The Signal Recognition Particle Pathway Is Required for Virulence in Streptococcus pyogenes

Strains, media, and culture conditions.Strains used included E. coli DH5α and S. pyogenes HSC5 (22). Unless otherwise indicated, routine culture of S. pyogenes was done at 37°C and employed Todd-Hewitt medium (BBL) supplemented with 0.2% yeast extract (Difco). For certain experiments, S. pyogenes was cultured in C medium under conditions previously described (32). Culture of E. coli utilized Luria-Bertani (LB) broth at 37°C with shaking. When required, antibiotics were used at the following concentrations: erythromycin at 750 μg/ml for E. coli and 1 μg/ml for S. pyogenes.

DNA and computational techniques.Plasmid DNA was isolated via standard techniques and used to transform S. pyogenes as described previously (10). Restriction endonucleases, ligases, and polymerases were used according to the manufacturer's recommendations. The fidelity of all constructs derived by PCR was confirmed by DNA sequencing analyses. All references to genomic loci are based on the genome of S. pyogenes strain SF370 (15). Gene assignments were based on the information available in the Kyoto Encyclopedia of Genes and Genomes (www.genome.jp ) and were supported by subsequent interrogation of the SF370 genome using BLAST (2) and query sequences derived from Bacillus subtilis gene products with experimentally confirmed activities, as noted in the text.

Construction of mutants.An in-frame deletion was constructed by an inverse-PCR technique (31) to delete an internal fragment encompassing 500 bp of the central portion of ffh (SPy_1200). The next downstream open reading frame (SPy_1198) is transcribed from the strand opposite ffh, suggesting that deletion mutations in ffh will not be polar. Allelic replacement proceeded as described previously (31), with confirmation of mutant genome structure by PCR using primers of the appropriate sequences. Primers for the deletion included Ffh deletion 1 (AAC CTA GGG GGC CTA TGG AAG ACC TCT TGA AAA TGA TTC CAG G) and Ffh deletion 2 (AAC CTA GGC AAA CCT TTT CTG ACA ATA TCG ACT GC). The resulting fragment of ffh has a deletion from amino acids spanning positions 180 to 340. The method for allelic replacement involves the construction of a merodiploid strain that contains both mutant and wild-type alleles in tandem on the chromosome that resolves to contain either the mutant or wild-type alleles (9, 31). Thus, to control for any unanticipated secondary mutations that may have arisen during the production of the mutant, all comparisons were made between a mutant and a matched wild-type sibling that arose from the same merodiploid.

Cellular fractionation and preparation of membranes.Protoplasts were prepared as previously described (40) and then lysed by multiple freeze-thaw cycles (−80°C/37°C) followed by agitation with glass beads (106 μm; Sigma) using a reciprocating shaking device (FastPrep; Qbiogene) at a speed setting of 4.5 for 45 s, repeated four to five times. Membranes were collected by centrifugation (120,000 × g, 4°C, 60 min) and resuspended in distilled water. Sodium dodecyl sulfate sample buffer was added to the membranes, followed by a 10-min boil. Membrane fractions were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to an Immobilon-PSQ membrane, and stained with Coomassie brilliant blue R. Protein identification was performed by N-terminal sequencing of Coomassie-stained bands (Midwest Analytical, St. Louis, MO).

Analysis and identification of proteins.Cell wall, membrane, and cytosolic fractions were prepared from cultures grown in Todd-Hewitt (THY) broth as previously described (43). Cell-free supernatants were subjected to trichloroacetic acid (TCA) precipitation for protein concentration. Cell wall fractions from protoplast preparations were utilized for M protein analysis, while culture supernatants were utilized for analysis of SpeB, streptolysin O (SLO), and S. pyogenes NAD glycohydrolase (SPN). Other fractions were consistently negative for the presence of these proteins. Protein samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Blocking and antibody binding were done in phosphate-buffered saline-Tween 20 with 5% nonfat dry milk. Antisera against SLO and SpeB have been described previously (31, 35). An antipeptide antiserum that recognizes multiple serotypes of M protein was generated as described previously (17). An antiserum that reacts specifically against SPN was generated using purified SPN (C. Smith, M. Caparon, and S. Hultgren, unpublished). Analyses of culture supernatant fluids for SpeB proteolytic activity and SLO hemolytic activity were conducted as described previously (32, 35). Relative protein concentrations were quantitated from digitized images of Western blots by using ImageJ software (http://rsb.info.nih.gov/ij/ ). Data are expressed as the ratio of mutant versus wild type and were derived from a minimum of three independent experiments.

