Inhibition of Cell Surface Export of Group A Streptococcal Anchorless Surface Dehydrogenase Affects Bacterial Adherence and Antiphagocytic P
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感染与免疫杂志 2005年第10期
Laboratory of Bacterial Pathogenesis, Public Health Research Institute at The International Center for Public Health, 225 Warren Street, Newark, New Jersey 07103-3535
ABSTRACT
Surface dehydrogenase (SDH) is an anchorless, multifunctional protein displayed on the surfaces of group A Streptococcus (GAS) organisms. SDH is encoded by a single gene, sdh (gap or plr) that is essential for bacterial survival. Hence, the resulting nonfeasibility of creating a knockout mutant is a major limiting factor in studying its role in GAS pathogenesis. An insertion mutagenesis strategy was devised in which a nucleotide sequence encoding a hydrophobic tail of 12 amino acids (337IVLVGLVMLLLS348) was added at the 3' end of the sdh gene, successfully creating a viable mutant strain (M1-SDHHBtail). In this mutant strain, the SDHHBtail protein was not secreted in the medium but was retained in the cytoplasm and to some extent trapped within the cell wall. Hence, SDHHBtail was not displayed on the GAS surface. The mutant strain, M1-SDHHBtail, grew at the same rate as the wild-type strain. The SDHHBtail protein displayed the same GAPDH activity as the wild-type SDH protein. Although the whole-cell extracts of the wild-type and mutant strains showed similar GAPDH activities, cell wall extracts of the mutant strain showed 5.5-fold less GAPDH activity than the wild-type strain. The mutant strain, M1-SDHHBtail, bound significantly less human plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity. These results indicate that the prevention of the cell surface export of SDH affects the virulence properties of GAS. The anchorless SDH protein, thus, is an important virulence factor.
INTRODUCTION
Surface proteins of pathogens play a variety of roles in virulence. Group A Streptococcus (GAS) (Streptococcus pyogenes), a human pathogen that is responsible for a variety of mild to severe invasive diseases of the pharynx and skin, displays an array of multifunctional proteins on its surface (5, 9, 11). In gram-positive pathogens, including GAS, classical surface proteins with defined structural components, such as a signal sequence, a proline/glycine-rich cell wall-associated domain, a hydrophobic membrane domain, and a cell wall-sorting hexapeptide motif (LPXTGX), have been characterized most extensively (15, 30). More recently, complete genome sequence analyses of six different GAS serotypes have revealed that lipoproteins also contribute significantly to the surface layer of GAS and in disease pathogenesis (1, 2, 13, 29, 43).
In the last decade, after reports of the presence of the glycolytic enzymes glycerladehyde-3-phosphate dehydrogenase (GAPDH) or streptococcal surface dehydrogenase (SDH) (27, 33) and streptococcal surface enolase (SEN) (37) on the surface of GAS, there has been an increasing awareness about the presence of similar glycolytic enzymes on the surfaces of a variety of human pathogens (for a review, see reference 32). Since these surface-located metabolic enzymes are generally found in the cytoplasm and lack C-terminal hydrophobic tails and N-terminal signal sequences required for their export to the cell surface, they are called anchorless surface proteins (32).
In addition to innate glycolytic activities, both SDH and SEN perform a variety of functions (27, 31, 33, 34, 36). Whereas some of the notable functions of SDH are its ability to bind various mammalian proteins, including plasminogen (Plg) (27, 33), ADP ribosylation (34), and host cell-signaling regulation (36), the function of SEN seems to be to serve as a major Plg-binding protein on the surface of GAS (31, 37). Although SDH in comparison to SEN is a weak Plg-binding protein (33), the C-terminal lysine residues of both SDH and SEN seem to play a crucial role in Plg binding (37). Like many other GAPDH molecules (4, 17, 18, 44), the predictive three-dimensional structure of SDH has indicated that the SDH molecule possesses two domains (24). The N-terminal NAD-binding domain (amino acid residues [aa] 1 to 149) consists of a six-stranded parallel -sheet flanked by helices. The C-terminal half of SDH contains the catalytic domain, which consists of a nine-stranded antiparallel sheet followed by three approximately parallel helices and a polypeptide chain that ends in a long helix. The region spanning amino acid residues 179 and 200, which forms an S-shaped antiparallel sheet, is also called the S-loop (18, 24, 44). Recently, we identified CD87/urokinase plasminogen activator receptor (uPAR) as the SDH-specific receptor on the surfaces of pharyngeal cells (24). The N-terminal domain of uPAR binds to the C-terminal alpha-helix and the flanking regions of the S-loop of the SDH molecule (24). This specific binding also plays a crucial role in GAS adherence to pharyngeal cells (24). Thus, the C-terminal portion of SDH is essential to perform a variety of biologically relevant functions. We also reported that ATP-producing glycolytic enzymes likely form a specialized complex on the surface of GAS (35). Although accumulated reports now indicate that anchorless proteins are present on the surfaces of many pathogens, including gram-positive bacteria, fungi, and parasites (32), our understanding of their biological functions and their roles in the pathogenesis of GAS and other bacterial pathogens is far from complete. Likewise, how they are exported to the cell surface is unknown.
To evaluate the role of virulence factors, the genetic approach to create a knockout mutant that can be tested in various in vitro and/or in vivo disease model systems as a distinct attenuated or nonpathogenic phenotype is highly desirable. However, the knockout mutagenesis approach is not achievable for a gene that is essential for microbial survival. In certain organisms, such as Escherichia coli, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, more than one GAPDH and enolase isoenzymes are present; each of these isoenzymes is encoded by a distinct gene or the organism is equipped with the Entner-Doudoroff alternate metabolic pathway (7, 20-22, 28, 46). Hence, it has been possible to create mutants defective in GAPDH, enolase, and other enzymes of glycolysis (19, 22, 23, 28). In GAS, glucose is metabolized solely by the glycolytic pathway, as GAS lacks the tricarbolicacetic acid (TCA)-metabolic cycle and the Entner-Doudoroff alternative metabolic pathway (1, 2, 13, 29, 43). The multifunctional SDH is encoded by a single essential gene (33, 45). The gene knockout approach to prevent SDH expression in GAS, therefore, is not feasible. The site-directed mutagenesis strategies that allow alteration in the specific functional domains without disturbing the conformational structure responsible for the catalytic function have been successfully employed to create Plg-binding enolase- and SDH/Plr-specific deletion mutants (3, 12, 45). Since the putative transport system responsible for exporting the anchorless metabolic surface enzymes is not known, it has not been possible to create a GAS mutant in which the anchorless enzyme is selectively retained in the cytoplasm and not expressed on the surface.
Since the SDH molecule does not possess the N-terminal signal sequence and the C-terminal hydrophobic tail, we hypothesized that the introduction of only a hydrophobic tail at the C-terminal end may result in its prevention of export to the surface and the SDH may be retained in the cytoplasm and/or in the membrane. Accordingly, in the present investigation, we altered the sdh gene by introducing a nucleotide sequence encoding a de novo C-terminal hydrophobic tail. The resulting mutated SDH protein remained largely restricted to the cytoplasm. This mutant strain behaved in a manner similar to that of the wild-type strain in terms of growth characteristics and GAPDH enzyme activity, but it bound significantly less Plg, adhered poorly to human pharyngeal cells, and lost the typical antiphagocytic activity associated with the wild-type strain. Our results also suggest that SDH may have a role in the regulation of the expression of a certain virulence factor-related gene(s), possibly at the transcriptional level.
MATERIALS AND METHODS
Bacterial strains, vectors, growth conditions, and DNA techniques. S. pyogenes wild-type strain M1-SF370 (M1-WT; ATCC 700294; American Type Culture Collection, Manassas, VA) was grown in Todd-Hewitt broth (Difco Laboratories) supplemented with 0.5% yeast extract or on proteose peptone-3 blood agar plates supplemented with spectinomycin (up to 500 μg/ml), when required. E. coli strain XL1-Blue was used for the cloning experiments and grown in Luria-Bertani (LB) broth or on LB agar plates. Vector pFW5 containing the spectinomycin resistance gene (aad-9) was used to transform the M1-SF370 strain (12, 39).
Reagents. Unless otherwise stated, all chemicals were procured from Sigma (St. Louis, MO).
Human pharyngeal cell lines. Detroit 562 (ATCC CCL138) pharyngeal cells were grown to confluence and maintained in a CO2 incubator as described previously (36).
Determination of the sequence of the hydrophobic tail. The prediction of hydrophobicity of a putative hydrophobic tail was determined based on the dense alignment surface (DAS) score as described at the website http://www.sbc.su.se/miklos/DAS/. The amino acid sequence of a hydrophobic tail to be inserted at the C-terminal end of SDH was based on the translated amino acid sequence of the epf gene in the M1 genome sequence database (13) and was edited using the DAS method to get an optimum DAS score (3.0), one indicative of a typical transmembrane location (8).
Construction of insertion and allelic-exchange sdhHBtail mutations in GAS. To create streptococcal mutant strains expressing SDH with a hydrophobic tail at the C-terminal end, the sdh gene (0.92-kb DNA fragment) and a 1.118-kb DNA fragment corresponding to the downstream region of the sdh gene in the M1-SF370 chromosome were PCR amplified using primer pairs SDH-SalI-F-/SDH-BamHI-R (5'-ACGCGTCGACATGGTAGTTAAAGT TGGTATTAACGG-3' and 5'-CGCGGATCCTTATTTAGCAATTTTTGCGAAGTACTCAAGAGTACG-3') and SDHDWN-PstI-F/SDHDWN-NdeI-R (5'-TTTTCTGCAGCTTGGTTTATGCTG AGTTCTTTTTCG-3 and 5'-GGGAATTCCATATGTATTGGAAAATGCTATCGTGCG-3'), respectively. The PCR-amplified products were cloned in the multiple cloning sites located upstream and downstream of the spectinomycin resistance gene, aad-9, in the suicide plasmid pFW5 (2.7 kb) to construct pFW5-sdh. To insert a DNA sequence encoding the putative hydrophobic tail at the 3' end of the sdh gene in the pFW5-sdh vector, a QuickChange II site-directed mutagenesis kit was used according to the manufacturer's instructions (Stratagene) with the plasmid pFW5-sdh as the template and a pair of complementary primers, SDHHBtail-F (5'-GTACTTCGCAAAAATTGCTAAAATTGTTCTTGTTGGCTTAGTTATGCTTCTTCTTTCTTAATAGGATCCTCGAGCTCTAG-3') and SDHHBtail-R (5'-CTAGAGCTCGAGGATCCTATTAAGAAAGAAGAAGCATAACTAAGCCAACAAGAACAATTTTAGCAATTTTTGCGAAGTAC-3'). The resulting plasmid, pFW5-sdhHBtail, was introduced into strain M1-WT by electroporation, using a Gene Pulser II electroporator (Bio-Rad) as described previously (12, 29). The mutant strain thus obtained was called M1-SDHHBtail. The confirmation of mutations and insertions at various stages of the experiments was obtained by one or more methods of PCR, DNA sequencing, and Southern hybridization. Using a similar strategy, a mutant strain lacking the M1 protein (M1Demm1) was created. The primers used to create this mutant were emm1-F (5'-ACGCGTCGACTAGGTCAAAAAGGTGGC-3'), emm1-R (5'-CGCGGATCCGCATTCTCTAAT CTCGCTT-3'), emm1DWN-F (5'-AACTGCAGGACTTGGACGCATCACGTGAA-3'), and emm1DWN-R (5'-GATTCCATATGCTGTCTCTTAGTTTCCTTCATTGGTGC-3'). The italicized sequences in these primers represent SalI, BamHI, PstI and NdeI restriction sites, respectively.
