M1T1 Group Research Articles

Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group A Streptococcus Is Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but Not in the Skin.

Group A Streptococcus (GAS) commonly causes pharyngitis and skin infections. Little is known why streptococcal pharyngitis usually does not lead to pneumonia and why the skin is a favorite niche for GAS. To partially address these questions, the effectiveness of neutrophils in clearing wild-type (wt) M1T1 GAS strain MGAS2221 from the lung and from the skin was examined in murine models of intratracheal pneumonia and subcutaneous infection. Ninety-nine point seven percent of the MGAS2221 inoculum was cleared from the lungs of C57BL/6J mice at 24 h after inoculation, while there was no MGAS2221 clearance from skin infection sites. The bronchial termini had robust neutrophil infiltration, and depletion of neutrophils abolished MGAS2221 clearance from the lung. Phagocyte NADPH oxidase but not myeloperoxidase was required for MGAS2221 clearance. Thus, wt M1T1 GAS can be cleared by neutrophils using an NADPH oxidase-dependent mechanism in the lung. MGAS2221 induced robust neutrophil infiltration at the edge of skin infection sites and throughout infection sites at 24 h and 48 h after inoculation, respectively. Neutrophils within MGAS2221 infection sites had no nuclear staining. Skin infection sites of streptolysin S-deficient MGAS2221 ΔsagA were full of neutrophils with nuclear staining, whereas MGAS2221 ΔsagA infection was not cleared. Gp91phox knockout (KO) and control mice had similar GAS numbers at skin infection sites and similar abilities to select SpeB activity-negative (SpeBA-) variants. These results indicate that phagocyte NADPH oxidase-mediated GAS killing is compromised in the skin. Our findings support a model for GAS skin tropism in which GAS generates an anoxic niche to evade phagocyte NADPH oxidase-mediated clearance.

Genome-wide discovery of novel M1T1 group A streptococcal determinants important for fitness and virulence during soft-tissue infection.

The Group A Streptococcus remains a significant human pathogen causing a wide array of disease ranging from self-limiting to life-threatening invasive infections. Epithelium (skin or throat) colonization with progression to the subepithelial tissues is the common step in all GAS infections. Here, we used transposon-sequencing (Tn-seq) to define the GAS 5448 genetic requirements for in vivo fitness in subepithelial tissue. A near-saturation transposon library of the M1T1 GAS 5448 strain was injected subcutaneously into mice, producing suppurative inflammation at 24 h that progressed to prominent abscesses with tissue necrosis at 48 h. The library composition was monitored en masse by Tn-seq and ratios of mutant abundance comparing the output (12, 24 and 48 h) versus input (T0) mutant pools were calculated for each gene. We identified a total of 273 subcutaneous fitness (scf) genes with 147 genes (55 of unknown function) critical for the M1T1 GAS 5448 fitness in vivo; and 126 genes (53 of unknown function) potentially linked to in vivo fitness advantage. Selected scf genes were validated in competitive subcutaneous infection with parental 5448. Two uncharacterized genes, scfA and scfB, encoding putative membrane-associated proteins and conserved among Gram-positive pathogens, were further characterized. Defined scfAB mutants in GAS were outcompeted by wild type 5448 in vivo, attenuated for lesion formation in the soft tissue infection model and dissemination to the bloodstream. We hypothesize that scfAB play an integral role in enhancing adaptation and fitness of GAS during localized skin infection, and potentially in propagation to other deeper host environments.

The Mga Regulon but Not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes to In Vivo Selection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms.

