi activity was found between 32–37°C, while activity was reduced to ∼80% at 27°C, ∼60% at 22°C, ∼40% at 17°C and 42°C and ∼25% at 47°C. Only marginal activity was observed at 12°C (<10%). This is only slightly lower than the optimal temperature range for rSpnA which was found to between 32 and 42°C (8). The enzymatic activity of rSpnAi under optimal conditions (1 mM CaCl2, 3 mM MgCl2, pH 7, 37°C) was determined by serial dilution of rSpnAi starting from 35 pM. As shown in Fig. 2F, 20 pmol of rSpnAi is the minimum amount of enzyme required to completely digest 1 µg of lambda DNA in 1 h. This is similar to rSpnA, for which 26 pmol was required for the same activity (8). All biochemical properties for rSpnAand rSpnAi are summarized in Table I. Table I. Summary of biochemical properties of rSpnAi and rSpnA Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  #without signal peptide and cell wall anchor regions. Table I. Summary of biochemical properties of rSpnAi and rSpnA Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  Biochemical properties  rSpnAi  rSpnA (8)  Molecular weight#  96280.61  94068.15  Cell wall anchor  +  +  Ca2+ and Mg2+ dependency  Yes  Yes  Optimum MgCl2  0.78–3.125 mM  1.56–3.125 mM  Optimum CaCl2  0.78–6.25 mM  0.78 mM  Optimum pH  pH 6.5–7.5  pH 5.5–7  Optimum temperature  32–37°C  32–42°C  Complete digest of 1µg of lambda DNA  20 pM  26 pM  #without signal peptide and cell wall anchor regions. Biochemical analysis of recombinant S5nAi We have previously shown that S5nA from S. pyogenes hydrolyses AMP and ADP, but not ATP, to produce the immunomodulatory product adenosine (9). We have tested rS5nAi for activity against the same components and found a similar substrate preference (Fig. 3A). The highest activity was observed against AMP, while ATP was not hydrolysed. In contrast to rS5nA, rS5nAi showed higher activity for ADP (9). There were also small differences in the activity for other nucleoside monophosphates. rS5nAi hydrolysed GMP, CMP and TMP with almost equal efficiency, but lower than for AMP, whereas rS5nA showed a high preference towards CMP (9). Both enzymes hydrolysed dAMP with similar efficiency as AMP, which is different from the NudP nucleosidase produced by Streptococcus agalactiae (Group B Streptococcus), which is unable to hydrolyse dAMP (35). Fig. 3 View largeDownload slide Biochemical analysis of rS5nAi. (A) Reaction mixtures containing 0.1 µM rS5nAi were incubated with 1 mM of various substrates for 20 min in the presence of 10 mM MgCl2 at 37°C. Hydrolysis of 1 mM AMP by 0.1 µM rS5nAi over 20 min was analysed in the presence of different divalent metal cations at varying concentrations (B), at different pH at 37°C (C) and at different temperature at pH 7 (D). (E) The time to reaction equilibrium was determined with 1 mM AMP and 0.1 µM rS5nAi in the presence of 10 mM MgCl2 at pH 7 and 37°C. (F) Enzyme kinetics of rS5nAi for AMP hydrolysis were determined by velocity measurements with various substrate concentrations in the presence of 10 mM MgCl2 at pH 7 and 37°C using GraphPad Prism V6.03 software. The Pi released in each experiment was quantified using a malachite green phosphate colorimetric assay kit. The error bars show the standard deviation of three independent experiments performed in triplicates. Fig. 3 View largeDownload slide Biochemical analysis of rS5nAi. (A) Reaction mixtures containing 0.1 µM rS5nAi were incubated with 1 mM of various substrates for 20 min in the presence of 10 mM MgCl2 at 37°C. Hydrolysis of 1 mM AMP by 0.1 µM rS5nAi over 20 min was analysed in the presence of different divalent metal cations at varying concentrations (B), at different pH at 37°C (C) and at different temperature at pH 7 (D). (E) The time to reaction