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Originally published In Press as doi:10.1074/jbc.M405434200 on August 10, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45909-45918, October 29, 2004
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Analysis of a Novel Prophage-encoded Group A Streptococcus Extracellular Phospholipase A2*

Michal J. Nagiec{ddagger}§, Benfang Lei§, Sarah K. Parker||, Michael L. Vasil||, Masakado Matsumoto§**, Robin M. Ireland§, Stephen B. Beres§, Nancy P. Hoe§, and James M. Musser{ddagger}§{ddagger}{ddagger}

From the {ddagger}Center for Human Bacterial Pathogenesis Research, Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, the §Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840, and the ||Department of Microbiology, University of Colorado Health Sciences Center, Denver, Colorado 80262

Received for publication, May 17, 2004 , and in revised form, August 2, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Group A Streptococcus (GAS) is an important human pathogen that causes many types of infections, including pharyngitis and severe invasive diseases. We recently sequenced the genome of a serotype M3 strain and identified a prophage-encoded secreted phospholipase A2 designated SlaA. To study SlaA structure-activity relationships, 20 site-specific mutants were constructed by alanine-replacement mutagenesis and purified to apparent homogeneity. Enzymatic activity was greatly reduced by alanine replacement of amino acid residues previously described as crucial in the catalytic mechanism of secreted phospholipase A2. Similarly, substitution of five residues in an inferred Ca2+-binding loop and three residues in the inferred active site region resulted in loss of activity of 76.5% or greater relative to the wild-type enzyme. Analysis of enzyme substrate specificity confirmed SlaA as a phospholipase A2, with activity against multiple phospholipid head groups and acyl chains located at the sn-2 position. PCR analysis of 1,189 GAS strains representing 48 M protein serotypes commonly causing human infections identified the slaA gene in 129 strains of nine serotypes (M1, M2, M3, M4, M6, M22, M28, M75, and st3757). Expression of SlaA by strains of these serotypes was confirmed by Western immunoblot. SlaA production increased rapidly and substantially on co-culture with Detroit 562 human pharyngeal epithelial cells. Together, these data provide new information about a novel extracellular enzyme that participates in GAS-human interactions.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Group A Streptococcus (GAS),1 a Gram-positive bacterial pathogen that causes diverse infections in humans, produces many extracellular molecules that contribute to host-pathogen interactions (1–3). Five genome sequences have been published for strains of this pathogen, including one strain each of serotype M1, M6, and M18, and two serotype M3 strains (4–8). Epidemiologic studies have documented that serotype M3 strains are the second most abundant cause of invasive infections in the United States, Canada, Western Europe, Japan, and Israel (8–12). In addition, prospective population-based surveillance studies have found that serotype M3 strains cause a higher rate of lethal infections than strains of other M types (13, 14). Serotype M1 strains are important because they are the most common cause of pharyngitis and invasive episodes (1).

One theme that has emerged from genome sequencing and related studies is that GAS strains contain multiple prophages that encode one or more proven or putative extracellular virulence factors (15, 16). For example, the sequenced strains of serotype M1, M3, M6, and M18 have 4–6 prophages or prophage-like elements. These prophages account for the great majority of strain-to-strain variation in gene content, and hence, are fundamental contributors to strain diversification and evolution.

Our analysis of the genome of serotype M3 strain MGAS315 identified a previously uncharacterized prophage-encoded extracellular phospholipase A2 (PLA2) designated SlaA (7). This GAS enzyme was the first secreted PLA2 (sPLA2) described for a bacterial pathogen. SlaA has a region of conserved amino acids residues found in several sPLA2 enzymes, including toxins made by venomous snakes, such as textilotoxin made by the Australian common brown snake (17). Of note, the prophage encoding SlaA was not present in serotype M3 GAS strains recovered before 1987, a time when resurgence of severe invasive disease episodes in North America, Europe, and Japan occurred (7). More recent studies have shown that SlaA expression was induced when strain MGAS315 was exposed to human pharyngeal epithelial cells (18), consistent with the observation that humans with pharyngitis and invasive episodes seroconvert to SlaA (7). In the aggregate, the data indicate that SlaA participates in host-pathogen interaction, perhaps as a factor that promotes colonization or virulence.

