What is the size of the group A streptococcal vir regulon? The Mga regulator affects expression of secreted and surface virulence factors

The vir regulon of group A streptococci (GAS) organizes the expression of several bacterial virulence factors under the control of the Mga regulator. Previously, the genes encoding the Mga regulator (mga), M and M-related proteins (emm, mrp, enn) and C5a peptidase (scpA) were reported to be clustered on the streptococcal genome in a core vir regulon. In the present study, the genomic regions of a serotype M49 strain upstream of mga and downstream of scpA were sequenced to assess the boundaries of the vir regulon. In the upstream region, an operon was identified that may be potentially involved in substrate transport and is independent from Mga regulation. In the downstream region, another Mga-controlled, scpA-cotranscribed gene was detected. This gene termed orfX encoded a 385-amino acid (aa) potential surface protein of unknown function. No binding of serum proteins to a recombinant ORFX was detectable and phagocytosis resistance of an orfX mutant remained unchanged. Downstream of orfX, another Mga-independent gene determined the 3′ end of the core vir regulon. Utilizing the M49 wild type, a mga ^– mutant and comparative Northern blot hybridization, genes encoding the capsule synthesis machinery, streptokinase and streptolysin O, as well as erythrogenic toxin A and DNase C were found to be Mga independent. In contrast, expression of the genes encoding the cysteine protease SpeB, streptococcin A and the oligopeptide permease was reduced in the mga ^– mutant. This indicated that in addition to the core vir regulon, Mga directly or indirectly controls a number of genes dispersed throughout the GAS genome. Received: 2 May 1996     

Lateral genetic transfers between group A and G streptococci for M-like genes are ongoing

Previously we described a long-polymerase chain reaction (PCR) method to amplify a 4–7 kb target containing most of the components of the vir regulon (mga,emm-like genes andscpA) in a number of group A streptococcus (GAS) isolates.¹In contrast to GAS, strains of human group G streptococcus (GGS) gave approximately 1.6 or 1.8 kb products. Sequence analysis of the amplified products issued from GGS templates revealed a mosaic consisting of upstream sequence frommga(the gene for positive regulator of vir regulon), an unidentified open reading frame, a short segment ofemm(the gene for M protein, an antiphagocytic molecule) and an upstream sequence ofscp(C5a-peptidase gene). A full lengthscpGis present immediately downstream from the mosaic segment in the human GGS genome. The GGS PCR fragment did not code formgaor full lengthemm. All human GGS isolates are known to code foremmbut the gene is separated fromscpGby at least 10 kb.²Our data, obtained using long-PCR and unrelated strains of GGS, confirm this. We could not detect a homologue ofmgain human GGS by hybridization analysis. The mosaic sequence suggests that enbloc transfer of the vir regulon from GAS to a GGS progenitor may have occurred, following which deletion and rearrangement events may have taken place. Partial nucleotide sequences ofemmcorresponding to the variable domain of M proteins from three local GGS isolates were determined. One sequence (emmGGS6) is 99% identical toemmfrom a geographicaly separated isolate of GGS recently described.³emmGGS6 also has significant homology withemmfrom a GAS strain (STDONALD) isolated from the same geographical area as was GGS6. The twoemmsequences (emmGGS6 andemmSTDONALD) revealed frameshift-compensatory frameshift mutations relative to each other, contributing to lower amino acid homology between the two predicted M proteins. SinceemmSTDONALD has no known relatives within the 80 or soemmsequences in the database, we speculate that it could have been laterally acquired from GGS. Horizontal transfers between GGS and GAS may be ongoing.     

Natural Variant of Collagen-Like Protein A in Serotype M3 Group A Streptococcus Increases Adherence and Decreases Invasive Potential

Group A Streptococcus (GAS) predominantly exists as a colonizer of the human oropharynx that occasionally breaches epithelial barriers to cause invasive diseases. Despite the frequency of GAS carriage, few investigations into the contributory molecular mechanisms exist. To this end, we identified a naturally occurring polymorphism in the gene encoding the streptococcal collagen-like protein A (SclA) in GAS carrier strains. All previously sequenced invasive serotype M3 GAS possess a premature stop codon in the sclA gene truncating the protein. The carrier polymorphism is predicted to restore SclA function and was infrequently identified by targeted DNA sequencing in invasive strains of the same serotype. We demonstrate that a strain with the carrier sclA allele expressed a full-length SclA protein, while the strain with the invasive sclA allele expressed a truncated variant. An isoallelic mutant invasive strain with the carrier sclA allele exhibited decreased virulence in a mouse model of invasive disease and decreased multiplication in human blood. Further, the isoallelic invasive strain with the carrier sclA allele persisted in the mouse nasopharynx and had increased adherence to cultured epithelial cells. Repair of the premature stop codon in the invasive sclA allele restored the ability to bind the extracellular matrix proteins laminin and cellular fibronectin. These data demonstrate that a mutation in GAS carrier strains increases adherence and decreases virulence and suggest selection against increased adherence in GAS invasive isolates.     