Invasive M1T1 group A Streptococcus (GAS) can have a mutation in the regulatory system CovRS, and this mutation can render strains hypervirulent. Interestingly, via mechanisms that are not well understood, the host innate immune system's neutrophils select spontaneous M1T1 GAS CovRS hypervirulent mutants, thereby enhancing the pathogen's ability to evade immune killing. It has been reported that the DNase Sda1 is critical for the resistance of M1T1 strain 5448 to killing in human blood and provides pressure for in vivo selection of CovRS mutations. We reexamined the role of Sda1 in the selection of CovRS mutations and in GAS innate immune evasion. Deletion of sda1 or all DNase genes in M1T1 strain MGAS2221 did not alter emergence of CovRS mutants during murine infection. Deletion of sda1 in strain 5448 resulted in Δsda1 mutants with (5448 Δsda1(M+) strain) and without (5448 Δsda1(M-) strain) M protein production. The 5448 Δsda1(M+) strain accumulated CovRS mutations in vivo and resisted killing in the bloodstream, whereas the 5448 Δsda1(M-) strain lost in vivo selection of CovRS mutations and was sensitive to killing. The deletion of emm and a spontaneous Mga mutation in MGAS2221 reduced and prevented in vivo selection for CovRS mutants, respectively. Thus, in contrast to previous reports, Sda1 is not critical for in vivo selection of invasive M1T1 CovRS mutants and GAS resistance to innate immune killing mechanisms. In contrast, M protein and other Mga-regulated proteins contribute to the in vivo selection of M1T1 GAS CovRS mutants. These findings advance the understanding of the progression of invasive M1T1 GAS infections.

Role for streptococcal collagen-like protein 1 in M1T1 group A Streptococcus resistance to neutrophil extracellular traps.

Streptococcal collagen-like protein 1 (Scl-1) is one of the most highly expressed proteins in the invasive M1T1 serotype group A Streptococcus (GAS), a globally disseminated clone associated with higher risk of severe invasive infections. Previous studies using recombinant Scl-1 protein suggested a role in cell attachment and binding and inhibition of serum proteins. Here, we studied the contribution of Scl-1 to the virulence of the M1T1 clone in the physiological context of the live bacterium by generating an isogenic strain lacking the scl-1 gene. Upon subcutaneous infection in mice, wild-type bacteria induced larger lesions than the Δscl mutant. However, loss of Scl-1 did not alter bacterial adherence to or invasion of skin keratinocytes. We found instead that Scl-1 plays a critical role in GAS resistance to human and murine phagocytic cells, allowing the bacteria to persist at the site of infection. Phenotypic analyses demonstrated that Scl-1 mediates bacterial survival in neutrophil extracellular traps (NETs) and protects GAS from antimicrobial peptides found within the NETs. Additionally, Scl-1 interferes with myeloperoxidase (MPO) release, a prerequisite for NET production, thereby suppressing NET formation. We conclude that Scl-1 is a virulence determinant in the M1T1 GAS clone, allowing GAS to subvert innate immune functions that are critical in clearing bacterial infections.

Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

Pathogen mutants arise during infections. Mechanisms of selection for pathogen variants are poorly understood. We tested whether neutrophils select mutations in the two-component regulatory system CovRS of group A Streptococcus (GAS) during infection using the lack of production of the protease SpeB (SpeB activity negative [SpeB(A-)]) as a marker. Depletion of neutrophils by antibodies RB6-8C5 and 1A8 reduced the percentage of SpeB(A-) variants (SpeB(A-)%) recovered from mice infected with GAS strain MGAS2221 by >76%. Neutrophil recruitment and SpeB(A-)% among recovered GAS were reduced by 95% and 92%, respectively, in subcutaneous MGAS2221 infection of CXCR2(-/-) mice compared with control mice. In air sac infection with MGAS2221, levels of neutrophils and macrophages in lavage fluid were reduced by 49% and increased by 287%, respectively, in CXCR2(-/-) mice compared with control mice, implying that macrophages play an insignificant role in the reduction of selection for SpeB(A-) variants in CXCR2(-/-) mice. One randomly chosen SpeB(A-) mutant outcompeted MGAS2221 in normal mice but was outcompeted by MGAS2221 in neutropenic mice and had enhancements in expression of virulence factors, innate immune evasion, skin invasion, and virulence. This and nine other SpeB(A-) variants from a mouse all had nonsynonymous covRS mutations that resulted in the SpeB(A-) phenotype and enhanced expression of the CovRS-controlled secreted streptococcal esterase (SsE). Our findings are consistent with a model that neutrophils select spontaneous covRS mutations that maximize the potential of GAS to evade neutrophil responses, resulting in variants with enhanced survival and virulence. To our knowledge, this is the first report of the critical contribution of neutrophils to the selection of pathogen variants.