The goal of the present study was to analyze enzyme structure-activity relationships in SlaA, assess the distribution of the slaA gene in strains of a wide variety of M protein serotypes, and examine in vitro expression of SlaA by GAS strains of diverse M protein serotypes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Streptococcal Strains—Serotype M3 GAS strain MGAS315 has been extensively characterized, and the genome of this strain has been sequenced (7, 19–24). Other strains used in this study are described in Table I.


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  		TABLE I.
Characteristics of Group A Streptococcus strains studied

 
Gene Cloning and Mutagenesis—The gene segment encoding the mature (i.e. without secretion signal sequence) form of SlaA was cloned in Escherichia coli strain pET21d (Novagen, Madison, WI) with the following primers: 5'-ACCATGGAAGGGATAAATGATAAAATGG-3' and 5'-CGAATTCTTAACATCCTATAGAACCTAC-3'. The source strain used was serotype M3 MGAS315 (SlaA). Site-specific mutagenesis was done with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), pET21d containing wild-type slaA, and oligonucleotide primers (Table II). The mutant genes were sequenced to confirm the presence of the desired mutations and rule out spurious mutations.


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  		TABLE II
SlaA amino acid replacements and primers

 
Protein Expression and Purification—Recombinant SlaA proteins were expressed in E. coli strain BL21 (Novagen, Madison, WI) containing a plasmid with the wild-type or mutant gene. An AKTA Explorer FPLC instrument (Amersham Biosciences) was used for all chromatography procedures. Recombinant SlaA (rSlaA) and SlaA mutants were purified from E. coli strains grown at 37 °C in 4 liters of Luria-Bertani broth supplemented with 100 mg of ampicillin per liter. Expression of recombinant protein was induced by adding isopropyl-1-thio-{beta}-D-galactopyranoside at OD600 nm of 0.5, and growth was continued until early stationary phase. The cells were harvested by centrifugation, suspended in 40 ml of 10 mM Tris-HCl buffer, pH 8.0, and sonicated for 15 min. Cell debris was removed by centrifugation, and the supernatant was loaded onto a DEAE Sepharose Fast Flow column (Amersham Biosciences) equilibrated with 10 mM Tris-HCl buffer, pH 8.0. Proteins were eluted with a linear gradient of 0 to 0.3 M NaCl in 10 mM Tris-HCl buffer, pH 8.0. Fractions were analyzed for the presence of rSlaA by SDS-PAGE, and peak fractions were pooled. rSlaA was precipitated by adding (NH4)2SO4 (70% saturation at 25 °C). The precipitate was collected by centrifugation, suspended in 10 ml of 10 mM Tris-HCl, pH 8.0, and loaded onto a Phenyl-Sepharose 6 Fast Flow (high sub) column (Amersham Biosciences) equilibrated with 0.8 M (NH4)2SO4 in 10 mM Tris-HCl buffer, pH 8.0. Protein was eluted with a step gradient of (NH4)2SO4 (0.16, 0.08, 0.016, and 0 M) in 10 mM Tris-HCl buffer, pH 8.0. Fractions containing rSlaA were pooled, (NH4)2SO4 was added to 1.0 M, and the material was applied to an Octyl-Sepharose 4 Fast Flow column (Amersham Biosciences) equilibrated with 1 M (NH4)2SO4 in 10 mM Tris-HCl buffer, pH 8.0. The protein was eluted with a step gradient of (NH4)2SO4 (0.23, 0.1, 0.02, and 0 M) in 10 mM Tris-HCl buffer, pH 8.0. Fractions containing rSlaA were pooled and dialyzed overnight at 4 °C against 4 liters of 10 mM Tris-HCl buffer, pH 8.0. The dialysate was loaded onto a DEAE Sepharose Fast Flow column (Amersham Biosciences) equilibrated with 10 mM Tris-HCl buffer, pH 8.0, and the protein was eluted with a step gradient of NaCl (0.20, 0.25, and 0.30 M). The fractions were analyzed by SDS-PAGE the fractions containing rSlaA were pooled, and the protein was concentrated with a 10,000 kDa Centricon Plus-20 filter (Millipore, Bedford MA) by centrifugation at 3,200 x g for 15 min at 4 °C. The purified rSlaA was stored at –20 °C. rSlaA prepared by these procedures was greater than 95% pure, as assessed by SDS-PAGE and Coomassie Blue staining (see below).