Analysis of transcription.RNA from various streptococcal strains was isolated and analyzed as described previously (7, 29). Briefly, strains grown overnight in THY medium were diluted 1:100 in fresh THY medium, followed by growth at 37°C to mid-logarithmic phase (optical density at 600 nm [OD₆₀₀] of 0.500). RNA was then isolated by using glass beads (Lysing Matrix A; Qbiogene) and a high-speed reciprocating shaking device (FP-120; Savant Instruments). RNA was further purified (RNeasy mini kit; Qiagen) and contaminating DNA was removed by DNase treatments according to the manufacturer's instructions (RNase-free DNase set [Qiagen] and DNase I, amplification grade [Invitrogen]). The A₂₆₀/A₂₈₀ ratio was used to determine the RNA concentration and purity. For cDNA synthesis, 5 μg total RNA was treated with 200 U Superscript II reverse transcriptase (Invitrogen) using 250 ng of oligonucleotide random primers (Invitrogen). Real-time reverse transcription-PCR (RT-PCR) was performed with an iCycler thermocycler (Bio-Rad) using iQ Sybr green supermix (Bio-Rad) and methods and primers described previously (29). Transcript abundance was normalized to that of recA as previously described (29), and data presented represent the means and standard deviations derived from at least three independent experiments that were performed on different days, with each individual sample analyzed in triplicate.

Zebrafish infections.Bacterial strains were back diluted 1:100 from overnight culture and grown in THY broth to an OD₆₀₀ of 0.30. Bacteria were then subjected to brief sonication to disrupt bacterial chains and used for intramuscular injection of zebrafish, as described previously (39). Experimental groups consisted of 10 zebrafish, each of which was injected with 10⁵ CFU in a volume of 10 μl or, in the case of mock infection, with an equivalent volume of sterile medium alone. Survival was monitored for 5 days, and any differences in survival between groups infected with wild-type or mutant strains were tested for significance as described previously (7). Data presented are pooled from three independent experiments that were conducted on different days.

Mouse subcutaneous infections.Bacterial strains were grown and prepared as outlined above and then used to infect mice subcutaneously as previously described (8). Experimental groups consisted of five mice, each of which received a subcutaneous injection of 10⁸ CFU in a volume of 100 μl into the right flank. Lesion development was monitored over a period of 96 h and recorded as described elsewhere (8). Differences between groups in lesion area and in number of mice developing an ulcer were tested for significance by the Mann-Whitney U test and the chi-square test with Yates' correction, respectively (8). For all test statistics, the null hypothesis was rejected when the P value was <0.05. Data presented are pooled from two independent experiments that were conducted on different days.

Ffh is not essential in Streptococcus pyogenes.Disruption of the SRP pathway leads to a variety of phenotypes in the various bacterial species examined. In E. coli and B. subtilis, deletion of Ffh is lethal (41). Furthermore, conditional depletion of Ffh and FtsY in B. subtilis results in a marked impairment of protein secretion (25), although the effect on the secretome may be more modest when the bacteria are cultured in rich media (49). In Streptococcus mutans, disruption of Ffh was first revealed in a screen for acid-sensitive mutants (19). It was subsequently shown that all components of the SRP pathway could be disrupted extensively in S. mutans with few phenotypic consequences apart from sensitivity to certain stresses (13). Interestingly, disruption of both the SRP and one of its two homologs of YidC (YidC2) resulted in S. mutans cells which were severely impaired in growth (23). We sought to ascertain whether SRP was also dispensable for the growth of S. pyogenes and to determine the effect of SRP disruption on the secretion of several important virulence factors. The SRP includes three critical components, a protein (Ffh), an RNA (scRNA), and a membrane receptor (FtsY) (45), and it has been shown that mutation of the genes encoding any of the components results in the same stress-sensitive phenotype in S. mutans (23). For analysis of S. pyogenes, we constructed an in-frame deletion in the allele of the gene encoding Ffh that removed 500 bp from the central portion of the Ffh open reading frame, which includes the region encoding conserved residues necessary for GTPase activity that are crucial for function (16). The resulting mutation in the allele [ffh(Δ180-340)] was successfully used to replace the wild-type gene in S. pyogenes HSC5 (Fig. 1A), and the resulting mutants grew normally and produced colonies on THY agar plates that were indistinguishable from wild-type colonies. Thus, similarly to results for S. mutans, the SRP pathway is dispensable for viability of S. pyogenes.