Cell fractionation and whole-cell extraction. M1-WT and M1-SDHHBtail (mutant) strains were grown to late log phase/early stationary phase (optical density at 600 nm [OD600], 0.8), and bacteria were harvested by centrifugation. The culture supernatants from these strains were saved, and the proteins therein were precipitated with 25% trichloroacetic acid (TCA). The resulting precipitates were separated by centrifugation (20,000 x g for 30 min at 4°C), washed twice with acetone, and suspended in 1/50 of the original volume of 50 mM Tris-HCl, pH 7.0. The harvested bacteria were subjected to mutanolysin treatment in a cell wall-extracting buffer (50 mM Tris-HCl, pH 7.0, 30% raffinose) containing 250 U/ml of mutanolysin (Sigma) as described previously to achieve nearly complete wall extraction (33). The resulting protoplasts were lysed in a hypotonic buffer, followed by repeated freezing and thawing. Bacterial membrane and cytoplasmic fractions were separated from the lysed protoplasts by ultracentrifugation (100,000 x g for 45 min at 4°C) as described previously (33). Whole-cell extracts from these two strains were obtained using the same procedures described above except that the cell wall-extracting buffer was used without 30% raffinose.
SDH and SDHHBtail purification. Clear whole lysates obtained as described above from 500 ml THY broth culture with each of the M1-WT and M1-SDHHBtail strains were dialyzed against 0.05 M NH4HCO3 and precipitated at 40%, 60%, and 80% ammonium sulfate saturation. The protein precipitates obtained from 60% to 80% ammonium sulfate saturation, which contained the major amount of SDH and SDHHBtail, were purified by Hi-trap Blue-HP column (1 ml) chromatography (Pharmacia) after equilibration with a starting buffer (50 mM Tris-HCl, pH 7.4). Proteins other than SDH and SDHHBtail were eluted first by the five-column volume of starting buffers containing 0.5 M NaCl followed by the five-column volume of the same buffer containing 1.0 M NaCl. The remaining column-bound proteins were eluted with the five-column volume of the starting buffer containing 5 mM NAD. The NAD-eluted fractions (1 ml each) contained only SDH or SDHHBtail, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The pooled fractions containing SDH or SDHHBtail were dialyzed against the starting buffer and stored at –70°C until further use.
Preparation of monospecific anti-SDH antibody. Anti-SDH specific immunoglobulin G (IgG) was purified from the SDH-immunized rabbit serum using an SDH-linked diaminodipropylamine M-phase (Pierce) affinity column as described previously (33, 37). The purified IgG was then treated with immobilized papain enzyme (Pierce) at 4°C overnight to cleave them between their Fab' and Fc regions. The latter was then removed by passing the cleaved products on a protein A column (Pierce) per the manufacturer's instructions. The unbound initial fall-through was used as Fab'-specific anti-SDH IgG.
Electrophoresis on SDS-polyacrylamide gels and Western blotting. Purified SDH, SDHHBtail, and cell wall-associated proteins were separated by SDS-PAGE and Western blotted onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the presence of SDH was detected by reacting PVDF membranes with anti-SDH monoclonal antibody (clone 13D5) (16) or monospecific rabbit anti-SDH IgG followed by corresponding alkaline phosphatase-labeled conjugate antibody (Fab' specific). SDH-specific protein bands on PVDF membranes were visualized using a chromogenic or a chemiluminescence-based substrate as described previously (16, 36, 37).
GAPDH activity. The ability of SDH and SDHHBtail to convert D-glyceraldehyde-3-phosphate (G-3-P) to 1,3-diphosphoglycerate in the presence of NAD+ was measured spectrometrically at 340 nm essentially as described before with some modifications (33). Briefly, the reaction was initiated by adding 3 μg of purified SDH or SDHHBtail proteins or 6 μg of each cell wall extract (strains M1-SF370 and M1-SDHHBtail) in a reaction buffer at a final volume of 600 μl containing 50 mM Tris-HCl, pH 7.4, 4 mM NaH2PO4, 4 mM NAD, 4 mM G-3-P, and 1 mM dithiothreitol. The conversion of NAD to NADH was monitored every 10 s spectrometrically (Beckman Coulter model DU640). To measure the GAPDH activity of intact bacteria, instead of a kinetic measurement as described above, only an endpoint OD340 was measured. M1-WT and M1HBtail strains were grown until late log phase (OD600 = 0.8) and washed twice in phosphate-buffered saline (PBS) to finally adjust the OD600 of the bacterial suspension to 1.0 (5 x 108 CFU). One milliliter of washed bacterial suspension was centrifuged, and the 600 μl of complete enzyme reaction was resuspended as described above for 5 min. At the end of incubation, the bacteria were removed by centrifugation and the absorbance of the supernatant was measured at 340 nm as described above.
Real-time quantitative PCR. Total RNA was extracted from M1-SF370 and M1-SDHHBtail strains grown to an OD of 0.8 using a commercially available RNA isolation kit (RNAwiz; Ambion). The total RNA (2 μg) isolated from the wild-type and mutant strains were converted to first-strand cDNA using an SYBR green iScript cDNA synthesis kit (Bio-Rad) in a final volume of 40 μl. Real-time PCR was performed in a reaction mixture (25 μl) containing 2.5 mM MgCl2, 250 μM deoxynucleoside triphosphate mix, 1.25 U of AmpliTaqGold DNA polymerase, 500 pM of each primer, and 1 μg of template. Gene-specific primer pairs used in this study were as follows: sdh (sdh-F, 5'-ATTAACGGTTTCGGTCGTATCGGACG-3'; sdh-R, 5'-GGATCACGTTCAGCAGAAACTTTG-3'), gyrA (gyrA-F, 5'-TTCGTATGGCTCAGTGGTTTAGTT-3'; gyrA-R, 5'-CTGGTTCTCTTTCGCTTCCATCGT-3'), and proS (proS-F, 5'-GGGTGGTTCTTGACAAGTCTATTGCG-3'; proS-R, 5'-TTCTGCCAAGGCATCTTCAGCA-3'). The mRNA copies specific to sdh were normalized with those specific to the housekeeping genes gyrA and proS for the M1-WT and M1-SDHHBtail strains, and data were evaluated as relative mRNA copy number ratios.
Different concentrations of genomic DNA were used to obtain a standard curve for each gene. The reaction mixtures were incubated for 10 min at 95°C to activate DNA polymerase, followed by 40 cycles each of denaturing at 95°C for 30 s, annealing at 58°C for 50 s, and extension at 72°C for 30 s. Fluorescence was measured in every well during each annealing step throughout the course of each reaction. Data were automatically analyzed by software of the real-time PCR instrument (Stratagene Mx-4000).
Immunofluorescence microscopy. The M1-WT and M1-SDHHBtail strains (106 CFU/ml of PBS) were attached to polylysine-treated eight-well glass slides and blocked with DAKO blocking buffer. Adherent bacteria were incubated with affinity-purified anti-SDH IgG (Fab' fragment, diluted 1:10 in DAKO dilution buffer) at 4°C for 8 h. The wells were washed several times with PBS and reacted with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (1:50 dilution) and incubated at room temperature for 1 h. The slides were washed several times with PBS, and the bacteria were stained with DAPI (4',6'-diamidino-2-phenylindole, 1 μg/ml) in SlowFade equilibration buffer (Molecular Probes) for 5 min. The stained bacteria were kept in SlowFade buffer containing glycerol and observed under a Nikon Eclipse C600 fluorescence microscope coupled with a Diagnostic RT color camera and the Spot software (v3.5.9). Bacterial cells stained with only conjugated antibody were treated as a control.
Fluorescence labeling of plasminogen. Human Plg was labeled with AlexaFluor-488-carboxylic acid succinimidyl ester (antibodies Em 495 and Em 519; Molecular Probes) using EDC (1-ethyl-3-[3-dimethylamino-propyl]carbodiimide) as per the manufacturer's instructions. Fluorescence of the labeled plasminogen was measured with a POLARstar Galaxy fluorimeter (BMG Labtechnologies GmbH, Offenburg, Germany). The values of fluorescence units versus different amounts of labeled plasminogen (1 to 5 μg) were plotted to obtain a standard curve.
Plasminogen-binding assay. Plg-binding activities of purified SDH proteins (SDH and SDHHBtail) were determined using a solid-phase ligand-binding assay and a slot blot apparatus (Bio-Rad) essentially as described before with some modifications (12). The binding of Plg to serially diluted SDH proteins was detected by rabbit anti-human Plg antibody (DAKO, Denmark), followed by corresponding alkaline-phosphatase-labeled conjugate. To determine Plg-binding activities of the intact M1-WT and M1-SDHHBtail strains, late-log-phase THY broth cultures (OD600, 0.8) were adjusted to an OD600 of 1.0 after two washes in 50 mM Tris, pH 8.0, buffer. The cultures were then centrifuged, and the resulting bacterial pellets were resuspended in TBST (Tris-buffered saline containing 0.5% Tween 20, 0.5% gelatin, and 0.5% bovine serum albumin) blocking buffer in a final volume that was 1/10 of the original volume. GAS strains (100 μl, 2 x109 CFU) were then mixed with twofold serially diluted labeled Plg in a deep-well 96-well plate for 2 h under constant mixing at room temperature. At the end of incubation, unbound labeled Plg was removed and the amount of bound Plg was determined with a fluorimeter as described above. Each dilution of Plg was tested in triplicate wells. Based on the standard curve, the exact amount of Plg bound to GAS strains was calculated. Data were statistically evaluated by unpaired t test with Welch's correction.
Phagocytosis/bactericidal assays. The ability of M1-WT and M1-SDHHBtail to resist phagocytosis and survive in heparinized human blood was determined by a bactericidal test as described previously (26). An overnight culture of each bacterial strain was diluted 1:50 in THY broth and grown without agitation to an OD600 of 0.15 at 37°C in a CO2 incubator. The bacterial suspension was diluted in THY broth (1:10,000) to obtain 4 x 103 CFU/ml for the initial inoculum of the assay. The exact number of bacteria added to the blood was determined by plating the bacterial culture and counting CFU on sheep blood agar plates. Fifty microliters of the suspension containing approximately 200 CFU was added to 1 ml of freshly heparinized blood in sterile glass tubes (100 by 10 mm) and was incubated with end-over-end rotation at 37°C for 3 h. The number of CFU in the inoculum used and those obtained after growth in human blood were quantified by the pour plate method using sheep blood agar plates. Blood for these experiments was drawn from healthy volunteers in 10-ml Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing 143 U of freeze-dried heparin. Opsonophagocytosis assays were performed essentially in the same way as the bactericidal assays except that the former assays were carried out in the presence of increasing amounts of purified anti-SDH antibody (SDH-specific IgG). Prior to these experiments, the necessary approval of the Institutional Review Board of UMDNJ, New Jersey, for human subject use was obtained.