SDS-PAGE and Native-PAGE—Samples for SDS-PAGE analysis were boiled for 3 min, loaded onto a Ready Gel 15% Tris-HCl gel (Bio-Rad) in Laemmli sample buffer containing 1% 2-mercaptoethanol, and resolved in running buffer (25 mM Tris, pH 8.3, 192 mM glycine, 0.1% SDS) at 200 V for 35 min. Samples for native-PAGE analysis were loaded onto a Ready Gel 15% Tris-HCl gel (Bio-Rad) in Native Sample Buffer (Bio-Rad) and resolved in native running buffer (25 mM Tris, pH 8.3, 192 mM glycine) at 90 V for 2 h. All gels were stained with Coomassie Blue.

Western Immunoblot Analysis—Purified recombinant proteins were resolved by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell, Keene, NH) with Towbin transfer buffer using a Trans-Blot S.D. semi-dry transfer cell (Bio-Rad) at 15 V for 40 min. The membrane was blocked with Amersham Biosciences Liquid Block (diluted 1:20 in 150 mM NaCl and 100 mM Tris-HCl, pH 8.0) for 1 h, and incubated for 1 h with affinity-purified rabbit polyclonal {alpha}-SlaA antibody (1:10,000 dilution) (Bethyl Laboratories, Montgomery, TX) added to the block solution. The membrane was rinsed twice and washed three times for 15 min each with 0.1% Tween 20 in PBS. The membrane was incubated for 1 h with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:4,000 in the block solution. The membrane was rinsed and washed as described above, and antibody-antigen interaction was visualized by enhanced chemiluminescence.

Monoclonal Antibody Production and Characterization—Three mouse monoclonal antibodies against purified rSlaA were generated by conventional methods (Lampire Biological Laboratories, Pipersville, PA). Linear epitope mapping was done with overlapping synthetic peptides (Mimotopes, Clayton, Victoria, Australia). Linear epitope mapping of monoclonal antibody 1D11 [PDB] –2H3, 1D11 [PDB] –3C3, and 9C3–2B12–11 identified reactivity with SlaA-specific peptide QNGELLKNYLILEGE, TQDKVSDDVLEMGML, and GQCQNHDSCYKWGAG, respectively.

PLA2 Enzyme Activity Assay—Purified wild-type and mutant proteins were assayed for PLA2 activity with the sPLA2 assay kit purchased from Cayman Chemical (Ann Arbor, MI). The assay measures the hydrolysis of phospholipids at the sn-1 and/or sn-2 position, yielding a free fatty acid and a lysophospholipid (25). All calculations and graphing were performed using GraphPad Prism 3.0 (San Diego, CA).

Analysis of Enzyme Substrate Specificity—Phospholipase A2 activity was detected using either fluorescent or radiolabeled substrates in mixed micelle form and radiolabeled dipalmitoyl [1,2-palmitoyl-1-14C]phosphatidylcholine (PC) in vesicle form (26). For radiolabel assays, 8 µg of SlaA was mixed with 0.454 nM radiolabeled substrate and 58 µM cold (unlabeled) dipalmitoyl PC in 50 mM Tris-HCl, pH 7.5, 2 mM CaCl2, and 0.25% Triton X-100. The radiolabeled substrates used included dipalmitoyl-[1,2-palmitoyl-1-14C]PC, 1-palmitoyl, 2-arachidonyl [arachidonyl-1-14C]PC, dipalmitoyl-[2-palmitoyl-1-14C]PC, 1-palmitoyl, 2-arachidonyl [arachidonyl-1-14C]phosphatidylethanolamine (PE), sphingomyelin [choline-methyl-14C] (SM, all from PerkinElmer Life Sciences), and dioleoyl phosphatidyl [3-14C]serine (PS, Amersham Biosciences). The test substrate was sonicated three times for 30 s and heated to 60 °C until clear before addition of the enzyme. The reaction mixtures were incubated at 37 °C for 2 h, and stopped by addition of chloroform-methanol-hydrochloric acid (50:50:0.3, v/v/v). For one-dimensional thin-layer chromatography, the lipids were analyzed with Silica Gel 60 plates, and separated with chloroform-methanol-water (14:6:1, v/v/v). Vesicle assays were done in the same manner with dipalmitoyl-[1, 2-palmitoyl-1-14C]PC, except without the addition of Triton X-100. Fluorescent mixed micelle assays were done using a range of 10–70 µg of SlaA mixed with 0.6 µg of FL C5, C16-phosphatidylinositol 3-phosphate (BODIPY, Molecular Probes, Eugene, OR) resuspended to 40 µM in 50 mM Tris-HCl with 2 mM CaCl2, 4.4 µM octylglucoside, and 0.02% bovine serum albumin. The reaction mixtures were incubated at 37 °C for 2 h, and the lipids were analyzed with Silica Gel 60 plates, in chloroform-acetone-methanol-water-ammonium hydroxide (45:35:8:2, v/v/v/v). Snake venom PLA2 from Naja mossambica mossambica (Sigma-Aldrich) was used as a control. The fluorescent and radiolabeled lipids were visualized and analyzed using Personal FX Phosphorimager and Quantity One software (Bio-Rad).