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FIG. 1.

Deletion of ffh results in specific nutritional requirements. Successful replacement of ffh (SPy_1200) in the wild-type strain with a deletion in the allele [ffh(Δ180-340)] produced Ffh⁻ strain FFH1. (A) Analysis of PCR products generated using ffh-specific primers by agarose gel electrophoresis revealed bands of the expected sizes for the wild-type (WT, open arrow) and mutant (Ffh⁻, filled arrow) strains as shown. Open and filled arrows at the left of the DNA size standards (Std.) (TrackIt DNA ladder; Invitrogen) indicate the 1,650- and 1,000-bp bands, respectively. The growth of the mutant (Ffh⁻) in liquid media was compared to that of a matched wild-type revertant (WT_rev) in THY medium (B), C medium (C), and C medium supplemented with glucose (D). Data shown are from a single experiment representative of a minimum of three independent experiments conducted on different days.

Medium-specific growth defect.The method for allelic replacement generates a tandem duplication of wild-type and mutant alleles that resolves by homologous recombination to produce strains carrying either the wild-type or the mutant allele (9). One Ffh⁻ mutant (FFH1) was chosen for further analysis, and its ability to grow in liquid media under various nutrient conditions was compared with that of a matched wild-type revertant (WT_rev). When grown in standard THY broth, a complex medium rich in glucose (18), the mutant showed a modest defect in its growth rate but obtained a growth yield equivalent to that of the wild type on the basis of culture densities (Fig. 1B). When grown in C medium, a more minimal medium rich in peptides but low in glucose, the Ffh⁻ mutant presented a pronounced defect in both growth rate and yield, with cultures reaching a final density that was less that 50% of that obtained with a strain with the wild-type gene (Fig. 1C). However, this defect could be rescued by the addition of more glucose to C medium, which produced a growth profile that was indistinguishable from that of THY medium (Fig. 1, compare panels D and B). Addition of NaCl (up to 150 mM) did not restore growth; however, buffering the pH of the medium to between pH 6.0 and pH 6.5 had the same effect as the addition of glucose and restored growth of the mutant to levels identical to those achieved with THY medium (data not shown).

Stress response activated.Membrane protein profiles of the wild-type revertant and the Ffh⁻ mutant were compared. Consistent with the relatively normal growth characteristics of the mutant in THY broth, these profiles were remarkably similar (Fig. 2). An exception was the presence of two bands that were consistently more intense in membranes of the mutant than in those of the wild-type revertant. Subjecting these bands to N-terminal sequencing revealed them to be DnaK and GroEL (Fig. 2). These proteins play prominent roles in several stress responses, particularly under conditions where misfolded proteins accumulate (38, 49). This observation suggests that, despite near-normal growth characteristics in this medium, the mutant is experiencing stress or may be accumulating some misfolded proteins.

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FIG. 2.

The majority of membrane proteins are targeted correctly in the absence of the SRP. Membranes purified from a mutant (Ffh⁻) and a matched wild-type revertant (WT_rev) were analyzed by SDS-PAGE and staining with Coomassie blue as shown. The identities of the bands labeled on the left of the figure were determined by N-terminal amino acid sequencing. The migration of several size standards (in kilodaltons) is indicated to the right of the figure.