Estimation of the hyaluronic acid of the bacterial capsule. The hyaluronic acid capsule produced by wild-type, mutant, and complemented mutant strains was measured essentially as described previously (40). Briefly, a standard curve was generated by the addition of 2 ml of a chromogenic reagent {20 mg of 1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naptho-[1,2-d]thiazolium bromide (Kodak) and 60 μl of glacial acetic acid in 100 ml of 50% formamide} into different concentrations of hyaluronic acid (0 to 30 μg/ml) in a 50-μl volume (ICN/MP Biomedicals LLC) and measuring absorbance at 640 nm. Bacterial cells from a 10-ml late-log-phase culture were washed with water and resuspended in 0.5 ml water. Capsule was released by shaking with 1 ml of chloroform and centrifuged. The aqueous phase (50 μl) was then processed as described above, and the hyaluronic acid content was determined by comparing the absorbance values with the standard curve as described above.
Streptococcal adherence assays. Streptococcal adherence to Detroit pharyngeal cells was carried out in 24-well tissue culture plates essentially as previously described (36, 38). Briefly, overnight cultures of M1-WT and M1-SDHHBtail were washed and adjusted to an OD600 of 1.0 in minimal essential medium without fetal bovine serum. Cells were infected with these strains (multiplicity of infection, 50 bacteria to 1 cell, 4 x 107 to 5 x 107 CFU/well) and incubated in a humidified CO2 incubator at 37°C for 3 h. Nonadherent bacteria were removed by repeated washing, Detroit cells with adherent bacteria were lysed, and the resulting cell lysates containing cell-associated bacteria were counted as CFU on sheep blood agar plates as described previously (24).
RESULTS
Fusion of a hydrophobic tail to SDH. Gram-positive bacterial surface proteins contain a signal sequence, an LPXTGX hexapeptide motif, and a hydrophobic tail for anchoring in the membrane lipid bilayer and subsequently for surface export (30). We, therefore, hypothesized that the addition of only a hydrophobic tail at the C-terminal end of SDH would allow SDH to anchor to the membrane without being exported to the cell surface, as the resulting protein would not have either a sorting signal for cell wall anchoring or a signal sequence for secretion. To create such a construct, we designed a putative hydrophobic tail based on the amino acid sequence of the hydrophobic tail region of the epf gene (spy0737) product in the M1-SF370 genome database (13). The translated amino acid sequence of the epf gene (2,045 aa, 221,959 Da) contains an LPXTGX motif and a hydrophobic tail with 18 amino acids (2020IVLVGLG2026VMS2029LLLG2033MVLY2037) with a predictable hydrophobicity score of 3.5 according to the DAS method (http://www.sbc.su.se/miklos/DAS/) (8). This score is well above the recommended optimum score (2.2) required for a transmembrane protein (8, 13). We edited this sequence and shortened the size of the de novo hydrophobic tail by removing Gly2026, Ser2029, and the last 5 amino acid residues (2033GMVLY2037). The resulting sequence (IVLVGLVMLLL) gave a DAS score of 3.1. The removal of the last 5 residues and only the Gly2026 residue gave DAS scores of 2.8 and 3.2, respectively. However, the deletion of the serine residue (Ser2029) of the sequence IVLVGLVMS2029LLL and its addition at the end of a resulting sequence, i.e., IVLVGLVMLLLS, increased the DAS value from 3.2 to 3.5. Thus, the amino acid sequence IVLVGLVMLLLS with a DAS score of 3.5 was considered the putative hydrophobic transmembrane region for SDH to be inserted at its C-terminal end (Fig. 1A and B).
Phenotypic characteristics of M1-SDHHBtail. PCR, DNA sequencing, and Southern hybridization assays with the mutant strain M1-SDHHBtail confirmed that the two-crossover allelic-exchange event between the M1-SF370 genome and pFW5-sdhHBtail occurred only at the desired locus without any polar effect (Fig. 2A).
Growth characteristics. M1-WT and M1-SDHHBtail strains grew similarly in THY broth throughout the lag and log phases of the growth period and until the stationary phase (Fig. 2B). Thus, the hydrophobic tail at the C-terminal end of SDH had no effect on bacterial growth.
SDH-specific mRNA and protein expression. To determine the effect of the insertion of a hydrophobic tail at the C-terminal end of SDH on protein expression, first SDH-specific mRNA expression was measured in wild-type and mutant strains grown to the late log phase (OD600 of 0.8) by real-time quantitative PCR. The expression of SDH-specific mRNAs with respect to housekeeping gene (gyrA and proS)-specific mRNAs in both the wild-type and mutant strains was found to be essentially the same without a statistically significant difference (gyrA, P = 0.137; proS, P = 0.213) (Fig. 2C). Western immunoblot analysis of whole-cell extract using anti-SDH antibody also indicated the presence of SDH and the SDHHBtail protein in their respective strains (Fig. 2D). We concluded that the introduction of hydrophobic residues at the C terminus of SDH does not affect metabolic functions or the growth of the resultant M1-SDHHBtail strain. The SDHHBtail protein, however, migrated faster than SDH during gel electrophoresis. The faster migration of the SDHHBtail protein could be attributed to the insertion of additional hydrophobic residues at the C terminus.
GAPDH activity of purified SDH and SDHHBtail. To determine the impact of the insertion of a hydrophobic tail in SDH on its enzymatic activity, the wild-type SDH and mutant SDHHBtail proteins were purified as described under Materials and Methods from whole-cell extracts and their GAPDH activities were determined using the same amount of protein (Fig. 3A). Both SDH and SDHHBtail demonstrated similar kinetics for the conversion of G-3-P to 1,3-diphosphoglycerate in the presence of NAD, indicating that the hydrophobic tail fused to the C-terminal part of SDH does not affect or interfere with the catalytic GAPDH activity of the SDHHBtail protein (Fig. 3A).
Surface localization of SDH and SDHHBtail. To demonstrate the effect of insertion mutagenesis in the sdh gene on the surface export of SDH, initially GAPDH activities per unit amount of total protein of the whole-cell extract of the wild-type and mutant strains were determined. In both cases, the ratios of the slopes of the kinetic activities were found to be similar, as shown in Fig. 3A (and data not shown). However, the GAPDH activity of the cell wall extract of the mutant M1-SDHHBtail strain was 5.5-fold or 76% less than in the cell wall extracts of the wild-type M1-SF370 strain, indicating that the insertion of the hydrophobic tail at the C-terminal end of SDH results in its retention in the cytoplasm, while significantly affecting its export to the surface and, hence, its association with the cell wall (Fig. 3B). GAPDH activity of the intact mutant M1-SDHHBtail strain was also found to be significantly less (approximately threefold or 60%) than that of the wild-type M1-WT strain, confirming the above results (Fig. 3C).
The presence of SDH on the surfaces of the wild-type and mutant strains was observed by immunofluorescence microscopy using the Fab' fragment of affinity-purified anti-SDH-antibody. An SDH-specific punctate green fluorescence pattern was seen on the surface of the wild-type strain (Fig. 3D). The M1-SDHHBtail mutant strain showed DAPI staining similar to that of the wild-type strain; however, it did not show any SDH-specific green fluorescence on its surface except where occasional lysed bacteria were observed (Fig. 3D). These data thus support our hypothesis and indicate that it is possible to retain an anchorless protein, SDH, in the cytoplasm and prevent its export to the cell surface by inserting a hydrophobic tail at its C terminus.
To confirm these findings, we determined the presence of SDH in the culture supernatants and other subcellular fractions, such as the cell wall, cytoplasm, and membrane fractions obtained from the M1-WT and M1-SDHHBtail bacterial suspensions, each possessing the same optical density. The presence of SDH was detected by Western immunoblot analysis using SDH-specific monoclonal antibody (13D5). The results showed the minimal amount of secreted SDH in the supernatant of both the wild-type and mutant strains (Fig. 3E). The immunoblot analysis of serially diluted samples derived from equal amounts of cell wall-associated proteins from these two strains showed that the cell wall fraction of the mutant strain contained approximately fourfold less SDH than the cell wall fraction of the wild-type strain (Fig. 3E). On the other hand, the cytoplasmic fraction of the mutant strain contained more SDH than that in the cytoplasmic fraction of the wild-type strain. These results corroborated those obtained by enzyme kinetic assay (Fig. 3B) and confirmed that the insertion of a hydrophobic tail at the C-terminal end of SDH results in the retention of SDH primarily in the bacterial cytoplasm and to some extent also within the bacterial cell walls. These results also showed that, contrary to our expectation, the SDHHBtail protein was not retained or trapped in the membrane, suggesting that the hydrophobic tail may interfere with the secretion of SDH.
Role of SDH in plasminogen binding. Both SDH/Plr (27, 33) and SEN (12, 37, 38) directly bind Plg; however, their relative contributions to the overall Plg-binding activity of GAS are not known. To determine the contribution of SDH to the Plg-binding activity of GAS, serially diluted purified SDH and SDHHBtail proteins (starting concentration, 2 μg each) were subjected to Plg binding and then detection of Plg binding by anti-Plg antibody in a slot blot-based direct ligand-binding Western blot assay. Plg-binding activities of serially diluted SDH and SDHHBtail revealed that both proteins bind to Plg equally (Fig. 4A), indicating that the insertion of hydrophobic tail at the C-terminal end of SDH does not interfere with the Plg-binding activity of the SDH molecule. To determine Plg-binding activities of the wild-type and mutant strains, each strain (109 CFU) was mixed with various concentrations of AlexaFluor-488-labeled Plg, and the amount of bound Plg was estimated based on the standard curve (labeled Plg versus fluorescence units) incorporated in each assay. At the highest concentration (300 nM) of exogenously added labeled Plg, the mutant strain acquired almost 60% less Plg than the wild-type (M1-WT) strain (2.4 pmol in the mutant strain versus 6.0 pmol in the wild type; P < 0.001), indicating that surface-expressed SDH plays a significant role in plasminogen-binding activity (Fig. 4B). Immunofluorescence microscopy showed the reduced Plg-binding activity of the mutant strain (Fig. 4C) and also confirmed the results obtained with the solid-phase ligand-binding assay (Fig. 4B). The residual plasminogen-binding activity of the mutant strain (Fig. 4C) is likely due to the presence of SEN, which is the major Plg-binding protein on the surface of GAS (12, 37, 38). The unique crescent-shaped Plg-binding pattern at the bacterial poles displayed by M1-SDHHBtail (Fig. 4C, right panel inset) indicates that in the absence of the surface display of SDH, the distribution of other plasminogen-binding proteins, such as SEN (37), is likely affected.