DNA Sequence Analysis of the slaA Gene among GAS Strains—The slaA gene was sequenced using PCR-amplified gene fragments and internal primers. All sequence data were collected with an Applied Biosystems model 3700 instrument.

Assessment of in Vitro Production of SlaA—GAS strains used to test for in vitro production of SlaA were cultured in protein-reduced Todd-Hewitt broth containing 0.2% (w/v) yeast extract (PR-THY; Difco Laboratories, Detroit, MI) in 5% CO2 at 37 °C (20). Bacteria were grown to mid-exponential, early stationary phase, and overnight, centrifuged, and the pellet was discarded. Culture supernatant proteins were precipitated with ethanol (final concentration, 75%), incubated for 1 h on ice and collected by centrifugation for 15 min at 12,000 x g. The precipitated proteins were suspended in 10 mM Tris-HCl, pH 8.0 and assayed for SlaA by Western immunoblot with rabbit {alpha}-SlaA polyclonal antisera, as described above.

Co-culture of GAS Strains with Detroit 562 Human Pharyngeal Epithelial Cells—Detroit 562 human pharyngeal epithelial cells (D562 cells) were grown on collagen-coated glass coverslips in 24-well tissue culture plates containing 1 ml of MEM plus 10% fetal bovine serum and phosphate-buffered saline-glucose and grown 2–3 days to 80% confluency. The growth medium was removed, and the cells were washed with 1 ml of MEM + 1 mM Ca2+ and Mg2+. 1 ml of fresh medium was added, and the cells were incubated for 3 h. During this time, overnight cultures of GAS test strains were inoculated into 25 ml of fresh THY medium and cultured for 3 h to an OD600 of 0.2–0.3. The bacteria were collected by centrifugation, washed once in phosphate-buffered saline, and suspended in 1 ml of phosphate-buffered saline. Bacteria (100 µl) were added to the D562 cells and incubated for 3 h at 37 °C. To test the time course of induction, one well was harvested immediately to serve as a 0 h control and then at 1, 2, and 3 h, respectively. The medium was removed and centrifuged at 4,000 x g to remove bacteria and host-cell debris. The supernatant was concentrated to 0.06 ml with a Nanosep 10,000 MWCO device (Pall Corporation, East Hills, NY). Each sample was mixed with 15 µl of 5x SDS sample buffer containing 1% 2-mercaptoethanol, boiled for 5 min, and an aliquot (10 µl) was resolved on a Ready Gel 15% Tris-HCl gel (Bio-Rad). Western immunoblot analysis was performed as described above with {alpha}-SlaA antibody diluted 1:100,000.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
slaA Gene Mutagenesis and Protein Purification—To identify amino acid residues in SlaA potentially important for structure-activity relationships, SlaA, textilotoxin, Naja naja atra venom PLA2, and bovine pancreatic PLA2 were aligned using Clustal W (Fig. 1). Textilotoxin, Naja naja atra venom PLA2, and bovine pancreatic PLA2 have been well studied and structure-activity relationships have been described (17, 27–31). Amino acid residues located in the putative active site and Ca2+-binding loop regions of SlaA were well conserved in these sPLA2 enzymes (Fig. 1). Conserved amino acid residues located in and around the putative active site and Ca2+-binding loop regions were replaced with alanine residues by site-specific mutagenesis. Additional conserved amino acids were replaced because of their putative structural importance based on known structure-activity relationships in Groups I and II sPLA2 enzymes (31). The twenty mutant proteins were overexpressed in E. coli and purified to apparent homogeneity (Fig. 2). Western immunoblot analysis using specific rabbit {alpha}-SlaA antibody and mouse monoclonal antibody confirmed that the correct protein had been purified (Fig. 2).