Shr is a broad-spectrum surface receptor that contributes to adherence and virulence in group A streptococcus

Group A streptococcus (GAS) is a common hemolytic pathogen that produces a range of suppurative infections and autoimmune sequelae in humans. Shr is an exported protein in GAS, which binds in vitro to hemoglobin, myoglobin, and the hemoglobin-haptoglobin complex. We previously reported that Shr is found in association with whole GAS cells and in culture supernatant. Here, we demonstrate that cell-associated Shr could not be released from the bacteria by the muralytic enzyme mutanolysin and was instead localized to the membrane. Shr was available, however, on the exterior of GAS, exposed to the extracellular environment. In vitro binding and competition assays demonstrated that in addition to hemoprotein binding, purified Shr specifically interacts with immobilized fibronectin and laminin. The absence of typical fibronectin-binding motifs indicates that a new protein pattern is involved in the binding of Shr to the extracellular matrix. Recombinant Lactococcus lactis cells expressing Shr on the bacterial surface gained the ability to bind to immobilized fibronectin, suggesting that Shr can function as an adhesin. The inactivation of shr resulted in a 40% reduction in the attachment to human epithelial cells in comparison to the parent strain. GAS infection elicited a high titer of Shr antibodies in sera from convalescent mice, demonstrating that Shr is expressed in vivo. The shr mutant was attenuated for virulence in an intramuscular zebrafish model system. In summary, this study identifies Shr as being a new microbial surface component recognizing adhesive matrix molecules in GAS that mediates attachment to epithelial cells and contributes to the infection process.     

The interaction between bacterial enolase and plasminogen promotes adherence of Streptococcus pneumoniae to epithelial and endothelial cells

Binding and conversion of the plasma protein plasminogen is an important pathogenesis mechanism of the human pathogen Streptococcus pneumoniae. Once converted into plasmin, the proteolytic activity of this major fibrinolysis component promotes degradation of extracellular matrix and the dissolution of fibrin clots. Here, we present the exploitation of plasminogen-binding as a further pivotal strategy of pneumococci facilitating adherence to eukaryotic cells. Flow cytometric measurements demonstrated the immobilization of plasminogen on host cell surfaces of human alveolar type II pneumocytes (A549), nasopharyngeal epithelium (Detroit 562) and brain-derived endothelial cells (HBMEC). These host-derived cells were employed in cell culture infection analyses followed by confocal microscopy to monitor the plasminogen-mediated adherence. Results of these studies revealed that host cell-bound plasminogen promotes pneumococcal adherence to human epithelial and endothelial cells in dose-dependent manner, whereas pneumococcal internalization was not enhanced. As an opposed effect pneumococcal-bound plasminogen reduced attachment to the epithelial and endothelial cells, and increased the interaction with neutrophil granulocytes. Moreover, the surface-displayed enolase, which serves as major pneumococcal plasminogen receptor, was identified as a key factor for plasminogen-mediated bacterial attachment in infection analyses with S. pneumoniae enolase mutants.     

Attenuation of virulence in multiple serotypes (M1, M3, and M28) of Group A Streptococcus after the loss of secreted esterase

IntroductionGroup A Streptococcus (GAS) can produce streptococcal secreted esterase (Sse), which inhibits neutrophil recruitment to the site of infection and is crucial for GAS pathogenesis. As an effective esterase, Sse hydrolyzes the sn-2 ester bond of human platelet-activating factor, inactivating it and abolishing its ability to recruit neutrophils.ObjectivesThe purpose of this study was to investigate the effects of sse deletion on the virulence of multiple serotypes of GAS.MethodsIsogenic strains that lack the sse gene (Δsse) were derived from the parent strains MGAS5005 (serotype M1, CovRS mutant), MGAS2221 (serotype M1, wild-type CovRS), MGAS315 (serotype M3, CovRS mutant) and MGAS6180 (serotype M28, wild-type CovRS) and were used to study the differences in virulence and pathogenicity of GAS serotypes.ResultsIn a subcutaneous infection model, mice infected with MGAS5005^Δsse exhibited higher survival rates but decreased dissemination to the organs compared with mice infected with MGAS5005. When mice were infected with the four Δsse mutants, the MPO activity and IFN-γ, TNF-α, IL-2 and IL-6 levels increased, but the skin lesion sizes decreased. In an intraperitoneal infection model, the absence of Sse significantly reduced the virulence of GAS, leading to increased mouse survival rates and decreased GAS burdens in the organs in most of the challenge experiments. In addition, the numbers of the four Δsse mutants were greatly reduced 60 min after incubation with isolated rat neutrophils.ConclusionOur results suggest that Sse participates in the pathogenesis of multiple GAS serotypes (MGAS5005, MGAS2221, MGAS315 and MGAS6180), particularly the hypervirulent CovS mutant strains MGAS5005 and MGAS315. These strain differences were positively correlated with the virulence of the serotype.     