Contribution of SDH to the antiphagocytic activity of GAS. Antiphagocytic properties of the wild-type and mutant strains were determined by measuring their abilities to multiply in fresh human blood during a 3-h incubation period as described in Materials and Methods. The numbers of live bacteria present before and after incubation were determined by counting CFU on blood agar plates. The results expressed in the form of multiplication factors (MF) (the number of CFU at 3 h over the number of CFU at time zero) revealed that, whereas the wild-type (M1-WT) strain survived in the blood (MF = 59.63 ± 6) as expected, the mutant strain (M1-SDHHBtail) were readily killed (MF = 0.58 ± 0.36), indicating that the antiphagocytic activity of the mutant strain was completely inhibited (99% inhibition; P < 0.0001) as was found for the M1emm1 strain, which lacks expression of a major virulence factor, the Emm1 protein (Fig. 5A). SDH, therefore, either directly or indirectly is involved in the antiphagocytic process of GAS. Since the M protein and the bacterial capsular polysaccharide are the major surface components responsible for the antiphagocytic activity of GAS, we questioned whether the loss of antiphagocytic activity in the mutant strain was due to the loss of the expression of the M1 protein and/or the polysaccharide capsule. To determine this, Western immunoblot analysis of the cell wall as well as cytoplasm-associated proteins of the wild-type and mutant strains was carried out using the class I M protein-specific 10B6 mouse monoclonal antibody (raised against the M6 protein [25]). The results demonstrated the absence of the expression of M1 protein in both fractions of the M1-SDHHBtail mutant strain (Fig. 5B). In a parallel study of capsular polysaccharide estimation, we observed no significant change (P > 0.05) in the capsule polysaccharide associated with the mutant M1-SDHHBtail strain (22.6 ± 0.1 μg/ml) from that in the wild-type strain (20.3 ± 0.6 μg/ml). Together, these results demonstrate that the insertion of the hydrophobic tail at the C-terminal end of SDH affects M1 protein expression but not capsule formation in GAS.
Alternatively, it is also conceivable that SDH also contributes to the antiphagocytic activity of GAS since in the absence of a surface display of SDH, the bacteria are readily phagocytosed and, as in the presence of anti-M protein antibody (14), the bacteria may readily be phagocytosed in the presence of anti-SDH antibody. To test this hypothesis, the wild-type GAS strain was subjected to bactericidal assay as described above in the absence and presence of increasing amounts of affinity-purified anti-SDH IgG (50 μg to 200 μg/per reaction mixture). The results showed a dose-dependent decrease in bacterial survival (numbers of CFU) and a corresponding increase in bacterial killing/phagocytosis of the wild-type GAS strain (Fig. 5D). With a 200-μg concentration of anti-SDH IgG, up to a 70% decrease in CFU was observed compared to the total number of bacteria grown under similar experimental conditions in the absence of any exogenously added anti-SDH antibody. These results indicate that either SDH is antiphagocytic in nature or the anti-SDH antibodies are opsonic in nature.
Contribution of SDH in GAS adherence. Since purified SDH recognizes CD87/uPAR as its receptor on the surfaces of pharyngeal cells and mediates GAS adherence to pharyngeal cells (24), we hypothesized that the prevention of the cell surface export of SDH might adversely affect GAS adherence to pharyngeal cells. To test this hypothesis, M1-WT and M1-SDHHBtail strains were examined for their ability to adhere to pharyngeal cells. The GAS adherence assay revealed that the mutant strain, M1-SDHHBtail, adhered to pharyngeal cells threefold less (70% less) than the wild-type strain (Fig. 6). Since the M1-SDHHBtail mutant strain does not express the M1 protein (Fig. 5B), we also included another mutant strain lacking the expression of the M1 protein (M1Demm1) in the bacterial adherence assay as an internal control. The results showed that the M1Demm1 mutant strain adhered in a way similar to that of the wild-type strain (P = 0.268), indicating that SDH and not the M protein plays an important role in GAS adherence to pharyngeal cells (Fig. 5).
DISCUSSION
SDH belongs to a novel class of anchorless cell wall-associated surface proteins (32). Several proteins, including SDH, that belong to this class of surface proteins are not only multifunctional but also essential for bacterial survival. Hence, the resulting nonfeasibility of the "gene knockout" strategy has been a major limiting factor in our progress in understanding their roles in disease pathogenesis. In the present investigation, we have addressed this limiting factor and devised a mutagenesis strategy that allows an insertion of a hydrophobic tail at the C-terminal end of SDH and prevents the export of SDH to the cell surface. This strategy is important for two main reasons. First, it does not affect the innate GAPDH activity of GAS. Second, this strategy also allows retaining this modified SDH in the cytoplasm and does not affect growth characteristics of bacteria.
SDH and other anchorless surface glycolytic enzymes from GAS and other microorganisms are exported to the cell surface in the absence of a signal sequence and hydrophobic domain. The precise mechanism of their export to the cell surface and subsequent secretion into the medium is not known. It is, therefore, interpreted that the surface association of this enzyme is possibly due to the reassociation of leaked cytoplasmic proteins as a result of the uncontrolled natural lysis of a few bacteria. We monitored the secretion of SDH in culture supernatant and its retention within bacteria by immunofluorescence, enzyme kinetics, and immunoblot methods. Whereas the immunofluorescence provided a visual proof of the location of wild-type and mutant SDH, the remaining two methods provided quantitative analysis of their association with the GAS cell wall, cytoplasm, and membranes. All methods indicated primarily that the mutated SDH is not secreted and is not displayed on the GAS surface. Further, in the wild-type strain, surface-displayed SDH is not derived from lysed bacteria. Based on the design of the experiment, we had expected a large amount of SDH to be trapped in the membrane. However, our results showed that the large amount of SDH was restricted to the cytoplasm, to some extent in the membrane, and also in the cell wall. The detection of some amount of SDHHBtail in the cell wall fraction of the intact mutant bacteria, albeit three to five times less than that in the wild type, suggest that while the hydrophobic tail primarily interferes with the secretion of SDH and helps to retain the SDHHBtail molecule within the cytoplasm, some molecules may escape during cell division and be retained and trapped within the cell wall while they transgress the cell envelope. These SDH molecules are likely accessible to the glyceraldehydes-3-phosphate substrate during enzyme kinetic assay of the intact bacteria but not accessible to anti-SDH antibody during the immunofluorescence assay. Thus, the presence of SDHHBtail buried within the GAS cell walls was detected in immunoblotting assays, and only the surface-located SDH was detected by the immunofluorescence method. Based on the latter method, more than 95% of the mutant bacterial population (M1-SDHHBtail) showed the absence of SDH on the surface.
SDH and many of the other anchorless proteins are multifunctional. Because of their essential nature, previous studies of mutational analyses of this and other anchorless proteins were restricted to functional regions, such as Plg-binding domains, that do not participate in essential catalytic activities (3, 12, 45). Further, the functional analyses of such domains that overlap catalytic domains were based on in vitro ligand-binding assays, i.e., measuring the Plg-binding activity of internal binding sites of SEN of GAS or a similar protein of S. pneumoniae, using truncated recombinant proteins or short relevant synthetic peptides bound to a solid phase (3, 12). In SDH/Plr, the second Plg-binding site is proposed to be located in the N-terminal region (45). The nature of this site is unknown. It is, however, likely that the putative second plasminogen-binding site contributes to the catalytic domain, since several N-terminally truncated recombinant SDH proteins representing deletions of the entire region in the first N-terminal half were catalytically inactive (24; V. Pancholi, unpublished data). It may be emphasized that although the C-terminal lysine residue plays an important role in Plg binding (45), the insertion of an additional hydrophobic tail did not interfere with the Plg-binding activity of the SDH molecule (Fig. 4). The mutagenesis approach described in the present study thus successfully allowed us to study the Plg-binding activity of GAS mediated by the SDH molecule as a whole. By using a similar mutagenesis approach, we propose that secretion of SEN and other anchorless surface molecules of GAS can also be prevented and that they can be localized or restrained to the cytoplasm without affecting their innate enzyme activities and other functions. The successful creation of such mutants may then help understand the contribution of anchorless surface proteins in GAS pathogenesis.
In the present study, the unexpected finding that the isogenic M1-SDHHBtail mutant strain lost the antiphagocytic activity of the wild-type strain raises an important question, namely, whether SDH is indeed antiphagocytic in nature. To understand the contribution of SDH to GAS antiphagocytic activity, we determined the antiphagocytic activity of the wild-type strain in the presence of anti-SDH polyclonal antibody. The increased phagocytosis of the wild-type GAS strain in the presence of anti-SDH antibody is likely due to its opsonic activity, as was also found for the antibody against another well-studied anchorless protein, SEN (16, 37). On the other hand, the absence of antiphagocytic activity in M1-SDHHBtail indeed seems due to the absence of the M1 protein as the expression of another antiphagocytic molecule, the polysaccharide capsule (14), was not affected in the mutant strain. Despite the facts that the sdh gene (SPy274) is monocistronic in the M1 genome as well as in other described GAS genomes (1, 2, 13, 29, 43) and hence that the insertion of a hydrophobic tail and the spectinomycin resistance gene aad-9 does not create any polar effect in the M1-SDHHBtail mutant, the expression of the emm1 gene (SPy 2018) product (M1 protein) is down regulated. Eukaryotic GAPDH has been shown to bind DNA and RNA, and this binding has been shown to play a role in the transcription regulation of eukaryotic genes (for reviews, see references 41 and 42). It is likely that SDH and its C-terminal portion in some way may have an important role in M1 expression, possibly at the transcriptional level.
We recently reported that SDH recognizes uPAR/CD87 as its receptor on the surfaces of pharyngeal cell and mediates GAS adherence (24). In that publication, we showed that the mutant GAS strain, displaying SDH with the C-terminal lysine residue (Lys336) deleted or with a C-terminal leucine residue instead of the lysine residue (Lys336Leu), adhered poorly to pharyngeal cells (24). It was, however, not possible to study those mutants to understand the contribution of other domains of the SDH molecule in the adherence process, as other domains contribute to the essential GAPDH activity of SDH (24). In the present study, this shortcoming was overcome by preventing the display of the whole SDH protein on the GAS surface. Our results showing the adherence ability of the M1emm1 mutant strain to pharyngeal cells in a way similar to that of the wild-type strain concur with the previously published reports, which indicated that the expression of the M protein has little effect on GAS adherence to human tonsillar/pharyngeal cells and keratinocytes (6, 10). In conjunction with this finding, the poor adherence ability of the M1-SDHHBtail strain, which does not display SDH, as well as of the M1 protein on its surface underscores the important contribution of SDH in GAS adherence to pharyngeal cells.
In summary, the mutagenesis approach used in the present study revealed new functions of SDH and its role in GAS pathogenesis, such as GAS adherence and antiphagocytic activity. It is not clear at present whether SDH is itself antiphagocytic or whether, in addition, it plays a role in the transcriptional or posttranscriptional control of GAS virulence factors. As the latter is true for the eukaryotic GAPDH (41, 42), it is an important question for future research. Evidence provided in the present study concludes that SDH is an important surface-expressed virulence factor and plays an important role in GAS pathogenesis.
ACKNOWLEDGMENTS
We thank Vincent A. Fischetti (The Rockefeller University, New York, NY) for providing 10B6 monoclonal antibody. We thank Carl Drlica and Issar Smith for critically reading the manuscript and Janet Hahn and Hedia Maamar for their initial help in immunofluorescence microscopy.
This research was supported by grant RO1-42827 (to V.P.) from the National Institutes of Health.