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  		FIG. 1.
Amino acid alignment of SlaA and related sPLA2. C-terminal region amino acid residues of SlaA, N. n. atra venom sPLA2, bovine pancreatic sPLA2, and the four subunits of textilotoxin were aligned with Clustal W using default parameters. Amino acid residues that match the consensus sequence are shaded in dark gray, and similar residues are shaded in light gray. The Ca2+ loop consensus sequence region is highlighted in blue, and the active site consensus sequence region is highlighted in red. Amino acid residues chosen for replacement are underlined. Conserved cysteine residues are indicated by green arrows. SlaC, phospholipase A2 from Group C Streptococcus.

 



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  		FIG. 2.
A, SDS-PAGE analysis of purified rSlaA and rSlaA mutant proteins (indicated by amino acid residue position of mature SlaA). B, Western immunoblot analysis of purified proteins using rabbit polyclonal anti-SlaA antisera. Small gel image shows Western immunoblot analysis of purified SlaA using monoclonal antibody 1D11 [PDB] –3C3. A 15% polyacrylamide gel was used.

 
Assessment of Mutant and Wild-type SlaA Enzymatic Activity—Mutant proteins with amino acid replacements located in the putative active site and Ca2+-binding loop regions had the greatest loss of enzymatic activity (Fig. 3). For example, replacement of the putative catalytic histidine residue (His-137) and aspartate residues (Asp-128, Asp-131, and Asp-138) in the putative active site region abolished activity (Fig. 3). In addition, replacement of amino acid residues located in the putative Ca2+-binding loop resulted in loss of activity ranging from 76.5% (G116A) to 100% (Y112A) (Fig. 3). In contrast, replacement of amino acid residues Val-126, Val-129, and Cys-151 resulted in mutant proteins with increased enzymatic activity (Fig. 3).



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  		FIG. 3.
Relative PLA2 activity of rSlaA mutant proteins. The following reaction conditions were used: 23 µg of enzyme and 1.66 mM 1,2-dithio analog of diheptanoyl phosphatidylcholine at 25 °C and pH 7.5. The data are presented as percent of wild-type activity.

 
Native-PAGE Analysis of SlaA and SlaA Mutant Proteins— Native-PAGE analysis was used to gain insight into the structural integrity of the mutant proteins. Three of the four mutant proteins with a cysteine residue replacement (C113A, C134A, and C151A) had substantially slower electrophoretic mobility than the wild-type enzyme, presumably due to altered protein conformation (Fig. 4). The electrophoretic mobility of the C140A mutant protein was not altered substantially, consistent with the hypothesis that C140 is located in the enzyme active site region and not involved in disulfide bond formation. The other mutant proteins lacked or had only modestly altered electrophoretic migration, suggesting that the observed changes in enzyme activity were not caused by gross changes in protein conformation.



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  		FIG. 4.
Native-PAGE analysis of rSlaA and mutant proteins (indicated by amino acid residue position of mature SlaA). The purified proteins were analyzed with a 15% polyacrylamide gel.

 
Assessment of SlaA Substrate Specificity—Use of radiolabeled substrates and 1D-TLC showed that SlaA had activity against phosphatidylcholine, phosphatidylserine, and phosphatidylethanoloamine. SlaA was not active against phosphatidylinositol 3P, as assessed with the fluorescent-labeled (BODIPY) 1D-TLC assay. With respect to the specificity of enzymatic activity against fatty acyl groups, SlaA-cleaved palmitic, oleic, and arachidonic acid present in the sn-2 position of the substrate. SlaA cleavage products were more readily released from substrates present in a mixed micelle assay than a PC vesicle assay, similar to other sPLA2s (26).

Incubation of SlaA with dipalmitoyl-[2-palmitoyl-1-14C]PC produced only palmitic acid as assessed by 1D-TLC. That is, no lysophosphatidylcholine (LPC) was detected, indicating that SlaA cleaved only at the sn-2 position. These results confirmed that SlaA has PLA2, rather than PLA1, activity. Moreover, incubation of SlaA and dipalmitoyl-[1,2-palmitoyl-1-14C]PC produced detectable palmitic acid and LPC, as analyzed by 1D-TLC. This result confirmed that SlaA has PLA, rather than phospholipase B (able to cleave at sn-1 and sn-2 positions at similar rates), activity.