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.     

Different alleles of the fcrA/mrp gene of Streptococcus pyogenes encode M-related proteins exhibiting an identical immunoglobulin-binding pattern

  The majority of group A streptococci (GAS, Streptococcus pyogenes) express immunoglobulin (Ig)-binding proteins. The genes encoding these proteins belong either to the emm or the emm-related (fcrA/mrp and enn) gene family and are located in close proximity on the GAS genome, where they form part of the vir regulon. In the present study analysis of sequence data of the 5′ terminal portions of the fcrA/mrp genes from GAS isolates representing 37 different M serotypes led to a classification of six different types. Thus, although fcrA/mrp genes exhibit an allelic polymorphism, they do not display the high degree of N-terminal sequence diversity found among emm genes. The nucleotide sequences of the fcrA/mrp genes from 3 GAS isolates, belonging to serotypes M8, M9, and M13 and representing newly characterized fcrA/mrp gene types, are reported. Analysis of the Ig-binding properties of recombinant FcrA/Mrp8, 9, and 13 proteins, demonstrated a similar Ig-binding profile being reactive with human IgG subclasses 1, 2, and 4. This pattern is identical to that previously described for other recombinant fcrA/mrp4, 49, 64/14 and 76 gene products, indicating that this property is not affected by the N-terminal variability. Evidence for recombination between an fcrA/mrp and an mga gene was observed in an M-type 33 strain isolate providing further support for the concept of gene rearrangement contributing to the diversity of vir regulon gene products. Received: 31 January 1996     

P27 (MBOV_RS03440) is a novel fibronectin binding adhesin of Mycoplasma bovis

Mycoplasma bovis, one of the major pathogens of bovine respiratory disease, binds to respiratory epithelial cells resulting in severe pneumonia and tissue damage. This study was designed to identify the adhesive function of a putative 27-kDa M. bovis lipoprotein, encoded by the gene MBOV_RS03440 and designated as P27. The gene was cloned and overexpressed to produce antibodies against the recombinant P27 (rP27). The western blot and flow cytometry assay confirmed P27 to be a surface-localized protein, while ELISA confirmed it to be an immunogenic protein. Confocal immunofluorescence microscopy demonstrated that rP27 bound to embryonic bovine lung (EBL) cell monolayers in a dose-dependent manner. Furthermore, anti-rP27 antiserum inhibited the attachment of M. bovis to EBL cells demonstrating the binding specificity of P27 to EBL cells. The attachment of rP27 to EBL cells was mediated by fibronectin (Fn), an extracellular matrix component. The interaction between rP27 and Fn was qualitatively and quantitatively monitored by ligand immunoblot assay, ELISA, and biolayer interferometry. Collectively, these results indicate that P27 is a novel Fn-binding, immunogenic adhesive protein of M. bovis, thereby contributing to the further understanding of the molecular pathogenesis of M. bovis.     

Cysteine protease SpeB expression in group A streptococci is influenced by the nutritional environment but SpeB does not contribute to obtaining essential nutrients

Group A streptococcal (GAS) cysteine protease is a major virulence factor involved in the pathogenesis of purulent and invasive infections. The secreted enzyme cleaves a number of different bacterial and host proteins which could contribute to different stages of the infective processes. It has been proposed that, among these functions, SpeB plays a role in obtaining nutrients during late growth phases. In the present study, speB mutants of various GAS serotypes were found to exhibit unaltered growth characteristics in several complex and chemically defined media (CDM). When amino acid-depleted CDM was prepared, neither SpeB activity on whole proteins added to the medium during incubation nor the addition of SpeB-digested proteins was able to support bacterial growth. SpeB also was unable to liberate iron from iron-containing protein sources added to iron-deficient CDM. However, SpeB levels in culture supernatants changed in response to the protein and glucose content of the media. Using a speB promoter-luciferase reporter, speB expression levels were found to correspond to peptide concentrations in the culture media. The effect appeared to be specific for peptides since addition of peptides derived from various proteins had an affect on expression, while addition of the whole proteins had no effect. Addition of glucose to CDM had no effect on speB expression, while glucose addition to complex medium decreased speB expression. Overall, SpeB did not appear to be directly involved in providing the bacteria with nutritional factors but expression of the speB gene responded to ratios of peptides and carbohydrates in the culture medium. Received: 10 April 1999