Streptococcal mitogenic exotoxin, SmeZ, is the most susceptible M1T1 streptococcal superantigen to degradation by the streptococcal cysteine protease, SpeB

Superantigens (SAgs) play an important role in the pathogenesis of severe invasive infections caused by Group A Streptococcus (GAS). We had shown earlier that the expression of streptococcal cysteine protease SpeB results in partial loss of the immune-stimulating activity of the native secreted GAS SAgs, namely the streptococcal pyrogenic exotoxins produced by the globally disseminated M1T1 GAS strain, associated with invasive infections worldwide. In this study, we examined the susceptibility of each of the M1T1 recombinant SAgs to degradation by rSpeB. Whereas SmeZ was degraded completely within 30 min of incubation with rSpeB, SpeG, and SpeA were more resistant and SpeJ was completely unaffected by the proteolytic effects of this protease. Proteomic analyses demonstrated that the order of susceptibility of the M1T1 SAgs to SpeB proteolysis is unaltered when they are present in a mixture that reflects their native physiological status. As expected, the degradation of SmeZ abolished its immune stimulatory activity. In silico sequence disorder and structural analyses revealed that SmeZ, unlike the three other structurally related SAgs, possesses a putative SpeB cleavage site within an area of the protein likely to be exposed to the surface. The study provides evidence for the effect of subtle structural differences between highly similar SAgs on their biological activity.     

The Fibrinogen-binding M1 Protein Reduces Pharyngeal Cell Adherence and Colonization Phenotypes of M1T1 Group A Streptococcus

Group A Streptococcus (GAS) is a leading human pathogen producing a diverse array of infections from simple pharyngitis (“strep throat”) to invasive conditions, including necrotizing fasciitis and toxic shock syndrome. The surface-anchored GAS M1 protein is a classical virulence factor that promotes phagocyte resistance and exaggerated inflammation by binding host fibrinogen (Fg) to form supramolecular networks. In this study, we used a virulent WT M1T1 GAS strain and its isogenic M1-deficient mutant to examine the role of M1-Fg binding in a proximal step in GAS infection-interaction with the pharyngeal epithelium. Expression of the M1 protein reduced GAS adherence to human pharyngeal keratinocytes by 2-fold, and this difference was increased to 4-fold in the presence of Fg. In stationary phase, surface M1 protein cleavage by the GAS cysteine protease SpeB eliminated Fg binding and relieved its inhibitory effect on GAS pharyngeal cell adherence. In a mouse model of GAS colonization of nasal-associated lymphoid tissue, M1 protein expression was associated with an average 6-fold decreased GAS recovery in isogenic strain competition assays. Thus, GAS M1 protein-Fg binding reduces GAS pharyngeal cell adherence and colonization in a fashion that is counterbalanced by SpeB. Inactivation of SpeB during the shift to invasive GAS disease allows M1-Fg binding, increasing pathogen phagocyte resistance and proinflammatory activities.     

Role of group A Streptococcus HtrA in the maturation of SpeB protease

The serine protease high-temperature requirement A (HtrA) (DegP) of the human pathogen Streptococcus pyogenes (group A Streptococcus; GAS) is localized to the ExPortal secretory microdomain and is reportedly essential for the maturation of cysteine protease streptococcal pyrogenic exotoxin B (SpeB). Here, we utilize HSC5 (M5 serotype) and the in-frame isogenic mutant HSC5DeltahtrA to determine whether HtrA contributes to the maturation of other GAS virulence determinants. Mutanolysin cell wall extracts and secreted proteins were arrayed by 2-DE and identified by MALDI-TOF PMF analysis. HSC5DeltahtrA had elevated levels of cell wall-associated M protein, whilst the supernatant had higher concentrations of M protein fragments and a reduced amount of mature SpeB protease, compared to wild-type (WT). Western blot analysis and protease assays revealed a delay in the maturation of SpeB in the HSC5DeltahtrA supernatant. HtrA was unable to directly process SpeB zymogen (proSpeB) to the active form in vitro. We therefore conclude that HtrA plays an indirect role in the maturation of cysteine protease SpeB.     

Post-proteomic identification of a novel phage-encoded streptodornase, Sda1, in invasive M1T1 Streptococcus pyogenes

The M1T1 strain remains the most frequently isolated strain from group A streptococcal (GAS) infection cases worldwide. We previously reported that M1T1 differs from the fully sequenced M1 SF370 strain. To better understand the reason for the persistence and increased virulence of M1T1, we analysed its secreted proteome and identified two virulence proteins that are not present in the sequenced M1 SF370 strain: streptococcal pyrogenic exotoxin A (SpeA) and a streptodornase D (SdaD) homologue. In the present study, we determined the nucleotide sequence of the M1T1 streptodornase and found that its deduced amino acid sequence is highly similar to other streptococcal streptodornases, and is most closely related to the SdaD of GAS strain M49. M1T1 Sda shares two highly conserved domains with several DNases and putative DNases in streptococci; however, it possesses a unique C-terminal amino acid sequence. Thus, we named the protein Sda1, and we detected the presence of the sda1 gene in 16 M1T1 clinical isolates. The cloned and expressed Sda1 degrades both streptococcal and mammalian DNA at physiological pH. Amino acid similarity analyses of known GAS deoxyribonucleases suggest that Sda1 may be a chimeric protein created through recombination events. Moreover, a natural mutation that resulted in longer Sda1 and SdaD as compared to other GAS DNases was found to confer increased activity on the protein. Analysis of the sequences flanking sda1 determined that it is carried by a prophage or a prophage-like element inserted in the tRNA-Ser gene of M1T1 GAS. Ongoing studies in our laboratory aim to determine the contribution of Sda1 to the virulence of this globally disseminated M1T1 strain.     