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ABSTRACT
Surface dehydrogenase (SDH) is an anchorless, multifunctional protein displayed on the surfaces of group A Streptococcus (GAS) organisms. SDH is encoded by a single gene, sdh (gap or plr) that is essential for bacterial survival. Hence, the resulting nonfeasibility of creating a knockout mutant is a major limiting factor in studying its role in GAS pathogenesis. An insertion mutagenesis strategy was devised in which a nucleotide sequence encoding a hydrophobic tail of 12 amino acids (337IVLVGLVMLLLS348) was added at the 3' end of the sdh gene, successfully creating a viable mutant strain (M1-SDHHBtail). In this mutant strain, the SDHHBtail protein was not secreted in the medium but was retained in the cytoplasm and to some extent trapped within the cell wall. Hence, SDHHBtail was not displayed on the GAS surface. The mutant strain, M1-SDHHBtail, grew at the same rate as the wild-type strain. The SDHHBtail protein displayed the same GAPDH activity as the wild-type SDH protein. Although the whole-cell extracts of the wild-type and mutant strains showed similar GAPDH activities, cell wall extracts of the mutant strain showed 5.5-fold less GAPDH activity than the wild-type strain. The mutant strain, M1-SDHHBtail, bound significantly less human plasminogen, adhered poorly to human pharyngeal cells, and lost its innate antiphagocytic activity. These results indicate that the prevention of the cell surface export of SDH affects the virulence properties of GAS. The anchorless SDH protein, thus, is an important virulence factor.
INTRODUCTION
Surface proteins of pathogens play a variety of roles in virulence. Group A Streptococcus (GAS) (Streptococcus pyogenes), a human pathogen that is responsible for a variety of mild to severe invasive diseases of the pharynx and skin, displays an array of multifunctional proteins on its surface (5, 9, 11). In gram-positive pathogens, including GAS, classical surface proteins with defined structural components, such as a signal sequence, a proline/glycine-rich cell wall-associated domain, a hydrophobic membrane domain, and a cell wall-sorting hexapeptide motif (LPXTGX), have been characterized most extensively (15, 30). More recently, complete genome sequence analyses of six different GAS serotypes have revealed that lipoproteins also contribute significantly to the surface layer of GAS and in disease pathogenesis (1, 2, 13, 29, 43).
In the last decade, after reports of the presence of the glycolytic enzymes glycerladehyde-3-phosphate dehydrogenase (GAPDH) or streptococcal surface dehydrogenase (SDH) (27, 33) and streptococcal surface enolase (SEN) (37) on the surface of GAS, there has been an increasing awareness about the presence of similar glycolytic enzymes on the surfaces of a variety of human pathogens (for a review, see reference 32). Since these surface-located metabolic enzymes are generally found in the cytoplasm and lack C-terminal hydrophobic tails and N-terminal signal sequences required for their export to the cell surface, they are called anchorless surface proteins (32).
In addition to innate glycolytic activities, both SDH and SEN perform a variety of functions (27, 31, 33, 34, 36). Whereas some of the notable functions of SDH are its ability to bind various mammalian proteins, including plasminogen (Plg) (27, 33), ADP ribosylation (34), and host cell-signaling regulation (36), the function of SEN seems to be to serve as a major Plg-binding protein on the surface of GAS (31, 37). Although SDH in comparison to SEN is a weak Plg-binding protein (33), the C-terminal lysine residues of both SDH and SEN seem to play a crucial role in Plg binding (37). Like many other GAPDH molecules (4, 17, 18, 44), the predictive three-dimensional structure of SDH has indicated that the SDH molecule possesses two domains (24). The N-terminal NAD-binding domain (amino acid residues [aa] 1 to 149) consists of a six-stranded parallel -sheet flanked by helices. The C-terminal half of SDH contains the catalytic domain, which consists of a nine-stranded antiparallel sheet followed by three approximately parallel helices and a polypeptide chain that ends in a long helix. The region spanning amino acid residues 179 and 200, which forms an S-shaped antiparallel sheet, is also called the S-loop (18, 24, 44). Recently, we identified CD87/urokinase plasminogen activator receptor (uPAR) as the SDH-specific receptor on the surfaces of pharyngeal cells (24). The N-terminal domain of uPAR binds to the C-terminal alpha-helix and the flanking regions of the S-loop of the SDH molecule (24). This specific binding also plays a crucial role in GAS adherence to pharyngeal cells (24). Thus, the C-terminal portion of SDH is essential to perform a variety of biologically relevant functions. We also reported that ATP-producing glycolytic enzymes likely form a specialized complex on the surface of GAS (35). Although accumulated reports now indicate that anchorless proteins are present on the surfaces of many pathogens, including gram-positive bacteria, fungi, and parasites (32), our understanding of their biological functions and their roles in the pathogenesis of GAS and other bacterial pathogens is far from complete. Likewise, how they are exported to the cell surface is unknown.
To evaluate the role of virulence factors, the genetic approach to create a knockout mutant that can be tested in various in vitro and/or in vivo disease model systems as a distinct attenuated or nonpathogenic phenotype is highly desirable. However, the knockout mutagenesis approach is not achievable for a gene that is essential for microbial survival. In certain organisms, such as Escherichia coli, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, more than one GAPDH and enolase isoenzymes are present; each of these isoenzymes is encoded by a distinct gene or the organism is equipped with the Entner-Doudoroff alternate metabolic pathway (7, 20-22, 28, 46). Hence, it has been possible to create mutants defective in GAPDH, enolase, and other enzymes of glycolysis (19, 22, 23, 28). In GAS, glucose is metabolized solely by the glycolytic pathway, as GAS lacks the tricarbolicacetic acid (TCA)-metabolic cycle and the Entner-Doudoroff alternative metabolic pathway (1, 2, 13, 29, 43). The multifunctional SDH is encoded by a single essential gene (33, 45). The gene knockout approach to prevent SDH expression in GAS, therefore, is not feasible. The site-directed mutagenesis strategies that allow alteration in the specific functional domains without disturbing the conformational structure responsible for the catalytic function have been successfully employed to create Plg-binding enolase- and SDH/Plr-specific deletion mutants (3, 12, 45). Since the putative transport system responsible for exporting the anchorless metabolic surface enzymes is not known, it has not been possible to create a GAS mutant in which the anchorless enzyme is selectively retained in the cytoplasm and not expressed on the surface.
Since the SDH molecule does not possess the N-terminal signal sequence and the C-terminal hydrophobic tail, we hypothesized that the introduction of only a hydrophobic tail at the C-terminal end may result in its prevention of export to the surface and the SDH may be retained in the cytoplasm and/or in the membrane. Accordingly, in the present investigation, we altered the sdh gene by introducing a nucleotide sequence encoding a de novo C-terminal hydrophobic tail. The resulting mutated SDH protein remained largely restricted to the cytoplasm. This mutant strain behaved in a manner similar to that of the wild-type strain in terms of growth characteristics and GAPDH enzyme activity, but it bound significantly less Plg, adhered poorly to human pharyngeal cells, and lost the typical antiphagocytic activity associated with the wild-type strain. Our results also suggest that SDH may have a role in the regulation of the expression of a certain virulence factor-related gene(s), possibly at the transcriptional level.
MATERIALS AND METHODS
Bacterial strains, vectors, growth conditions, and DNA techniques. S. pyogenes wild-type strain M1-SF370 (M1-WT; ATCC 700294; American Type Culture Collection, Manassas, VA) was grown in Todd-Hewitt broth (Difco Laboratories) supplemented with 0.5% yeast extract or on proteose peptone-3 blood agar plates supplemented with spectinomycin (up to 500 μg/ml), when required. E. coli strain XL1-Blue was used for the cloning experiments and grown in Luria-Bertani (LB) broth or on LB agar plates. Vector pFW5 containing the spectinomycin resistance gene (aad-9) was used to transform the M1-SF370 strain (12, 39).
Reagents. Unless otherwise stated, all chemicals were procured from Sigma (St. Louis, MO).
Human pharyngeal cell lines. Detroit 562 (ATCC CCL138) pharyngeal cells were grown to confluence and maintained in a CO2 incubator as described previously (36).
Determination of the sequence of the hydrophobic tail. The prediction of hydrophobicity of a putative hydrophobic tail was determined based on the dense alignment surface (DAS) score as described at the website http://www.sbc.su.se/miklos/DAS/. The amino acid sequence of a hydrophobic tail to be inserted at the C-terminal end of SDH was based on the translated amino acid sequence of the epf gene in the M1 genome sequence database (13) and was edited using the DAS method to get an optimum DAS score (3.0), one indicative of a typical transmembrane location (8).
Construction of insertion and allelic-exchange sdhHBtail mutations in GAS. To create streptococcal mutant strains expressing SDH with a hydrophobic tail at the C-terminal end, the sdh gene (0.92-kb DNA fragment) and a 1.118-kb DNA fragment corresponding to the downstream region of the sdh gene in the M1-SF370 chromosome were PCR amplified using primer pairs SDH-SalI-F-/SDH-BamHI-R (5'-ACGCGTCGACATGGTAGTTAAAGT TGGTATTAACGG-3' and 5'-CGCGGATCCTTATTTAGCAATTTTTGCGAAGTACTCAAGAGTACG-3') and SDHDWN-PstI-F/SDHDWN-NdeI-R (5'-TTTTCTGCAGCTTGGTTTATGCTG AGTTCTTTTTCG-3 and 5'-GGGAATTCCATATGTATTGGAAAATGCTATCGTGCG-3'), respectively. The PCR-amplified products were cloned in the multiple cloning sites located upstream and downstream of the spectinomycin resistance gene, aad-9, in the suicide plasmid pFW5 (2.7 kb) to construct pFW5-sdh. To insert a DNA sequence encoding the putative hydrophobic tail at the 3' end of the sdh gene in the pFW5-sdh vector, a QuickChange II site-directed mutagenesis kit was used according to the manufacturer's instructions (Stratagene) with the plasmid pFW5-sdh as the template and a pair of complementary primers, SDHHBtail-F (5'-GTACTTCGCAAAAATTGCTAAAATTGTTCTTGTTGGCTTAGTTATGCTTCTTCTTTCTTAATAGGATCCTCGAGCTCTAG-3') and SDHHBtail-R (5'-CTAGAGCTCGAGGATCCTATTAAGAAAGAAGAAGCATAACTAAGCCAACAAGAACAATTTTAGCAATTTTTGCGAAGTAC-3'). The resulting plasmid, pFW5-sdhHBtail, was introduced into strain M1-WT by electroporation, using a Gene Pulser II electroporator (Bio-Rad) as described previously (12, 29). The mutant strain thus obtained was called M1-SDHHBtail. The confirmation of mutations and insertions at various stages of the experiments was obtained by one or more methods of PCR, DNA sequencing, and Southern hybridization. Using a similar strategy, a mutant strain lacking the M1 protein (M1Demm1) was created. The primers used to create this mutant were emm1-F (5'-ACGCGTCGACTAGGTCAAAAAGGTGGC-3'), emm1-R (5'-CGCGGATCCGCATTCTCTAAT CTCGCTT-3'), emm1DWN-F (5'-AACTGCAGGACTTGGACGCATCACGTGAA-3'), and emm1DWN-R (5'-GATTCCATATGCTGTCTCTTAGTTTCCTTCATTGGTGC-3'). The italicized sequences in these primers represent SalI, BamHI, PstI and NdeI restriction sites, respectively.