equilibrium was determined with 1 mM AMP and 0.1 µM rS5nAi in the presence of 10 mM MgCl2 at pH 7 and 37°C. (F) Enzyme kinetics of rS5nAi for AMP hydrolysis were determined by velocity measurements with various substrate concentrations in the presence of 10 mM MgCl2 at pH 7 and 37°C using GraphPad Prism V6.03 software. The Pi released in each experiment was quantified using a malachite green phosphate colorimetric assay kit. The error bars show the standard deviation of three independent experiments performed in triplicates. Divalent cations are important as co-factors for nucleosidase activity. We analysed the activity of rS5nAi for AMP with either Mg2+, Mn2+, Ca2+ or Zn2+ at 37°C and pH 7 (Fig. 3B). The activity of rS5nAi was slightly higher in the presence of Mg2+ compared to Ca2+ in a dose-dependent manner with highest activity at 10 mM. Highest activity was also achieved with lower concentrations of Mn2+ (0.1 and 1 mM), whereas a higher concentration of 10 mM had an inhibitory effect. In contrast, rS5nAi hydrolysis of AMP was strongly decreased with Zn2+, in particular at 10 mM. These results are very similar to those observed for rS5nA (9). As observed for the nuclease enzyme, the optimal pH range was higher for S5nAi (pH 5–7.5) compared to S5nA (pH 5–6.6) (Fig. 3C), which might reflect an adaptation to the higher pH of fish blood compared to human blood (33, 34). The temperature requirements for rS5nA and S5nAi are very similar (9). Like rS5nA, rS5nAi shows maximum activity at 42°C, but is active over a wide range of temperatures (Fig. 3D). Even at 21 and 52°C, the enzymatic activity was found to be over 25% suggesting strong thermostability. Finally we tested the enzymatic activity against AMP at 37°C, pH 7 and 10 mM MgCl2. A time course showed that AMP hydrolysis with 0.1 μM rS5nAi reached equilibrium after 30 min (Fig. 3E), which is comparable to 25 min for rS5nA (9). The reaction kinetics followed the Michaelis–Menten model for a single substrate with a Km of 121.3 ± 1.683 μM and a Vmax of 7,808 ± 78.3 nmol of released Pi/mg enzyme/min (Fig. 3F). This corresponds well with the kinetics previously reported for rS5nA (Km of 168.3 ± 38 μM and a Vmax of 7,550 ± 326 nmol of released Pi/mg enzyme/min) (9). All biochemical properties for rS5nA and rS5nAi are summarized in Table II. Table II. Summary of biochemical properties of rS5nAi and rS5nA Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  aWithout signal peptide and cell wall anchor regions. bAMP hydrolysis with 0.1 μM rS5nAi at 37°C, pH 7 and 10 mM MgCl2. Table II. Summary of biochemical properties of rS5nAi and rS5nA Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  Biochemical properties  rS5nAi  rS5nA (9)  Molecular weighta  66935.19  66922.8  Cell wall anchor  +  +  Optimum pH  pH 5–7.5  pH 5–6.5  Optimum temperature  42°C  42°C  Substrate preference  AMP > dAMP > CMP = TMP = GMP > ADP ≫ ATP  AMP = dAMP = CMP > GMP= TMP > ADP > ATP  Enzyme kinetics      Time to reaction equilibriumb  30 min  25 min  Vmax (nmol Pi/mg/min)  7,808 ± 78.3  7,550 ± 326  Km  121.3 ± 1.683 µM  168.3 ± 38 µM  Activating cations  Mg2+, Ca2+, Mn2+  Mg2+, Ca2+, Mn2+  Inhibiting cations  Zn2+  Zn2+  aWithout signal peptide and cell wall anchor regions. bAMP hydrolysis with 0.1 μM rS5nAi at 37°C, pH 7 and 10 mM MgCl2. Synergy between SpnAi and S5nAi Based on work conducted with Staphylococus aureus, it has previously been suggested that nucleases and nucleotidases might work in synergy to evade the host immune system. It was proposed that the nuclease would hydrolyze DNA, including NETs, to generate the nucleotidase substrate dAMP, which would then be hydrolyzed to produce deoxyadenosine (dAdo) to trigger the caspase-3-mediated death of macrophages