Distribution of the slaA Gene among GAS Strains and Analysis of slaA Allelic Variation—The slaA gene was discovered as a consequence of sequencing the genome of a serotype M3 GAS strain. We showed previously that the slaA gene and the prophage encoding it were not present in serotype M3 strains until the mid-1980s, whereas the vast majority of serotype M3 strains recovered since the mid-1980s have the slaA gene and prophage (7). However, it is not known if the slaA gene is present in strains other than serotype M3 GAS. To determine if slaA was widely distributed in natural populations of GAS, 1,189 strains representing 48 emm types were studied by PCR. The slaA gene was present in 129 of the 1,189 strains, including emm1, emm2, emm3, emm4, emm6, emm22, emm28, emm75, and st3757 (Table I).

To test the hypothesis that allelic variation existed in the slaA gene present in these emm types, slaA was sequenced in the 129 GAS strains (Table I). 128 of 129 strains had the same slaA allele, designated slaA1. Only one additional allelic variant was identified (designated slaA2), and this variant was found in only one emm28 strain (MGAS9233). The slaA2 allele differed from slaA1 by one synonymous (silent) nucleotide change.

Analysis of in Vitro Expression of SlaA—Previous study of serotype M3 strain MGAS315 showed that SlaA was actively secreted into the culture supernatant of this organism (18). However, in vitro production by other GAS strains has not been studied. To test the hypothesis that organisms with the slaA gene expressed SlaA in vitro, 51 strains of diverse emm types were studied. As assessed using culture supernatants obtained at three phases of growth, relatively few (n = 17) strains produced immunoreactive SlaA (Table I). This result was unexpected given that we previously found that patients infected with serotype M3 strains seroconverted to SlaA (7). In addition, the slaA gene sequencing data did not reveal nucleotide variation that would account for lack of in vitro expression of immunoreactive SlaA.

Banks et al. (18) recently reported that serotype M3 strain MGAS315 significantly up-regulated SlaA production when grown in vitro in the presence of D562 human pharyngeal epithelial cells. Hence, we tested the hypothesis that analogous up-regulation of SlaA production would occur in the 51 GAS strains tested. Consistent with the hypothesis, a strikingly increased amount of immunoreactive SlaA was detected in the tissue culture medium (Fig. 5). Moreover, virtually all GAS strains produced detectable immunoreactive SlaA in this assay (Table I).



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  		FIG. 5.
Western immunoblot analysis showing up-regulation of in vitro expression of SlaA occurring on co-culture of GAS with D562 human epithelial cells. Serotype M3 strain MGAS1563 was used as a representative organism. M.E., mid-exponential phase of growth; L.E., late-exponential phase of growth; Stationary, stationary phase of growth; Co-culture, strain MGAS1563 co-cultured with D562 human epithelial cells. Culture supernatants were analyzed using purified rabbit anti-SlaA antibody raised against purified recombinant SlaA.

 
To further probe the characteristics of the interaction between GAS and D562 pharyngeal cells, we conducted a time course experiment to assess the rapidity with which increased immunoreactive SlaA occurred on initiation of co-culture. Genetically representative strains selected from four M-serotypes causing abundant episodes of pharyngitis and invasive disease were used, including M2 strain MGAS12508, M3 strain MGAS1563, M6 strain MGAS10394, and M28 strain MGAS6180. The bacteria were co-cultured with D562 cells for a maximum of 3 h. Western immunoblot analysis (Fig. 6) showed that immunoreactive SlaA was absent in the culture supernatant at the zero hour time point for all strains. Immunoreactive SlaA increased rapidly over the 3 h of co-culture for all strains analyzed. The greatest increase in immunoreactive SlaA occurred between 1 and 2 h, indicating rapid induction of SlaA.



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  		FIG. 6.
Western immunoblot analysis showing time course of up-regulation of in vitro expression of SlaA occurring on coculture of GAS with D562 human epithelial cells. Supernatants obtained at 0, 1, 2, and 3 h after co-culture are shown. A, GAS serotype M2 strain MGAS12508. B, GAS serotype M3 strain MGAS1563. C, GAS serotype M6 strain MGAS10394. D, GAS serotype M28 strain MGAS6180.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The protein we characterized in this study was the first secreted PLA2 enzyme to be isolated from a pathogenic bacterium, making SlaA an important addition to the PLA2 superfamily. Although the biologic function of SlaA has not been elucidated, it is reasonable to hypothesize that it participates in host-pathogen interaction. Secreted PLA2 enzymes can have a wide range of functions, including cellular toxicity, anticoagulant activity, proinflammatory effects, and bactericidal activity (30). The presence of anti-SlaA antibodies in convalescent sera obtained from patients with infections caused by serotype M3 GAS strains and the substantial up-regulation of SlaA production in response to interaction with human epithelial cells support the idea that SlaA participates in GAS pathogenesis (7, 18).