Peroxide Responsive Regulator PerR of group A Streptococcus Is Required for the Expression of Phage-Associated DNase Sda1 under Oxidative Stress

The peroxide regulator (PerR) is a ferric uptake repressor-like protein, which is involved in adaptation to oxidative stress and iron homeostasis in group A streptococcus. A perR mutant is attenuated in surviving in human blood, colonization of the pharynx, and resistance to phagocytic clearance, indicating that the PerR regulon affects both host environment adaptation and immune escape. Sda1 is a phage-associated DNase which promotes M1T1 group A streptococcus escaping from phagocytic cells by degrading DNA-based neutrophil extracellular traps. In the present study, we found that the expression of sda1 is up-regulated under oxidative conditions in the wild-type strain but not in the perR mutant. A gel mobility shift assay showed that the recombinant PerR protein binds the sda1 promoter. In addition, mutation of the conserved histidine residue in the metal binding site of PerR abolished sda1 expression under hydrogen peroxide treatment conditions, suggesting that PerR is directly responsible for the sda1 expression under oxidative stress. Our results reveal PerR-dependent sda1 expression under oxidative stress, which may aid innate immune escape of group A streptococcus.

Study of streptococcal hemoprotein receptor (Shr) in iron acquisition and virulence of M1T1 group A streptococcus

Streptococcus pyogenes (group A streptococcus, GAS) is a human bacterial pathogen of global significance, causing severe invasive diseases associated with serious morbidity and mortality. To survive within the host and establish an infection, GAS requires essential nutrients, including iron. The streptococcal hemoprotein receptor (Shr) is a surface-localized GAS protein that binds heme-containing proteins and extracellular matrix components. In this study, we employ targeted allelic exchange mutagenesis to investigate the role of Shr in the pathogenesis of the globally disseminated serotype M1T1 GAS. The shr mutant exhibited a growth defect in iron-restricted medium supplemented with ferric chloride, but no significant differences were observed in neutrophil survival, antimicrobial peptide resistance, cell surface charge, fibronectin-binding or adherence to human epithelial cells and keratinocytes, compared with wild-type. However, the shr mutant displayed a reduction in human blood proliferation, laminin-binding capacity and was attenuated for virulence in in vivo models of skin and systemic infection. We conclude that Shr augments GAS adherence to laminin, an important extracellular matrix attachment component. Furthermore, Shr-mediated iron uptake contributes to GAS growth in human blood, and is required for full virulence of serotype M1T1 GAS in mouse models of invasive disease.

Genetic Switch to Hypervirulence Reduces Colonization Phenotypes of the Globally Disseminated Group AStreptococcusM1T1 Clone

The recent resurgence of invasive group A streptococcal disease has been paralleled by the emergence of the M1T1 clone. Recently, invasive disease initiation has been linked to mutations in the covR/S 2-component regulator. We investigated whether a fitness cost is associated with the covS mutation that counterbalances hypervirulence. Wild-type M1T1 group A Streptococcus and an isogenic covS-mutant strain derived from animal passage were compared for adherence to human laryngeal epithelial cells, human keratinocytes, or fibronectin; biofilm formation; and binding to intact mouse skin. Targeted mutagenesis of capsule expression of both strains was performed for analysis of its unique contribution to the observed phenotypes. The covS-mutant bacteria showed reduced capacity to bind to epithelial cell layers as a consequence of increased capsule expression. The covS-mutant strain also had reduced capacity to bind fibronectin and to form biofilms on plastic and epithelial cell layers. A defect in skin adherence of the covS-mutant strain was demonstrated in a murine model. Reduced colonization capacity provides a potential explanation for why the covS mutation, which confers hypervirulence, has not become fixed in the globally disseminated M1T1 group A Streptococcus clone, but rather may arise anew under innate immune selection in individual patients.