Cell fractionation and whole-cell extraction. M1-WT and M1-SDHHBtail (mutant) strains were grown to late log phase/early stationary phase (optical density at 600 nm [OD600], 0.8), and bacteria were harvested by centrifugation. The culture supernatants from these strains were saved, and the proteins therein were precipitated with 25% trichloroacetic acid (TCA). The resulting precipitates were separated by centrifugation (20,000 x g for 30 min at 4°C), washed twice with acetone, and suspended in 1/50 of the original volume of 50 mM Tris-HCl, pH 7.0. The harvested bacteria were subjected to mutanolysin treatment in a cell wall-extracting buffer (50 mM Tris-HCl, pH 7.0, 30% raffinose) containing 250 U/ml of mutanolysin (Sigma) as described previously to achieve nearly complete wall extraction (33). The resulting protoplasts were lysed in a hypotonic buffer, followed by repeated freezing and thawing. Bacterial membrane and cytoplasmic fractions were separated from the lysed protoplasts by ultracentrifugation (100,000 x g for 45 min at 4°C) as described previously (33). Whole-cell extracts from these two strains were obtained using the same procedures described above except that the cell wall-extracting buffer was used without 30% raffinose.
SDH and SDHHBtail purification. Clear whole lysates obtained as described above from 500 ml THY broth culture with each of the M1-WT and M1-SDHHBtail strains were dialyzed against 0.05 M NH4HCO3 and precipitated at 40%, 60%, and 80% ammonium sulfate saturation. The protein precipitates obtained from 60% to 80% ammonium sulfate saturation, which contained the major amount of SDH and SDHHBtail, were purified by Hi-trap Blue-HP column (1 ml) chromatography (Pharmacia) after equilibration with a starting buffer (50 mM Tris-HCl, pH 7.4). Proteins other than SDH and SDHHBtail were eluted first by the five-column volume of starting buffers containing 0.5 M NaCl followed by the five-column volume of the same buffer containing 1.0 M NaCl. The remaining column-bound proteins were eluted with the five-column volume of the starting buffer containing 5 mM NAD. The NAD-eluted fractions (1 ml each) contained only SDH or SDHHBtail, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The pooled fractions containing SDH or SDHHBtail were dialyzed against the starting buffer and stored at –70°C until further use.
Preparation of monospecific anti-SDH antibody. Anti-SDH specific immunoglobulin G (IgG) was purified from the SDH-immunized rabbit serum using an SDH-linked diaminodipropylamine M-phase (Pierce) affinity column as described previously (33, 37). The purified IgG was then treated with immobilized papain enzyme (Pierce) at 4°C overnight to cleave them between their Fab' and Fc regions. The latter was then removed by passing the cleaved products on a protein A column (Pierce) per the manufacturer's instructions. The unbound initial fall-through was used as Fab'-specific anti-SDH IgG.
Electrophoresis on SDS-polyacrylamide gels and Western blotting. Purified SDH, SDHHBtail, and cell wall-associated proteins were separated by SDS-PAGE and Western blotted onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the presence of SDH was detected by reacting PVDF membranes with anti-SDH monoclonal antibody (clone 13D5) (16) or monospecific rabbit anti-SDH IgG followed by corresponding alkaline phosphatase-labeled conjugate antibody (Fab' specific). SDH-specific protein bands on PVDF membranes were visualized using a chromogenic or a chemiluminescence-based substrate as described previously (16, 36, 37).
GAPDH activity. The ability of SDH and SDHHBtail to convert D-glyceraldehyde-3-phosphate (G-3-P) to 1,3-diphosphoglycerate in the presence of NAD+ was measured spectrometrically at 340 nm essentially as described before with some modifications (33). Briefly, the reaction was initiated by adding 3 μg of purified SDH or SDHHBtail proteins or 6 μg of each cell wall extract (strains M1-SF370 and M1-SDHHBtail) in a reaction buffer at a final volume of 600 μl containing 50 mM Tris-HCl, pH 7.4, 4 mM NaH2PO4, 4 mM NAD, 4 mM G-3-P, and 1 mM dithiothreitol. The conversion of NAD to NADH was monitored every 10 s spectrometrically (Beckman Coulter model DU640). To measure the GAPDH activity of intact bacteria, instead of a kinetic measurement as described above, only an endpoint OD340 was measured. M1-WT and M1HBtail strains were grown until late log phase (OD600 = 0.8) and washed twice in phosphate-buffered saline (PBS) to finally adjust the OD600 of the bacterial suspension to 1.0 (5 x 108 CFU). One milliliter of washed bacterial suspension was centrifuged, and the 600 μl of complete enzyme reaction was resuspended as described above for 5 min. At the end of incubation, the bacteria were removed by centrifugation and the absorbance of the supernatant was measured at 340 nm as described above.
Real-time quantitative PCR. Total RNA was extracted from M1-SF370 and M1-SDHHBtail strains grown to an OD of 0.8 using a commercially available RNA isolation kit (RNAwiz; Ambion). The total RNA (2 μg) isolated from the wild-type and mutant strains were converted to first-strand cDNA using an SYBR green iScript cDNA synthesis kit (Bio-Rad) in a final volume of 40 μl. Real-time PCR was performed in a reaction mixture (25 μl) containing 2.5 mM MgCl2, 250 μM deoxynucleoside triphosphate mix, 1.25 U of AmpliTaqGold DNA polymerase, 500 pM of each primer, and 1 μg of template. Gene-specific primer pairs used in this study were as follows: sdh (sdh-F, 5'-ATTAACGGTTTCGGTCGTATCGGACG-3'; sdh-R, 5'-GGATCACGTTCAGCAGAAACTTTG-3'), gyrA (gyrA-F, 5'-TTCGTATGGCTCAGTGGTTTAGTT-3'; gyrA-R, 5'-CTGGTTCTCTTTCGCTTCCATCGT-3'), and proS (proS-F, 5'-GGGTGGTTCTTGACAAGTCTATTGCG-3'; proS-R, 5'-TTCTGCCAAGGCATCTTCAGCA-3'). The mRNA copies specific to sdh were normalized with those specific to the housekeeping genes gyrA and proS for the M1-WT and M1-SDHHBtail strains, and data were evaluated as relative mRNA copy number ratios.
Different concentrations of genomic DNA were used to obtain a standard curve for each gene. The reaction mixtures were incubated for 10 min at 95°C to activate DNA polymerase, followed by 40 cycles each of denaturing at 95°C for 30 s, annealing at 58°C for 50 s, and extension at 72°C for 30 s. Fluorescence was measured in every well during each annealing step throughout the course of each reaction. Data were automatically analyzed by software of the real-time PCR instrument (Stratagene Mx-4000).
Immunofluorescence microscopy. The M1-WT and M1-SDHHBtail strains (106 CFU/ml of PBS) were attached to polylysine-treated eight-well glass slides and blocked with DAKO blocking buffer. Adherent bacteria were incubated with affinity-purified anti-SDH IgG (Fab' fragment, diluted 1:10 in DAKO dilution buffer) at 4°C for 8 h. The wells were washed several times with PBS and reacted with fluorescein isothiocyanate-conjugated anti-rabbit IgG antibody (1:50 dilution) and incubated at room temperature for 1 h. The slides were washed several times with PBS, and the bacteria were stained with DAPI (4',6'-diamidino-2-phenylindole, 1 μg/ml) in SlowFade equilibration buffer (Molecular Probes) for 5 min. The stained bacteria were kept in SlowFade buffer containing glycerol and observed under a Nikon Eclipse C600 fluorescence microscope coupled with a Diagnostic RT color camera and the Spot software (v3.5.9). Bacterial cells stained with only conjugated antibody were treated as a control.
Fluorescence labeling of plasminogen. Human Plg was labeled with AlexaFluor-488-carboxylic acid succinimidyl ester (antibodies Em 495 and Em 519; Molecular Probes) using EDC (1-ethyl-3-[3-dimethylamino-propyl]carbodiimide) as per the manufacturer's instructions. Fluorescence of the labeled plasminogen was measured with a POLARstar Galaxy fluorimeter (BMG Labtechnologies GmbH, Offenburg, Germany). The values of fluorescence units versus different amounts of labeled plasminogen (1 to 5 μg) were plotted to obtain a standard curve.
Plasminogen-binding assay. Plg-binding activities of purified SDH proteins (SDH and SDHHBtail) were determined using a solid-phase ligand-binding assay and a slot blot apparatus (Bio-Rad) essentially as described before with some modifications (12). The binding of Plg to serially diluted SDH proteins was detected by rabbit anti-human Plg antibody (DAKO, Denmark), followed by corresponding alkaline-phosphatase-labeled conjugate. To determine Plg-binding activities of the intact M1-WT and M1-SDHHBtail strains, late-log-phase THY broth cultures (OD600, 0.8) were adjusted to an OD600 of 1.0 after two washes in 50 mM Tris, pH 8.0, buffer. The cultures were then centrifuged, and the resulting bacterial pellets were resuspended in TBST (Tris-buffered saline containing 0.5% Tween 20, 0.5% gelatin, and 0.5% bovine serum albumin) blocking buffer in a final volume that was 1/10 of the original volume. GAS strains (100 μl, 2 x109 CFU) were then mixed with twofold serially diluted labeled Plg in a deep-well 96-well plate for 2 h under constant mixing at room temperature. At the end of incubation, unbound labeled Plg was removed and the amount of bound Plg was determined with a fluorimeter as described above. Each dilution of Plg was tested in triplicate wells. Based on the standard curve, the exact amount of Plg bound to GAS strains was calculated. Data were statistically evaluated by unpaired t test with Welch's correction.
Phagocytosis/bactericidal assays. The ability of M1-WT and M1-SDHHBtail to resist phagocytosis and survive in heparinized human blood was determined by a bactericidal test as described previously (26). An overnight culture of each bacterial strain was diluted 1:50 in THY broth and grown without agitation to an OD600 of 0.15 at 37°C in a CO2 incubator. The bacterial suspension was diluted in THY broth (1:10,000) to obtain 4 x 103 CFU/ml for the initial inoculum of the assay. The exact number of bacteria added to the blood was determined by plating the bacterial culture and counting CFU on sheep blood agar plates. Fifty microliters of the suspension containing approximately 200 CFU was added to 1 ml of freshly heparinized blood in sterile glass tubes (100 by 10 mm) and was incubated with end-over-end rotation at 37°C for 3 h. The number of CFU in the inoculum used and those obtained after growth in human blood were quantified by the pour plate method using sheep blood agar plates. Blood for these experiments was drawn from healthy volunteers in 10-ml Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ) containing 143 U of freeze-dried heparin. Opsonophagocytosis assays were performed essentially in the same way as the bactericidal assays except that the former assays were carried out in the presence of increasing amounts of purified anti-SDH antibody (SDH-specific IgG). Prior to these experiments, the necessary approval of the Institutional Review Board of UMDNJ, New Jersey, for human subject use was obtained.