Predictions of Structural Features Derived from the Amino Acid Sequence—Historically, sPLA2s that have been characterized have been obtained mainly from mammalian tissue, blood, and vertebrate and insect venoms (30). Secreted PLA2s have a histidine residue as their catalytic amino acid residue and, with a few exceptions, a highly conserved active site region (DXCCXXHDXCY) and Ca2+-binding loop (XYCGXGGXG) (31, 32, 33). Comparison of SlaA with other sPLA2s revealed some notable differences. As expected, SlaA has diverged considerably from sPLA2s made by eukaryotic organisms, but the active site and Ca2+-binding loop regions are well conserved. One striking difference between SlaA and eukaryotic sPLA2s is the small number of cysteine residues in SlaA relative to the other sPLA2s. sPLA2s are generally cysteine-rich and contain 5–8 disulfide bonds that contribute to structural integrity (30). Among PLA2s assigned to group I, II, V, or X, six disulfide bonds are absolutely conserved. In contrast, SlaA has only six cysteine residues, and only one putative disulfide pair (Cys-113–Cys-134) is conserved relative to the enzymes in the groups listed above.

Recently a new group (XIV) of PLA2s was described that includes enzymes made by the soil bacterium Streptomyces violaceoruber and Tuber borchii, a symbiotic fungus (34, 35). These discoveries showed that sPLA2s are not produced exclusively by higher eukaryotes. These two enzymes have high amino acid sequence homology to one another, especially in their active site and Ca2+-binding regions. Compared with groups I, II, III, V, IX, X, XI, and XII enzymes, group XIV enzymes have relatively few cysteine residues, only one conserved residue (Cys) in the Ca2+-binding loop, and very low sequence conservation in the active site region. Of note, the crystal structure of the Streptomyces enzyme showed conservation of the His-Asp catalytic center motif, but absence of a loop architecture in the Ca2+-binding region. The SlaA enzyme appears to represent a unique type of sPLA2 in that it is made by a prokaryote, has low cysteine content like group XIV enzymes, but has an active site and Ca2+-binding loop sequence most similar to group I and II enzymes. SlaA also differs from other sPLA2s in molecular mass. The great majority of previously described PLA2s have an inferred molecular mass (mature protein) of 12.4–15 kDa (30), whereas the inferred molecular mass of mature SlaA is 18.6 kDa. The additional amino acids responsible for this increase in inferred molecular mass are located at the N-terminal region immediately after the secretion signal. Based on these aggregate amino acid sequence features and the assessment that SlaA hydrolyzes the ester bond of a range of phospholipids at the sn-2 position, we propose that SlaA be designated as the first member of a novel group of the PLA2 superfamily of enzymes.

Insights into Substrate Specificity—The number of phospholipid species is too numerous to investigate the ability of SlaA to cleave all possible substrates. Hence, we focused on potential physiologically relevant substrates. Specifically, we addressed whether SlaA distinguished between the sn-1 and sn-2 positions, cleaved saturated and/or unsaturated fatty acyl groups, and used substrates with varying physiologically relevant head groups. Our analysis revealed that SlaA functions on saturated, monounsaturated, and polyunsaturated fatty acyl groups. Most significantly, we found that SlaA was able to cleave arachidonic acid, a known precursor to ecosanoids participating in the inflammatory cascade. Furthermore, we found that SlaA was able to cleave three common phospholipid head groups (Table III).


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  		TABLE III
Activity of SlaA against select substrates

 
Insights Into SlaA Structure-Activity Relationships Revealed by Site-directed Mutagenesis—Alanine replacement mutagenesis provided important insights into structure-activity relationships in SlaA, particularly with respect to amino acid residues putatively involved in enzyme catalysis and Ca2+ binding. The crystal structure of SlaA has not been solved, and molecular modeling using SWISS-MODEL was unsuccessful due to the low sequence identity between SlaA and sPLA2 enzymes with structures deposited in the Protein Data Bank. The results of the site-directed mutagenesis studies support the idea that H137 is the catalytic residue, consistent with amino acid sequence alignments (Fig. 1) and known structure-activity relationships in other sPLA2 enzymes (30). In addition, with the exception of Gln-135, all targeted conserved amino acid residues within the putative active site region had greatly decreased or total loss of enzyme activity. We note that Gln-135 has no known involvement in the catalytic function of sPLA2s enzymes (31).