Integrating neighbor clustering, coexpression clustering and subsystems analysis to define dynamic changes in regulatory networks associated with group A streptococcal sociomicrobiology and niche adaptation

Background Bacterial colonies often consist of heterogeneous communities rather than genetically identical cells with harmonized gene expression profiles[1]. Dramatic changes, such as the onset of infection, may perturb a colony’s sociomicrobiology leading a minor subpopulation with a mutant phenotype to prevail in the host; however, capturing such transitions in real time is difficult. While differential microarray analysis has become a method of choice for comparing the transcriptomes of bacterial subpopulations, current microarray analysis tools are more optimized to the study of eukaryotic organisms. Here, we set out to develop a systems biology model for studying the transcriptional reprogramming underlying the transition of M1T1 group A streptococci[2] from a virulent to a hypervirulent phenotype [3-5]. In addition, we aimed at integrating and optimizing microarray analysis strategies to better understand bacterial regulatory networks.

Streptococcal Inhibitor of Complement Promotes Innate Immune Resistance Phenotypes of Invasive M1T1 Group A Streptococcus

Streptococcal inhibitor of complement (SIC) is a highly polymorphic extracellular protein and putative virulence factor secreted by M1 and M57 strains of group A Streptococcus (GAS). The sic gene is highly upregulated in invasive M1T1 GAS isolates following selection of mutations in the covR/S regulatory locus in vivo. Previous work has shown that SIC (allelic form 1.01) binds to and inactivates complement C5b67 and human cathelicidin LL-37. We examined the contribution of SIC to innate immune resistance phenotypes of GAS in the intact organism, using (1) targeted deletion of sic in wild-type and animal-passaged (covS mutant) M1T1 GAS harboring the sic 1.84 allele and (2) heterologous expression of sic in M49 GAS, which does not possess the sic genein its genome. We find that M1T1 SIC production is strongly upregulated upon covS mutation but that the sic gene is not required for generation and selection of covS mutants in vivo. SIC 1.84 bound both human and murine cathelicidins and was necessary and sufficient to promote covS mutant M1T1 GAS resistance to LL-37, growth in human whole blood and virulence in a murine model of systemic infection. Finally, the sic knockout mutant M1T1 GAS strain was deficient in growth in human serum and intracellular macrophage survival. We conclude that SIC contributes to M1T1 GAS immune resistance and virulence phenotypes.

M1T1 group A streptococcal pili promote epithelial colonization but diminish systemic virulence through neutrophil extracellular entrapment

Group A Streptococcus is a leading human pathogen associated with a diverse array of mucosal and systemic infections. Cell wall anchored pili were recently described in several species of pathogenic streptococci, and in the case of GAS, these surface appendages were demonstrated to facilitate epithelial cell adherence. Here we use targeted mutagenesis to evaluate the contribution of pilus expression to virulence of the globally disseminated M1T1 GAS clone, the leading agent of both GAS pharyngitis and severe invasive infections. We confirm that pilus expression promotes GAS adherence to pharyngeal cells, keratinocytes, and skin. However, in contrast to findings reported for group B streptococcal and pneumococcal pili, we observe that pilus expression reduces GAS virulence in murine models of necrotizing fasciitis, pneumonia and sepsis, while decreasing GAS survival in human blood. Further analysis indicated the systemic virulence attenuation associated with pilus expression was not related to differences in phagocytic uptake, complement deposition or cathelicidin antimicrobial peptide sensitivity. Rather, GAS pili were found to induce neutrophil IL-8 production, promote neutrophil transcytosis of endothelial cells, and increase neutrophil release of DNA-based extracellular traps, ultimately promoting GAS entrapment and killing within these structures.

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.

Genetic Characterization and Virulence Role of the RALP3/LSA Locus Upstream of the Streptolysin S Operon in Invasive M1T1 Group AStreptococcus