Selective defects in secretion of proteins with cleavable signal sequences.A prominent characteristic of S. pyogenes is its ability to secrete a large number of proteins during growth in culture and in tissues that have cleavable signal sequences. The fate of a subset of these proteins, including the M protein, is to become covalently associated with the cell wall (34). Others have well-characterized toxic activities and are known to have distinctly different trafficking fates when the bacterium is growing while attached to the surface of host cells. For example, many secreted proteins, such as the SpeB cysteine protease, are trafficked into the extracellular spaces. At least one, SPN (also known as Nga), is translocated across the host cell membrane into its cytosol by a process that involves a second secreted translocator protein, SLO, that is itself delivered to the host cell membrane (33, 35, 37). Since M protein, SpeB, SPN, and SLO have different trafficking fates, the consequence of the loss of Ffh on their secretion was assessed. Following secretion of the M protein via the Sec pathway, the signal sequence of the M protein is cleaved and subsequently processed at a site toward its carboxy-terminal end and covalently cross-linked to peptidoglycan by the enzyme sortase (4). Examination of cell walls revealed that the amount of M protein in the mutant was similar to or smaller than the amount of M protein in the wild-type parental strain (Fig. 3), indicating that Ffh is not essential for the secretion of M protein and its delivery to the sortase pathway. In contrast, the loss of Ffh had marked effects on the secretion of SPN and SLO. Analysis of culture supernatants revealed that SPN and SLO were undetectable or present at very low levels even after the proteins in these supernatants were concentrated by TCA precipitation (Fig. 3). Additionally, culture supernatants from Ffh⁻ mutants lacked detectable SLO hemolytic activity and SPN activity (Table 1), confirming a defect for secretion of these proteins. The genes encoding SPN and SLO are cotranscribed from a common promoter (36), and the failure to observe the secreted forms of either protein was not due to a defect in transcription from this promoter, as analysis of the slo and spn transcripts by real-time RT-PCR at the time of maximal expression in logarithmically growing cultures indicated that these transcripts were as abundant in the mutant as in the wild-type revertant (Table 1). A similar analysis of the transcript levels of the gene encoding M protein at a time point corresponding to the period of maximal expression also showed no differences between the Ffh⁻ mutant strain and the wild-type revertant (Table 1). No intracellular pool of SLO was detected when the mutant was subjected to lysis at this time point (data not shown), suggesting that the polypeptide was degraded rapidly in the absence of secretion, although translational arrest of the message could also account for this observation. Taken together, these data show that the SRP pathway does make a critical contribution to secretion of at least a subset of the exported virulence factors of S. pyogenes that possess cleavable signal sequences.

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FIG. 3.

The SRP is required for the secretion of a distinct subset of virulence factors. Analysis of the secretion of a selection of virulence factors that have both cleavable signal sequences and distinct trafficking fates during infection is shown. Included are the sortase pathway substrate M protein, the SpeB cysteine protease that is released into the extracellular compartment, SLO that is trafficked to the host cell membrane, and SPN that is translocated into the host cell cytosol. The mutant (Ffh⁻) and a matched wild-type revertant (WT_rev) were compared by Western blot analyses using antisera specific for each indicated protein. Samples analyzed for M protein were cell walls from protoplast preparations, cell-free overnight culture supernatant fluids for SpeB, and cell-free overnight supernatant fluids that were concentrated by TCA precipitation for SLO and SPN. The migration of the zymogen (proSpeB) and mature (SpeB) forms of SpeB are indicated to the right of the SpeB panel. The times shown on the right of the figure indicate the onset of stationary phase (0 h) and subsequent time points.

Medium-dependent effect on secretion of SpeB.The absence of Ffh had a different effect on secretion of SpeB, as it was observed that the Ffh⁻ mutant secreted the SpeB polypeptide at levels higher than those observed for the wild-type revertant during growth in THY medium (Fig. 3). Much of this effect resulted from an alteration in the temporal pattern of speB transcription. In the wild type, speB is not transcribed during the log phase of growth, and transcription is initiated at high levels during the transition into stationary phase (28, 29). However, in the mutant, high levels of speB transcription were observed at the late log phase of growth (Table 1), and these began to decline by the time transcription in the wild type was at peak levels (t = 2 h) (Table 1). This increased level of SpeB protein did not result in greater expression of protease activity in the Ffh⁻ mutant, as SpeB-dependent cysteine protease activity in culture supernatant from the mutant was somewhat reduced from levels observed for the wild-type revertant (Table 1). This phenotype was medium dependent, as the supernatant fluids from the Ffh⁻ mutant lacked both detectable cysteine protease activity (data not shown) and detectable levels of the SpeB polypeptide following culture in the glucose-deficient C medium, which is a medium that supports robust expression of SpeB by the wild-type revertant (Fig. 4). This defect was not due to a failure of the mutant to express SpeB, as the intracellular fraction of the mutant contained detectable levels of the SpeB polypeptide (Fig. 4, “cytoplasmic”). Glucose has a repressive effect on expression of SpeB (28, 29), and as expected, supplementing C medium with glucose resulted in repression of SpeB expression in the wild-type revertant (Fig. 4, compare WT_rev supernatant in “C medium + Glc” column to that in “C medium” column). However, the addition of glucose resulted in increased SpeB expression in the Ffh⁻ mutant, as levels in the supernatant fraction for both the SpeB polypeptide (Fig. 4, “C medium + Glc”) and cysteine protease activity (data not shown) were elevated to the levels observed in the wild-type revertant in unsupplemented C medium. Similarly to the effects on growth in C medium (see above), buffering to pH 6.5, but not the addition of NaCl, resulted in enhanced levels of SpeB in the supernatant fluids of the mutant (data not shown).