DNase Sda1 provides selection pressure for a switch to invasive group A streptococcal infection

Most invasive bacterial infections are caused by species that more commonly colonize the human host with minimal symptoms. Although phenotypic or genetic correlates underlying a bacterium's shift to enhanced virulence have been studied, the in vivo selection pressures governing such shifts are poorly understood. The globally disseminated M1T1 clone of group A Streptococcus (GAS) is linked with the rare but life-threatening syndromes of necrotizing fasciitis and toxic shock syndrome. Mutations in the GAS control of virulence regulatory sensor kinase (covRS) operon are associated with severe invasive disease, abolishing expression of a broad-spectrum cysteine protease (SpeB) and allowing the recruitment and activation of host plasminogen on the bacterial surface. Here we describe how bacteriophage-encoded GAS DNase (Sda1), which facilitates the pathogen's escape from neutrophil extracellular traps, serves as a selective force for covRS mutation. The results provide a paradigm whereby natural selection exerted by the innate immune system generates hypervirulent bacterial variants with increased risk of systemic dissemination.     

Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci

Primary infection of the human host by group A streptococci (GAS) most often involves either the epidermis of the skin or the oropharyngeal mucosa. A humanized in vivo model for impetigo was used to investigate the basis for host tissue tropism among GAS. Disruption of the speB gene (encoding for a secreted cysteine proteinase) led to a loss of virulence for two impetigo-derived strains (M-types 33 and 53), as evidenced by a diminution in tissue damage and a lack of reproductive growth. The level of cysteine proteinase activity in overnight cultures was associated with the extent of gross pathological changes induced by strains displaying varied degrees of virulence in the impetigo model. Moreover, high levels of secreted cysteine proteinase activity correlated with a genetic marker for preferred tissue site of infection at the skin (emm pattern D). The addition of exogenous SpeB to a speB mutant (emm pattern D) or to an avirulent throat-like strain (emm pattern A) led to increased bacterial reproduction at the skin. The data provide both experimental and epidemiological evidence for a critical role of a secreted bacterial protease in promoting host tissue-specific infection.     

The role played by the group A streptococcal negative regulator Nra on bacterial interactions with epithelial cells

Group A streptococci (GAS) specifically attach to and internalize into human epithelial host cells. In some GAS isolates, fibronectin-binding proteins were identified as being responsible for these virulence traits. In the present study, the previously identified global negative regulator Nra was shown to control the binding of soluble fibronectin probably via regulation of protein F2 and/or SfbII expression in the serotype M49 strain 591. According to results from a conventional invasion assay based on the recovery of viable intracellular bacteria, the increased fibronectin binding did not affect bacterial adherence to HEp-2 epithelial cells, but was associated with a reduction in the internalization rates. However, when examined by confocal and electron microscopy techniques, the nra-mutant bacteria were shown to exhibit higher adherence and internalization rates than the corresponding wild type. The mutant bacteria escaped from the phagocytic vacuoles much faster, promoting consistent morphological changes which resulted in severe host cell damage. The apoptotic and lytic processes observed in nra-mutant infected host cells were correlated with an increased expression of the genes encoding superantigen SpeA, the cysteine protease SpeB, and streptolysin S in the nra-mutant bacteria. Adherence and internalization rates of a nra/speB-double mutant at wild-type levels indicated that the altered speB expression in the nra mutant contributed to the observed changes in both processes. The Nra-dependent effects on bacterial virulence were confined to infections carried out with stationary growth phase bacteria. In conclusion, the obtained results demonstrated that the global GAS regulator Nra modulates virulence genes, which are involved in host cell damage. Thus, by helping to achieve a critical balance of virulence factor expression that avoids the injury of target cells, Nra may facilitate GAS persistence in a safe intracellular niche.     