Group A Streptococcus (GAS) is a leading human pathogen associated with a wide spectrum of mucosal and invasive infections. GAS expresses a large number of virulence determinants whose expression is under the control of several transcriptional regulatory networks. Here we performed the first mutational analysis of a genetic locus immediately upstream of the streptolysin S biosynthetic operon in several GAS genome sequences, including that of the M1T1 serotype, the leading isolates associated with serious invasive disease. The locus consists of a predicted RofA-like stand-alone transcriptional regulator (RALP3) and the largest open reading frame in the GAS genome, encoding a predicted LPXSG motif cell wall-anchored protein we have named LSA (for "large surface-anchored" protein). Comparative reverse transcription-PCR analysis of wild-type M1T1 GAS and an isogenic RALP3-deficient mutant identifies RALP3 as a global transcriptional regulator affecting expression of numerous virulence factor genes, including those for strong repression of the hyaluronic acid capsule and cysteine protease production. RALP3 contributed to GAS epithelial cell invasion and bloodstream survival. LSA was found to be under negative regulation by RALP3 and to influence GAS-epithelial cell interactions and GAS antimicrobial peptide sensitivity. Isogenic M1T1 GAS mutants lacking either RALP3 or LSA were attenuated in a murine model of systemic infection, indicating that this locus plays a role in the virulence potential of the organism.

Invasive M1T1 group A Streptococcus undergoes a phase-shift in vivo to prevent proteolytic degradation of multiple virulence factors by SpeB.

A globally disseminated strain of M1T1 group A Streptococcus (GAS) has been associated with severe infections in humans including necrotizing fasciitis and toxic shock syndrome. Recent clinicoepidemiologic data showed a striking inverse relationship between disease severity and the degree to which M1T1 GAS express the streptococcal cysteine protease, SpeB. Electrophoretic 2-D gel analysis of the secreted M1T1 proteome, coupled with MALDI-TOF mass spectroscopy, revealed that expression of active SpeB caused the degradation of the vast majority of secreted GAS proteins, including several known virulence factors. Injection of a SpeB+/SpeA- M1T1 GAS strain into a murine subcutanous chamber model of infection selected for a stable phase-shift to a SpeB-/SpeA+ phenotype that expressed a full repertoire of secreted proteins and possessed enhanced lymphocyte-stimulating capacity. The proteome of the SpeB-in vivo phase-shift form closely matched the proteome of an isogenic speB gene deletion mutant of the original M1T1 isolate. The absence or the inactivation of SpeB allowed proteomic identification of proteins in this M1T1 clone that are not present in the previously sequenced M1 genome including SpeA and another bacteriophage-encoded novel streptodornase allele. Further proteomic analysis of the M1T1 SpeB+ and SpeB- phase-shift forms in the presence of a cysteine protease inhibitor demonstrated differences in the expression of several proteins, including the in vivo upregulation of SpeA, which occurred independently of SpeB inactivation.

Reciprocal, temporal expression of SpeA and SpeB by invasive M1T1 group a streptococcal isolates in vivo.

The streptococcal pyrogenic exotoxins (Spes) play a central role in the pathogenesis of invasive group A streptococcal (GAS) infections. The majority of recent invasive GAS infections have been caused by an M1T1 strain that harbors the genes for several streptococcal superantigens, including speA, speB, speF, speG, and smeZ. However, considerable variation in the expression of Spe proteins among clonal M1 isolates has been found, and many of the speA-positive M1 strains do not produce detectable amounts of SpeA in vitro. This study was designed to test the hypothesis that speA gene expression can be induced in vivo. A mouse infection chamber model that allows sequential sampling of GAS isolates at various time points postinfection was developed and used to monitor the kinetics of Spe production in vivo. Micropore Teflon diffusion chambers were implanted subcutaneously in BALB/c mice, and after 3 weeks the pores became sealed with connective tissue and sterile fluid containing a white blood cell infiltrate accumulated inside the infection chambers. Representative clonal M1T1 isolates expressing no detectable SpeA were inoculated into the implanted chambers, and the expression of SpeA in the aspirated aliquots of the chamber fluid was analyzed on successive days postinfection. Expression of SpeA was detected in the chamber fluid as early as days 3 to 5 postinfection in most animals, with a significant increase in expression by day 7 in all infected mice. Isolates recovered from the chamber and grown in vitro continued to produce SpeA even after 21 passages in vitro, suggesting stable switch on of the speA gene. A temporal relation between the upregulation of SpeA expression and the downregulation of SpeB expression was observed in vivo. These data suggest that in vivo host and/or environmental signals induced speA gene expression and suppressed speB gene expression. This underscores the role of the host-pathogen interaction in regulating the expression of streptococcal virulence factors in vivo. The model described here should facilitate such studies.