and monocytes restricting macrophage influx into abscesses (36, 37). To test the possibility that such a synergy also exists between SpnAi and S5nAi, we tested both enzymes together using salmon sperm DNA as substrate. A strong production of inorganic phosphate (Pi) was observed when both enzymes were mixed together. In contrast, addition of either SpnAi or S5nAi alone (or no enzyme) did not generate detectable Pi (Fig. 4). Fig. 4 View largeDownload slide Synergy between rSpnAi and rS5nAi. Incubation of salmon sperm DNA with both enzymes, but not with individual enzymes, results in the generation of Pi which was quantified using a malachite green phosphate colorimetric assay kit. This suggests that SpnAi hydrolyses dsDNA to produce deoxynucleotide monophosphates including dAMP, which can then be used as a substrate by S5nAi to generate deoxyadenosine (dAdo) and Pi. The error bars show the standard deviation of three independent experiments performed in triplicates. Fig. 4 View largeDownload slide Synergy between rSpnAi and rS5nAi. Incubation of salmon sperm DNA with both enzymes, but not with individual enzymes, results in the generation of Pi which was quantified using a malachite green phosphate colorimetric assay kit. This suggests that SpnAi hydrolyses dsDNA to produce deoxynucleotide monophosphates including dAMP, which can then be used as a substrate by S5nAi to generate deoxyadenosine (dAdo) and Pi. The error bars show the standard deviation of three independent experiments performed in triplicates. Conclusion The biochemical properties of the S. iniae enzymes SpnAi and S5nAi are very similar to those reported for S. pyogenes SpnA and S5nA, respectively. Together with similar protein lengths, domain structures and amino acid similarities, it is highly likely that these proteins are true orthologues and act together as immune evasion factors. Future work will include the generation of spnAi and s5nAi deletion mutants in S. iniae and their use in a zebrafish infection model (28, 31, 32). Investigation into bacterial dissemination in the host, NET degradation, and migration of neutrophils and macrophages to the site of infection will provide new insights into the function of the two enzymes. This might allow us to draw conclusions on similar virulence mechanisms used by S. pyogenes in the human host. Acknowledgements We thank Dr. Sarah Highlander, JCVI, La Jolla, C.A. for sending us the S. iniae 9117 strain. Funding This study was financially supported by a Graduate Student fund from the University of Auckland. K.Y.S. was supported by an University of Auckland International Doctoral Scholarship. J.M.S.L. is the recipient of a New Zealand National Heart Foundation Research Fellowship. Conflict of Interest None declared. References 1 Stevens D.L., Bryant A.E. ( 2016) Severe group A streptococcal infections in Streptococcus pyogenes: Basic Biology to Clinical Manifestations  ( Ferretti J.J., Stevens D.L., Fischetti V.A., eds.), University of Oklahoma Health Sciences Center, Oklahoma City (OK) 2 Cunningham M.W. ( 2012) Streptococcus and rheumatic fever. Curr. Opin. Rheumatol.  24, 408 Google Scholar CrossRef Search ADS PubMed  3 Martin W.J., Steer A.C., Smeesters P.R., Keeble J., Inouye M., Carapetis J., Wicks I.P. ( 2015) Post-infectious group A streptococcal autoimmune syndromes and the heart. Autoimmun. Rev . 14, 710– 725 Google Scholar CrossRef Search ADS PubMed  4 Carapetis J.R., Steer A.C., Mulholland E.K., Weber M. ( 2005) The global burden of group A streptococcal diseases. Lancet Infect. Dis.  5, 685– 694 Google Scholar CrossRef Search ADS PubMed  5 Bisno A.L., Brito M.O., Collins C. ( 2003) Molecular basis of group A streptococcal virulence. Lancet Infect. Dis . 