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FIG. 4.

Medium-dependent secretion defect. A Western blot analysis of SpeB expression from a mutant (Ffh⁻) and a wild-type revertant (WT_rev) following culture in C medium or C medium supplemented with glucose (C medium + Glc) is shown. As indicated in the figure, both supernatant and cytoplasmic fractions were analyzed.

The SRP pathway is required for pathogenesis in a zebrafish model of necrotic myositis.Since the Ffh⁻ mutant had both a medium-specific growth defect and a selective deficiency in its ability to export several toxins, it was of interest to determine whether the mutant was also altered in its ability to cause disease. The virulence of the Ffh⁻ mutant was evaluated in a zebrafish model of necrotic myositis that reproduces several features commonly observed in streptococcal infection of human muscle, including local growth in muscle tissue, extensive necrosis of the infected tissue, and diminished inflammation (39). Infection of zebrafish with the wild-type revertant at approximately 10-fold the 50% lethal dose produced characteristic survival curves in which greater than 90% of infected zebrafish did not survive past day 3 postinfection (Fig. 5). Also, all fish infected with the wild-type revertant demonstrated a characteristic discolored lesion at the site of infection apparent 24 h postinfection that reflects the extent of the underlying necrosis in muscle (39), and all zebrafish that developed a lesion did not survive. In contrast, greater than 95% of zebrafish infected with the Ffh⁻ mutant were still viable at day 3 (Fig. 5), demonstrating that the loss of the SRP rendered S. pyogenes significantly less virulent (P < 0.0001). Although lesions in zebrafish infected with the mutant were not as intense as or of equivalent area to those in zebrafish infected with the wild-type revertant, approximately 25% of the zebrafish infected with the mutant did develop lesions by day 2, and these animals typically survived and their lesions resolved. Lesion development had not been associated with any of the attenuated mutants analyzed previously (8, 39).

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FIG. 5.

The SRP is required for virulence in an animal model of necrotic myositis. Groups of zebrafish were challenged intramuscularly with 1 × 10⁵ CFU of a mutant (Ffh⁻) or a matched wild-type revertant (WT_rev) or were injected with sterile media alone (Mock). Survival was monitored for 72 h, and the data are presented as a Kaplan-Meier plot. Data are pooled from three independent experiments, each of which was conducted using 10 zebrafish per group. These data indicate that the mutant was significantly less lethal than the wild-type revertant (P < 0.0001).

The SRP pathway is required for pathogenesis in a murine model of subcutaneous infection.Virulence of the Ffh⁻ mutant was also examined in a murine model of subcutaneous infection, in which challenge with S. pyogenes HSC5 has been observed to cause visible ulcer-like lesions following injection into subcutaneous tissue (8). Consistent with previous reports, all mice injected with the wild-type revertant had visible lesions by 48 h postinfection, while most mice infected with the Ffh⁻ mutant failed to develop any evidence of a visible ulcer even following 72 h (Fig. 6) and 96 h (data not shown) of examination, demonstrating that the mutant was significantly less virulent (P < 0.02). A subset of mice infected with the mutant did develop ulcers (Fig. 6). However, the wild-type revertant caused lesions that were significantly larger in size than those caused by the mutant when compared at a time when lesion formation by the wild-type revertant had reached a maximum (72 h) (P < 0.005) (Fig. 6) and at subsequent time points over the duration of the experiment (96 h). The attenuation of the mutant is not due to its failure to secrete SLO and SPN, as mutants of this wild-type strain (HSC5) defective for expression of these two proteins are not attenuated in this model (J. Ghosh and M. G. Caparon, unpublished). These data provide additional evidence to suggest that Ffh plays an important role in S. pyogenes virulence.

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FIG. 6.

The SRP is required for virulence in the murine subcutaneous ulcer model. Groups of female SKH1 hairless mice received a subcutaneous injection of 10⁷ CFU of a mutant (Ffh⁻) or a matched wild-type revertant (WT_rev) into the hind flank. Following 72 h, the area of any visible ulcer that formed at the site of injection was determined as described previously (8). Data shown are pooled from two independent experiments. Each symbol represents the area of the ulcer observed in an individual mouse, and the horizontal bar indicates the mean value obtained for the pooled set of 10 mice. As indicated in the figure, lesions caused by the WT_rev were significantly larger than those caused by the Ffh⁻ mutant.