Mosaic prophages with horizontally acquired genes account for the emergence and diversification of the globally disseminated M1T1 clone of Streptococcus pyogenes

The recrudescence of severe invasive group A streptococcal (GAS) diseases has been associated with relatively few strains, including the M1T1 subclone that has shown an unprecedented global spread and prevalence and high virulence in susceptible hosts. To understand its unusual epidemiology, we aimed to identify unique genomic features that differentiate it from the fully sequenced M1 SF370 strain. We constructed DNA microarrays from an M1T1 shotgun library and, using differential hybridization, we found that both M1 strains are 95% identical and that the 5% unique M1T1 clone sequences more closely resemble sequences found in the M3 strain, which is also associated with severe disease. Careful analysis of these unique sequences revealed three unique prophages that we named M1T1.X, M1T1.Y, and M1T1.Z. While M1T1.Y is similar to phage 370.3 of the M1-SF370 strain, M1T1.X and M1T1.Z are novel and encode the toxins SpeA2 and Sda1, respectively. The genomes of these prophages are highly mosaic, with different segments being related to distinct streptococcal phages, suggesting that GAS phages continue to exchange genetic material. Bioinformatic and phylogenetic analyses revealed a highly conserved open reading frame (ORF) adjacent to the toxins in 18 of the 21 toxin-carrying GAS prophages. We named this ORF paratox, determined its allelic distribution among different phages, and found linkage disequilibrium between particular paratox alleles and specific toxin genes, suggesting that they may move as a single cassette. Based on the conservation of paratox and other genes flanking the toxins, we propose a recombination-based model for toxin dissemination among prophages. We also provide evidence that a minor population of the M1T1 clonal isolates have exchanged their virulence module on phage M1T1.Y, replacing it with a different module identical to that found on a related M3 phage. Taken together, the data demonstrate that mosaicism of the GAS prophages has contributed to the emergence and diversification of the M1T1 subclone.     

Dispersal of Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease severity in a murine model

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.     

A Naturally Occurring Mutation in ropB Suppresses SpeB Expression and Reduces M1T1 Group A Streptococcal Systemic Virulence

Epidemiological studies of group A streptococcus (GAS) have noted an inverse relationship between SpeB expression and invasive disease. However, the role of SpeB in the course of infection is still unclear. In this study we utilize a SpeB-negative M1T1 clinical isolate, 5628, with a naturally occurring mutation in the gene encoding the regulator RopB, to elucidate the role of RopB and SpeB in systemic virulence. Allelic exchange mutagenesis was used to replace the mutated ropB allele in 5628 with the intact allele from the well characterized isolate 5448. The inverse allelic exchange was also performed to replace the intact ropB in 5448 with the mutated allele from 5628. An intact ropB was found to be essential for SpeB expression. While the ropB mutation was shown to have no effect on hemolysis of RBC''s, extracellular DNase activity or survival in the presence of neutrophils, strains with the mutated ropB allele were less virulent in murine systemic models of infection. An isogenic SpeB knockout strain containing an intact RopB showed similarly reduced virulence. Microarray analysis found genes of the SpeB operon to be the primary target of RopB regulation. These data show that an intact RopB and efficient SpeB production are necessary for systemic infection with GAS.     

The ExPortal: an organelle dedicated to the biogenesis of secreted proteins in Streptococcus pyogenes

The Gram-positive pathogen Streptococcus pyogenes secretes proteins through the ExPortal, a unique single microdomain of the cellular membrane specialized to contain the Sec translocons. It has been proposed that the ExPortal functions as an organelle to promote the biogenesis of secreted proteins by coordinating interactions between nascent unfolded secretory proteins and membrane-associated chaperones. In this study we provide evidence to support this model. It was found that HtrA (DegP), a surface anchored accessory factor required for maturation of the secreted SpeB cysteine protease, was localized exclusively to the ExPortal. Furthermore, the ATP synthase beta subunit was not localized to the ExPortal, suggesting that retention is likely restricted to a specific subset of exported proteins. Mutations that disrupted the anchoring, but not the protease activity, of HtrA, also altered the maturation kinetics of SpeB demonstrating that localization to the ExPortal was important for HtrA function. These data indicate that the ExPortal provides a mechanism by which Gram-positive bacteria can coordinate protein secretion and subsequent biogenesis in the absence of a specialized protein-folding compartment.     

Consideration of cysteine protease activity for serological M-typing of clinical Streptococcus pyogenes isolates

Clinical isolates of Streptococcus pyogenes were classified by serological typing of their surface M protein. Non-M typeable strains with the emm1 gene were characterized as the degradation of M protein caused by overproduction of the extracellular cysteine protease, SpeB. These events are dependent on the growth phase. M protein produced prior to expression of SpeB is degraded in the stationary phase when the active form of SpeB is detected. The proteolytic degradation of M protein should be considered for precise M typing analysis.     

Application of immunoproteomics to analysis of post-translational processing of the antiphagocytic M protein of Streptococcus

Post-translational modification of the antiphagocytic M1 protein of Streptococcus pyogenes can influence its binding properties for human immunoglobulin G subclasses and its invasive potential. Current methods of monitoring this modification event involve N-terminal sequencing and are cumbersome, slow and not amenable to routine analysis. In this study we demonstrate that surface enhanced laser desorption/ionization-time of flight mass spectrometry can be used to monitor modification of the M1 protein by the secreted bacterial cysteine protease, SpeB. This method, when combined with a specific antibody capture step provides a specific, rapid and sensitive assay for key virulence factors of the important human pathogen Streptococcus pyogenes.