3, 191– 200 Google Scholar CrossRef Search ADS PubMed  6 Cole J.N., Barnett T.C., Nizet V., Walker M.J. ( 2011) Molecular insight into invasive group A streptococcal disease. Nat. Rev. Micro.  9, 724– 736 Google Scholar CrossRef Search ADS   7 Courtney H.S., Hasty D.L., Dale J.B. ( 2002) Molecular mechanisms of adhesion, colonization, and invasion of group A streptococci. Ann. Med.  34, 77– 87 Google Scholar CrossRef Search ADS PubMed  8 Chang A., Khemlani A., Kang H., Proft T. ( 2011) Functional analysis of Streptococcus pyogenes nuclease A (SpnA), a novel group A streptococcal virulence factor. Mol. Microbiol . 79, 1629– 1642 Google Scholar CrossRef Search ADS PubMed  9 Zheng L., Khemlani A., Lorenz N., Loh J.M., Langley R.J., Proft T. ( 2015) Streptococcal 5′-nucleotidase A (S5nA), a novel Streptococcus pyogenes virulence factor that facilitates immune evasion. J. Biol. Chem.  290, 31126– 31137 Google Scholar CrossRef Search ADS PubMed  10 Papayannopoulos V., Zychlinsky A. ( 2009) NETs: a new strategy for using old weapons. Trends Immunol.  30, 513– 521 Google Scholar CrossRef Search ADS PubMed  11 Brinkmann V., Reichard U., Goosmann C., Fauler B., Uhlemann Y., Weiss D.S., Weinrauch Y., Zychlinsky A. ( 2004) Neutrophil extracellular traps kill bacteria. Science  303, 1532– 1535 Google Scholar CrossRef Search ADS PubMed  12 Hasegawa T., Minami M., Okamoto A., Tatsuno I., Isaka M., Ohta M. ( 2010) Characterization of a virulence-associated and cell-wall-located DNase of Streptococcus pyogenes. Microbiology  156, 184– 190 Google Scholar CrossRef Search ADS PubMed  13 Chalmers C., Khemlani A.H.J., Sohn C.R., Loh J.M.S., Tsai C.J., Proft T. ( 2017) Streptococcus pyogenes nuclease A (SpnA) mediated virulence does not exclusively depend on nuclease activity. J. Microbiol. Immunol. Infect . pii: S1684-1182(17)30236-0; doi: 10.1016/j.jmii.2017.09.006 [Epub ahead of print]. 14 Gorini S., Gatta L., Pontecorvo L., Vitiello L., la Sala A. ( 2013) Regulation of innate immunity by extracellular nucleotides. Am. J. Blood Res.  3, 14– 28 Google Scholar PubMed  15 Hasko G., Cronstein B.N. ( 2004) Adenosine: an endogenous regulator of innate immunity. Trends Immunol.  25, 33– 39 Google Scholar CrossRef Search ADS PubMed  16 Vitiello L., Gorini S., Rosano G., la Sala A. ( 2012) Immunoregulation through extracellular nucleotides. Blood  120, 511– 518 Google Scholar CrossRef Search ADS PubMed  17 Idzko M., Ferrari D., Eltzschig H.K. ( 2014) Nucleotide signalling during inflammation. Nature  509, 310– 317 Google Scholar CrossRef Search ADS PubMed  18 Xaus J., Mirabet M., Lloberas J., Soler C., Lluis C., Franco R., Celada A. ( 1999) IFN-gamma up-regulates the A2B adenosine receptor expression in macrophages: a mechanism of macrophage deactivation. J. Immunol . 162, 3607– 3614 Google Scholar PubMed  19 Cronstein B.N., Kramer S.B., Weissmann G., Hirschhorn R. ( 1983) Adenosine: a physiological modulator of superoxide anion generation by human neutrophils. J. Exp. Med . 158, 1160– 1177 Google Scholar CrossRef Search ADS PubMed  20 Edwards C.K.3rd, Watts L.M., Parmely M.J., Linnik M.D., Long R.E., Borcherding D.R. ( 1994) Effect of the carbocyclic nucleoside analogue MDL 201, 112 on inhibition of interferon-gamma-induced priming of Lewis (LEW/N) rat macrophages for enhanced respiratory burst and MHC class II Ia+ antigen expression. J. Leukoc. Biol.  56, 133– 144 Google Scholar CrossRef Search ADS PubMed  21 Hasko G., Szabo C., Nemeth Z.H., Kvetan V., Pastores S.M., Vizi E.S. ( 1996) Adenosine receptor agonists differentially regulate IL-10, TNF-alpha, and nitric oxide production in RAW 264.7 macrophages and in endotoxemic mice. J. Immunol . 