If SlaA has a His-Asp catalytic dyad like other sPLA2, based on amino acid sequence alignments it is possible that Asp-177 represents the catalytic dyad Asp residue. To test this idea, we replaced Asp-177 with an Ala residue. However, despite extensive attempts, we were unable to purify sufficient recombinant mutant protein for conducting enzyme assays.

Replacement of amino acid residues thought to bind Ca2+ (Gly-114 and Asp-138) or enhance the structural integrity of the Ca2+-binding loop (Y112, C113, G114, and G119) resulted in loss of virtually all enzyme activity. This loss of enzyme activity could not be reversed by the addition of excess Ca2+ to the reaction mixture (data not shown). These results are consistent with the hypothesis that SlaA requires calcium for maximum enzymatic activity (30, 36).

Previous studies have shown that tyrosine residues are located in the catalytic network of PLA2 enzymes. Site-directed mutational studies have demonstrated that tyrosines function primarily to provide structural support (27). Based on amino acid sequence alignments, the most likely residues serving this function in SlaA are Tyr-141 and Tyr-160. Consistent with the idea that these two tyrosine residues are functionally important, the Y141A and Y160A mutant proteins had greatly decreased PLA2 activity (Fig. 3).

Molecular Population Genetics of the slaA Gene—The slaA gene was discovered as a consequence of sequencing the genome of a contemporary serotype M3 GAS strain. The slaA gene was found to be encoded by a prophage which was recently shown to be inducible (18). These observations raised the possibility that slaA was more widely distributed in GAS than simply among serotype M3 strains. We found that the slaA gene was present in 10.8% of 1,189 GAS strains tested, including emm1, emm2, emm3, emm4, emm22, emm28, emm75, and st3757 strains. DNA sequence analysis of slaA in 129 strains revealed that with a single exception, all isolates had the identical allele of this gene. The presence of the same allele of a prophage-encoded gene in GAS strains that are otherwise highly differentiated in overall chromosomal character is strong evidence that slaA has been horizontally transferred in nature, presumably by transduction. Moreover, the lack of nucleotide variation in slaA suggests that the dissemination of slaA to diverse GAS strains has occurred very recently.

In Vitro Expression of SlaA—There is considerable emerging evidence that environmental signals have an important role in influencing the expression of prophage-encoded extracellular proven or putative virulence factors in GAS (15, 16, 18). By showing that the interaction of four GAS strains representing commonly occurring M protein serotypes with D562 pharyngeal cells resulted in greatly increased SlaA expression, we provided additional evidence supporting this idea. Furthermore, our data indicated that 92% of GAS strains tested upregulated SlaA expression in vitro in response to interaction with D562 cells. As assessed by a time-course assay, up-regulation of production of immunoreactive SlaA occurred rapidly on co-culture with D562 pharyngeal epithelial cells. Together, the data support the hypothesis that SlaA production confers an enhanced survival capacity to GAS during human interaction in the upper respiratory tract, and perhaps other anatomic sites. The results of studies conducted with an isogenic mutant strain in which the slaA gene was inactivated are consistent with the idea that SlaA production enhances GAS survival.2


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Current address: Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717. Back

** Current address: Dept. of Microbiology, Aichi Prefectural Institute of Public Health, Nagoya 462-8576, Japan. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Dept. of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Tel.: 713-798-3823; Fax: 713-798-5838; E-mail: musser{at}bcm.tmc.edu.

1 The abbreviations used are: GAS, Group A Streptococcus; PLA2, phospholipase A2; sPLA2, secreted phospholipase A2; Musser Group A Streptococcus MGAS, strain; SlaA, Group A streptococcal phospholipase A2; rSlaA, recombinant SlaA; PC, phosphatidylcholine; SM, sphingomyelin; PS, phosphatidylserine; PE, phosphatidylethanolamine; FA, fatty acid; LPC, lysophosphatidylcholine; LPS, lysophosphatidylserine; PI3P, phosphatidylinositol 3-phosphate; PL, phospholipid. Back

2 I. Sitkiewicz, R. Ireland, and J.M. Musser, manuscript in preparation. Back


   ACKNOWLEDGMENTS
 
We thank K. Krause and S. Shelburne for helpful discussion and critical reading of the manuscript, and G. Sylva for technical assistance.



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TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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