Estimation of the hyaluronic acid of the bacterial capsule. The hyaluronic acid capsule produced by wild-type, mutant, and complemented mutant strains was measured essentially as described previously (40). Briefly, a standard curve was generated by the addition of 2 ml of a chromogenic reagent {20 mg of 1-ethyl-2-[3-(1-ethylnaphtho-[1,2-d]thiazolin-2-ylidene)-2-methylpropenyl]naptho-[1,2-d]thiazolium bromide (Kodak) and 60 μl of glacial acetic acid in 100 ml of 50% formamide} into different concentrations of hyaluronic acid (0 to 30 μg/ml) in a 50-μl volume (ICN/MP Biomedicals LLC) and measuring absorbance at 640 nm. Bacterial cells from a 10-ml late-log-phase culture were washed with water and resuspended in 0.5 ml water. Capsule was released by shaking with 1 ml of chloroform and centrifuged. The aqueous phase (50 μl) was then processed as described above, and the hyaluronic acid content was determined by comparing the absorbance values with the standard curve as described above.
Streptococcal adherence assays. Streptococcal adherence to Detroit pharyngeal cells was carried out in 24-well tissue culture plates essentially as previously described (36, 38). Briefly, overnight cultures of M1-WT and M1-SDHHBtail were washed and adjusted to an OD600 of 1.0 in minimal essential medium without fetal bovine serum. Cells were infected with these strains (multiplicity of infection, 50 bacteria to 1 cell, 4 x 107 to 5 x 107 CFU/well) and incubated in a humidified CO2 incubator at 37°C for 3 h. Nonadherent bacteria were removed by repeated washing, Detroit cells with adherent bacteria were lysed, and the resulting cell lysates containing cell-associated bacteria were counted as CFU on sheep blood agar plates as described previously (24).
RESULTS
Fusion of a hydrophobic tail to SDH. Gram-positive bacterial surface proteins contain a signal sequence, an LPXTGX hexapeptide motif, and a hydrophobic tail for anchoring in the membrane lipid bilayer and subsequently for surface export (30). We, therefore, hypothesized that the addition of only a hydrophobic tail at the C-terminal end of SDH would allow SDH to anchor to the membrane without being exported to the cell surface, as the resulting protein would not have either a sorting signal for cell wall anchoring or a signal sequence for secretion. To create such a construct, we designed a putative hydrophobic tail based on the amino acid sequence of the hydrophobic tail region of the epf gene (spy0737) product in the M1-SF370 genome database (13). The translated amino acid sequence of the epf gene (2,045 aa, 221,959 Da) contains an LPXTGX motif and a hydrophobic tail with 18 amino acids (2020IVLVGLG2026VMS2029LLLG2033MVLY2037) with a predictable hydrophobicity score of 3.5 according to the DAS method (http://www.sbc.su.se/miklos/DAS/) (8). This score is well above the recommended optimum score (2.2) required for a transmembrane protein (8, 13). We edited this sequence and shortened the size of the de novo hydrophobic tail by removing Gly2026, Ser2029, and the last 5 amino acid residues (2033GMVLY2037). The resulting sequence (IVLVGLVMLLL) gave a DAS score of 3.1. The removal of the last 5 residues and only the Gly2026 residue gave DAS scores of 2.8 and 3.2, respectively. However, the deletion of the serine residue (Ser2029) of the sequence IVLVGLVMS2029LLL and its addition at the end of a resulting sequence, i.e., IVLVGLVMLLLS, increased the DAS value from 3.2 to 3.5. Thus, the amino acid sequence IVLVGLVMLLLS with a DAS score of 3.5 was considered the putative hydrophobic transmembrane region for SDH to be inserted at its C-terminal end (Fig. 1A and B).
Phenotypic characteristics of M1-SDHHBtail. PCR, DNA sequencing, and Southern hybridization assays with the mutant strain M1-SDHHBtail confirmed that the two-crossover allelic-exchange event between the M1-SF370 genome and pFW5-sdhHBtail occurred only at the desired locus without any polar effect (Fig. 2A).
Growth characteristics. M1-WT and M1-SDHHBtail strains grew similarly in THY broth throughout the lag and log phases of the growth period and until the stationary phase (Fig. 2B). Thus, the hydrophobic tail at the C-terminal end of SDH had no effect on bacterial growth.
SDH-specific mRNA and protein expression. To determine the effect of the insertion of a hydrophobic tail at the C-terminal end of SDH on protein expression, first SDH-specific mRNA expression was measured in wild-type and mutant strains grown to the late log phase (OD600 of 0.8) by real-time quantitative PCR. The expression of SDH-specific mRNAs with respect to housekeeping gene (gyrA and proS)-specific mRNAs in both the wild-type and mutant strains was found to be essentially the same without a statistically significant difference (gyrA, P = 0.137; proS, P = 0.213) (Fig. 2C). Western immunoblot analysis of whole-cell extract using anti-SDH antibody also indicated the presence of SDH and the SDHHBtail protein in their respective strains (Fig. 2D). We concluded that the introduction of hydrophobic residues at the C terminus of SDH does not affect metabolic functions or the growth of the resultant M1-SDHHBtail strain. The SDHHBtail protein, however, migrated faster than SDH during gel electrophoresis. The faster migration of the SDHHBtail protein could be attributed to the insertion of additional hydrophobic residues at the C terminus.
GAPDH activity of purified SDH and SDHHBtail. To determine the impact of the insertion of a hydrophobic tail in SDH on its enzymatic activity, the wild-type SDH and mutant SDHHBtail proteins were purified as described under Materials and Methods from whole-cell extracts and their GAPDH activities were determined using the same amount of protein (Fig. 3A). Both SDH and SDHHBtail demonstrated similar kinetics for the conversion of G-3-P to 1,3-diphosphoglycerate in the presence of NAD, indicating that the hydrophobic tail fused to the C-terminal part of SDH does not affect or interfere with the catalytic GAPDH activity of the SDHHBtail protein (Fig. 3A).
Surface localization of SDH and SDHHBtail. To demonstrate the effect of insertion mutagenesis in the sdh gene on the surface export of SDH, initially GAPDH activities per unit amount of total protein of the whole-cell extract of the wild-type and mutant strains were determined. In both cases, the ratios of the slopes of the kinetic activities were found to be similar, as shown in Fig. 3A (and data not shown). However, the GAPDH activity of the cell wall extract of the mutant M1-SDHHBtail strain was 5.5-fold or 76% less than in the cell wall extracts of the wild-type M1-SF370 strain, indicating that the insertion of the hydrophobic tail at the C-terminal end of SDH results in its retention in the cytoplasm, while significantly affecting its export to the surface and, hence, its association with the cell wall (Fig. 3B). GAPDH activity of the intact mutant M1-SDHHBtail strain was also found to be significantly less (approximately threefold or 60%) than that of the wild-type M1-WT strain, confirming the above results (Fig. 3C).
The presence of SDH on the surfaces of the wild-type and mutant strains was observed by immunofluorescence microscopy using the Fab' fragment of affinity-purified anti-SDH-antibody. An SDH-specific punctate green fluorescence pattern was seen on the surface of the wild-type strain (Fig. 3D). The M1-SDHHBtail mutant strain showed DAPI staining similar to that of the wild-type strain; however, it did not show any SDH-specific green fluorescence on its surface except where occasional lysed bacteria were observed (Fig. 3D). These data thus support our hypothesis and indicate that it is possible to retain an anchorless protein, SDH, in the cytoplasm and prevent its export to the cell surface by inserting a hydrophobic tail at its C terminus.
To confirm these findings, we determined the presence of SDH in the culture supernatants and other subcellular fractions, such as the cell wall, cytoplasm, and membrane fractions obtained from the M1-WT and M1-SDHHBtail bacterial suspensions, each possessing the same optical density. The presence of SDH was detected by Western immunoblot analysis using SDH-specific monoclonal antibody (13D5). The results showed the minimal amount of secreted SDH in the supernatant of both the wild-type and mutant strains (Fig. 3E). The immunoblot analysis of serially diluted samples derived from equal amounts of cell wall-associated proteins from these two strains showed that the cell wall fraction of the mutant strain contained approximately fourfold less SDH than the cell wall fraction of the wild-type strain (Fig. 3E). On the other hand, the cytoplasmic fraction of the mutant strain contained more SDH than that in the cytoplasmic fraction of the wild-type strain. These results corroborated those obtained by enzyme kinetic assay (Fig. 3B) and confirmed that the insertion of a hydrophobic tail at the C-terminal end of SDH results in the retention of SDH primarily in the bacterial cytoplasm and to some extent also within the bacterial cell walls. These results also showed that, contrary to our expectation, the SDHHBtail protein was not retained or trapped in the membrane, suggesting that the hydrophobic tail may interfere with the secretion of SDH.
Role of SDH in plasminogen binding. Both SDH/Plr (27, 33) and SEN (12, 37, 38) directly bind Plg; however, their relative contributions to the overall Plg-binding activity of GAS are not known. To determine the contribution of SDH to the Plg-binding activity of GAS, serially diluted purified SDH and SDHHBtail proteins (starting concentration, 2 μg each) were subjected to Plg binding and then detection of Plg binding by anti-Plg antibody in a slot blot-based direct ligand-binding Western blot assay. Plg-binding activities of serially diluted SDH and SDHHBtail revealed that both proteins bind to Plg equally (Fig. 4A), indicating that the insertion of hydrophobic tail at the C-terminal end of SDH does not interfere with the Plg-binding activity of the SDH molecule. To determine Plg-binding activities of the wild-type and mutant strains, each strain (109 CFU) was mixed with various concentrations of AlexaFluor-488-labeled Plg, and the amount of bound Plg was estimated based on the standard curve (labeled Plg versus fluorescence units) incorporated in each assay. At the highest concentration (300 nM) of exogenously added labeled Plg, the mutant strain acquired almost 60% less Plg than the wild-type (M1-WT) strain (2.4 pmol in the mutant strain versus 6.0 pmol in the wild type; P < 0.001), indicating that surface-expressed SDH plays a significant role in plasminogen-binding activity (Fig. 4B). Immunofluorescence microscopy showed the reduced Plg-binding activity of the mutant strain (Fig. 4C) and also confirmed the results obtained with the solid-phase ligand-binding assay (Fig. 4B). The residual plasminogen-binding activity of the mutant strain (Fig. 4C) is likely due to the presence of SEN, which is the major Plg-binding protein on the surface of GAS (12, 37, 38). The unique crescent-shaped Plg-binding pattern at the bacterial poles displayed by M1-SDHHBtail (Fig. 4C, right panel inset) indicates that in the absence of the surface display of SDH, the distribution of other plasminogen-binding proteins, such as SEN (37), is likely affected.