157, 4634– 4640 Google Scholar PubMed  22 Bouma M.G., Jeunhomme T.M., Boyle D.L., Dentener M.A., Voitenok N.N., van den Wildenberg F.A., Buurman W.A. ( 1997) Adenosine inhibits neutrophil degranulation in activated human whole blood: involvement of adenosine A2 and A3 receptors. J. Immunol . 158, 5400– 5408 Google Scholar PubMed  23 Weinstein M.R., Litt M., Kertesz D.A., Wyper P., Rose D., Coulter M., McGeer A., Facklam R., Ostach C., Willey B.M., Borczyk A., Low D.E. ( 1997) Invasive infections due to a fish pathogen, Streptococcus iniae. N Engl. J. Med.  337, 589– 594 Google Scholar CrossRef Search ADS PubMed  24 Lau S.K., Woo P.C., Luk W.-K., Fung A.M., Hui W.-T., Fong A.H., Chow C.-W., Wong S.S., Yuen K.-Y. ( 2006) Clinical isolates of Streptococcus iniae from Asia are more mucoid and β-hemolytic than those from North America. Diagn. Microbiol. Infect. Dis . 54, 177– 181 Google Scholar CrossRef Search ADS PubMed  25 Baiano J.C., Barnes A.C. ( 2009) Towards control of Streptococcus iniae. Emerg. Infect. Dis.  15, 1891 Google Scholar CrossRef Search ADS PubMed  26 Pier G.B., Madin S.H. ( 1976) Streptococcus iniae sp-nov, a beta-hemolytic streptococcus isolated from an amazon freshwater dolphin, Inia Geoffrensis. Int. J. Syst. Bacteriol . 26, 545– 553 Google Scholar CrossRef Search ADS   27 Miller J.D., Neely M.N. ( 2005) Large-scale screen highlights the importance of capsule for virulence in the zoonotic pathogen Streptococcus iniae. Infect. Immun . 73, 921– 934 Google Scholar CrossRef Search ADS PubMed  28 Rowe H.M., Withey J.H., Neely M.N. ( 2014) Zebrafish as a model for zoonotic aquatic pathogens. Dev. Comp. Immunol.  46, 96– 107 Google Scholar CrossRef Search ADS PubMed  29 Facklam R., Elliott J., Shewmaker L., Reingold A. ( 2005) Identification and characterization of sporadic isolates of Streptococcus iniae isolated from humans. J. Clin. Microbiol.  43, 933– 937 Google Scholar CrossRef Search ADS PubMed  30 Zlotkin A., Hershko H., Eldar A. ( 1998) Possible transmission of Streptococcus iniae from wild fish to cultured marine fish. Appl. Environ. Microbiol . 64, 4065– 4067 Google Scholar PubMed  31 Neely M.N., Pfeifer J.D., Caparon M. ( 2002) Streptococcus-zebrafish model of bacterial pathogenesis. Infect. Immunity  70, 3904– 3914 Google Scholar CrossRef Search ADS   32 Saralahti A., Ramet M. ( 2015) Zebrafish and streptococcal infections. Scand. J. Immunol.  82, 174– 183 Google Scholar CrossRef Search ADS PubMed  33 Borvinskaya E., Gurkov A., Shchapova E., Baduev B., Shatilina Z., Sadovoy A., Meglinski I., Timofeyev M. ( 2017) Parallel in vivo monitoring of pH in gill capillaries and muscles of fishes using microencapsulated biomarkers. Biol. Open  6, 673– 677 Google Scholar CrossRef Search ADS PubMed  34 Evans D., Claiborne J. ( 1997) The Physiology of Fishes , CRC Press, Boca Raton 35 Firon A., Dinis M., Raynal B., Poyart C., Trieu-Cuot P., Kaminski P.A. ( 2014) Extracellular nucleotide catabolism by the Group B Streptococcus ectonucleotidase NudP increases bacterial survival in blood. J. Biol. Chem.  289, 5479– 5489 Google Scholar CrossRef Search ADS PubMed  36 Thammavongsa V., Missiakas D.M., Schneewind O. ( 2013) Staphylococcus aureus degrades neutrophil extracellular traps to promote immune cell death. Science  342, 863– 866 Google Scholar CrossRef Search ADS PubMed  37 Thammavongsa V., Schneewind O., Missiakas D.M. ( 2011) Enzymatic properties of Staphylococcus aureus adenosine synthase (AdsA). BMC Biochem.  12, 56 Google Scholar CrossRef Search ADS PubMed  Abbreviations Abbreviations S5nA Streptococcal 5′-nucleotidase A SpnA Streptococcus pyogenes nuclease A © The Author(s) 2018. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

The Journal of BiochemistryOxford University Press

Published: Apr 5, 2018

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