Contribution of SDH to the antiphagocytic activity of GAS. Antiphagocytic properties of the wild-type and mutant strains were determined by measuring their abilities to multiply in fresh human blood during a 3-h incubation period as described in Materials and Methods. The numbers of live bacteria present before and after incubation were determined by counting CFU on blood agar plates. The results expressed in the form of multiplication factors (MF) (the number of CFU at 3 h over the number of CFU at time zero) revealed that, whereas the wild-type (M1-WT) strain survived in the blood (MF = 59.63 ± 6) as expected, the mutant strain (M1-SDHHBtail) were readily killed (MF = 0.58 ± 0.36), indicating that the antiphagocytic activity of the mutant strain was completely inhibited (99% inhibition; P < 0.0001) as was found for the M1emm1 strain, which lacks expression of a major virulence factor, the Emm1 protein (Fig. 5A). SDH, therefore, either directly or indirectly is involved in the antiphagocytic process of GAS. Since the M protein and the bacterial capsular polysaccharide are the major surface components responsible for the antiphagocytic activity of GAS, we questioned whether the loss of antiphagocytic activity in the mutant strain was due to the loss of the expression of the M1 protein and/or the polysaccharide capsule. To determine this, Western immunoblot analysis of the cell wall as well as cytoplasm-associated proteins of the wild-type and mutant strains was carried out using the class I M protein-specific 10B6 mouse monoclonal antibody (raised against the M6 protein [25]). The results demonstrated the absence of the expression of M1 protein in both fractions of the M1-SDHHBtail mutant strain (Fig. 5B). In a parallel study of capsular polysaccharide estimation, we observed no significant change (P > 0.05) in the capsule polysaccharide associated with the mutant M1-SDHHBtail strain (22.6 ± 0.1 μg/ml) from that in the wild-type strain (20.3 ± 0.6 μg/ml). Together, these results demonstrate that the insertion of the hydrophobic tail at the C-terminal end of SDH affects M1 protein expression but not capsule formation in GAS.
Alternatively, it is also conceivable that SDH also contributes to the antiphagocytic activity of GAS since in the absence of a surface display of SDH, the bacteria are readily phagocytosed and, as in the presence of anti-M protein antibody (14), the bacteria may readily be phagocytosed in the presence of anti-SDH antibody. To test this hypothesis, the wild-type GAS strain was subjected to bactericidal assay as described above in the absence and presence of increasing amounts of affinity-purified anti-SDH IgG (50 μg to 200 μg/per reaction mixture). The results showed a dose-dependent decrease in bacterial survival (numbers of CFU) and a corresponding increase in bacterial killing/phagocytosis of the wild-type GAS strain (Fig. 5D). With a 200-μg concentration of anti-SDH IgG, up to a 70% decrease in CFU was observed compared to the total number of bacteria grown under similar experimental conditions in the absence of any exogenously added anti-SDH antibody. These results indicate that either SDH is antiphagocytic in nature or the anti-SDH antibodies are opsonic in nature.
Contribution of SDH in GAS adherence. Since purified SDH recognizes CD87/uPAR as its receptor on the surfaces of pharyngeal cells and mediates GAS adherence to pharyngeal cells (24), we hypothesized that the prevention of the cell surface export of SDH might adversely affect GAS adherence to pharyngeal cells. To test this hypothesis, M1-WT and M1-SDHHBtail strains were examined for their ability to adhere to pharyngeal cells. The GAS adherence assay revealed that the mutant strain, M1-SDHHBtail, adhered to pharyngeal cells threefold less (70% less) than the wild-type strain (Fig. 6). Since the M1-SDHHBtail mutant strain does not express the M1 protein (Fig. 5B), we also included another mutant strain lacking the expression of the M1 protein (M1Demm1) in the bacterial adherence assay as an internal control. The results showed that the M1Demm1 mutant strain adhered in a way similar to that of the wild-type strain (P = 0.268), indicating that SDH and not the M protein plays an important role in GAS adherence to pharyngeal cells (Fig. 5).
DISCUSSION
SDH belongs to a novel class of anchorless cell wall-associated surface proteins (32). Several proteins, including SDH, that belong to this class of surface proteins are not only multifunctional but also essential for bacterial survival. Hence, the resulting nonfeasibility of the "gene knockout" strategy has been a major limiting factor in our progress in understanding their roles in disease pathogenesis. In the present investigation, we have addressed this limiting factor and devised a mutagenesis strategy that allows an insertion of a hydrophobic tail at the C-terminal end of SDH and prevents the export of SDH to the cell surface. This strategy is important for two main reasons. First, it does not affect the innate GAPDH activity of GAS. Second, this strategy also allows retaining this modified SDH in the cytoplasm and does not affect growth characteristics of bacteria.
SDH and other anchorless surface glycolytic enzymes from GAS and other microorganisms are exported to the cell surface in the absence of a signal sequence and hydrophobic domain. The precise mechanism of their export to the cell surface and subsequent secretion into the medium is not known. It is, therefore, interpreted that the surface association of this enzyme is possibly due to the reassociation of leaked cytoplasmic proteins as a result of the uncontrolled natural lysis of a few bacteria. We monitored the secretion of SDH in culture supernatant and its retention within bacteria by immunofluorescence, enzyme kinetics, and immunoblot methods. Whereas the immunofluorescence provided a visual proof of the location of wild-type and mutant SDH, the remaining two methods provided quantitative analysis of their association with the GAS cell wall, cytoplasm, and membranes. All methods indicated primarily that the mutated SDH is not secreted and is not displayed on the GAS surface. Further, in the wild-type strain, surface-displayed SDH is not derived from lysed bacteria. Based on the design of the experiment, we had expected a large amount of SDH to be trapped in the membrane. However, our results showed that the large amount of SDH was restricted to the cytoplasm, to some extent in the membrane, and also in the cell wall. The detection of some amount of SDHHBtail in the cell wall fraction of the intact mutant bacteria, albeit three to five times less than that in the wild type, suggest that while the hydrophobic tail primarily interferes with the secretion of SDH and helps to retain the SDHHBtail molecule within the cytoplasm, some molecules may escape during cell division and be retained and trapped within the cell wall while they transgress the cell envelope. These SDH molecules are likely accessible to the glyceraldehydes-3-phosphate substrate during enzyme kinetic assay of the intact bacteria but not accessible to anti-SDH antibody during the immunofluorescence assay. Thus, the presence of SDHHBtail buried within the GAS cell walls was detected in immunoblotting assays, and only the surface-located SDH was detected by the immunofluorescence method. Based on the latter method, more than 95% of the mutant bacterial population (M1-SDHHBtail) showed the absence of SDH on the surface.
SDH and many of the other anchorless proteins are multifunctional. Because of their essential nature, previous studies of mutational analyses of this and other anchorless proteins were restricted to functional regions, such as Plg-binding domains, that do not participate in essential catalytic activities (3, 12, 45). Further, the functional analyses of such domains that overlap catalytic domains were based on in vitro ligand-binding assays, i.e., measuring the Plg-binding activity of internal binding sites of SEN of GAS or a similar protein of S. pneumoniae, using truncated recombinant proteins or short relevant synthetic peptides bound to a solid phase (3, 12). In SDH/Plr, the second Plg-binding site is proposed to be located in the N-terminal region (45). The nature of this site is unknown. It is, however, likely that the putative second plasminogen-binding site contributes to the catalytic domain, since several N-terminally truncated recombinant SDH proteins representing deletions of the entire region in the first N-terminal half were catalytically inactive (24; V. Pancholi, unpublished data). It may be emphasized that although the C-terminal lysine residue plays an important role in Plg binding (45), the insertion of an additional hydrophobic tail did not interfere with the Plg-binding activity of the SDH molecule (Fig. 4). The mutagenesis approach described in the present study thus successfully allowed us to study the Plg-binding activity of GAS mediated by the SDH molecule as a whole. By using a similar mutagenesis approach, we propose that secretion of SEN and other anchorless surface molecules of GAS can also be prevented and that they can be localized or restrained to the cytoplasm without affecting their innate enzyme activities and other functions. The successful creation of such mutants may then help understand the contribution of anchorless surface proteins in GAS pathogenesis.
In the present study, the unexpected finding that the isogenic M1-SDHHBtail mutant strain lost the antiphagocytic activity of the wild-type strain raises an important question, namely, whether SDH is indeed antiphagocytic in nature. To understand the contribution of SDH to GAS antiphagocytic activity, we determined the antiphagocytic activity of the wild-type strain in the presence of anti-SDH polyclonal antibody. The increased phagocytosis of the wild-type GAS strain in the presence of anti-SDH antibody is likely due to its opsonic activity, as was also found for the antibody against another well-studied anchorless protein, SEN (16, 37). On the other hand, the absence of antiphagocytic activity in M1-SDHHBtail indeed seems due to the absence of the M1 protein as the expression of another antiphagocytic molecule, the polysaccharide capsule (14), was not affected in the mutant strain. Despite the facts that the sdh gene (SPy274) is monocistronic in the M1 genome as well as in other described GAS genomes (1, 2, 13, 29, 43) and hence that the insertion of a hydrophobic tail and the spectinomycin resistance gene aad-9 does not create any polar effect in the M1-SDHHBtail mutant, the expression of the emm1 gene (SPy 2018) product (M1 protein) is down regulated. Eukaryotic GAPDH has been shown to bind DNA and RNA, and this binding has been shown to play a role in the transcription regulation of eukaryotic genes (for reviews, see references 41 and 42). It is likely that SDH and its C-terminal portion in some way may have an important role in M1 expression, possibly at the transcriptional level.
We recently reported that SDH recognizes uPAR/CD87 as its receptor on the surfaces of pharyngeal cell and mediates GAS adherence (24). In that publication, we showed that the mutant GAS strain, displaying SDH with the C-terminal lysine residue (Lys336) deleted or with a C-terminal leucine residue instead of the lysine residue (Lys336Leu), adhered poorly to pharyngeal cells (24). It was, however, not possible to study those mutants to understand the contribution of other domains of the SDH molecule in the adherence process, as other domains contribute to the essential GAPDH activity of SDH (24). In the present study, this shortcoming was overcome by preventing the display of the whole SDH protein on the GAS surface. Our results showing the adherence ability of the M1emm1 mutant strain to pharyngeal cells in a way similar to that of the wild-type strain concur with the previously published reports, which indicated that the expression of the M protein has little effect on GAS adherence to human tonsillar/pharyngeal cells and keratinocytes (6, 10). In conjunction with this finding, the poor adherence ability of the M1-SDHHBtail strain, which does not display SDH, as well as of the M1 protein on its surface underscores the important contribution of SDH in GAS adherence to pharyngeal cells.
In summary, the mutagenesis approach used in the present study revealed new functions of SDH and its role in GAS pathogenesis, such as GAS adherence and antiphagocytic activity. It is not clear at present whether SDH is itself antiphagocytic or whether, in addition, it plays a role in the transcriptional or posttranscriptional control of GAS virulence factors. As the latter is true for the eukaryotic GAPDH (41, 42), it is an important question for future research. Evidence provided in the present study concludes that SDH is an important surface-expressed virulence factor and plays an important role in GAS pathogenesis.
ACKNOWLEDGMENTS
We thank Vincent A. Fischetti (The Rockefeller University, New York, NY) for providing 10B6 monoclonal antibody. We thank Carl Drlica and Issar Smith for critically reading the manuscript and Janet Hahn and Hedia Maamar for their initial help in immunofluorescence microscopy.
This research was supported by grant RO1-42827 (to V.P.) from the National Institutes of Health.
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