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Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.

Group A Streptococcus (GAS; Streptococcus pyogenes ) causes a wide range of infections, including pharyngitis, impetigo, and necrotizing fasciitis, and results in over half a million deaths annually. GAS ScpC (SpyCEP), a 180-kDa surface-exposed, subtilisin-like serine protease, acts as an essential virulence factor that helps S. pyogenes evade the innate immune response by cleaving and inactivating C-X-C chemokines. ScpC is thus a key candidate for the development of a vaccine against GAS and other pathogenic streptococcal species. Here, we report the crystal structures of full-length ScpC wild-type, the inactive mutant, and the ScpC-AEBSF inhibitor complex. We show ScpC to be a multi-domain, modular protein consisting of nine structural domains, of which the first five constitute the PR + A region required for catalytic activity. The four unique C-terminal domains of this protein are similar to collagen-binding and pilin proteins, suggesting an additional role for ScpC as an adhesin that might mediate the attachment of S. pyogenes to various host tissues. The Cat domain of ScpC is similar to subtilisin-like proteases with significant difference to dictate its specificity toward C-X-C chemokines. We further show that ScpC does not undergo structural rearrangement upon maturation. In the ScpC-inhibitor complex, the bound inhibitor breaks the hydrogen bond between active-site residues, which is essential for catalysis. Guided by our structure, we designed various epitopes and raised antibodies capable of neutralizing ScpC activity. Collectively, our results demonstrate the structure, maturation process, inhibition, and substrate recognition of GAS ScpC, and reveal the presence of functional domains at the C-terminal region., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In the last two decades an increasing number of local outbreaks of invasive group A streptococcus (GAS) infections including necrotizing fasciitis (NF) have been reported. We identified the streptococcal invasion locus (sil) which is essential for virulence of the M14 strain JS95 isolated from an NF patient. This locus contains six genes: silA/B and silD/E encoding two-component system (TCS) and ABC transporter, respectively, homologous to the corresponding entities in the regulon of Streptococcus pneumoniae involved in genetic competence. Situated between these two units are silC and silCR, which highly overlap and are transcribed from the complementing strand at opposite directions. SilCR is a putative competence stimulating peptide, but in the M14 strain it has a start codon mutation. Deletion of silC or addition of synthetic SilCR attenuates virulence of the M14 strain. Here we found that silC and silCR form a novel regulatory circuit that controls the sil locus transcription. Under non-inducing conditions silC represses the silCR promoter. Externally added SilCR peptide activates the TCS, which in turn stimulates silCR transcription. Ongoing silCR transcription mediates the repression of the converging and overlapping silC transcript. Transcription of bacteriocin-like peptide (blp) operon mirrors the inverse relationships between the silC and silCR transcripts. It is upregulated by either addition of SilCR or deletion of silC. Moreover, expression of silC from a plasmid in a silC deleted-mutant significantly represses blp transcription. Finally, we show that 18% of clinically relevant GAS isolates possess sil and produce SilCR. Based on these results we propose a working model for regulation gene expression and virulence in GAS by the SilCR signalling peptide.

Group A streptococcus (GAS) causes diseases ranging from benign to severe infections such as necrotizing fasciitis (NF). The reasons for the differences in severity of streptococcal infections are unexplained. We developed the polymorphic-tag-lengths-transposon-mutagenesis (PTTM) method to identify virulence genes in vivo. We applied PTTM on an emm14 strain isolated from a patient with NF and screened for mutants of decreased virulence, using a mouse model of human soft-tissue infection. A mutant that survived in the skin but was attenuated in its ability to reach the spleen and to cause a lethal infection was identified. The transposon was inserted into a small open reading frame (ORF) in a locus termed sil, streptococcal invasion locus. sil contains at least five genes (silA-E) and is highly homologous to the quorum-sensing competence regulons of Streptococcus pneumoniae. silA and silB encode a putative two-component system whereas silD and silE encode two putative ABC transporters. silC is a small ORF of unknown function preceded by a combox promoter. Insertion and deletion mutants of sil had a diminished lethality in the animal model. Virulence of a deletion mutant of silC was restored when injected together with the avirulent emm14-deletion mutant, but not when these mutants were injected into opposite flanks of a mouse. DNA transfer between these mutants occurred in vivo but could not account for the complementation of virulence. DNA exchange between the emm14-deletion mutant and mutants of sil occurred also in vitro, at a frequency of approximately 10-8 for a single antibiotic marker. Whereas silC and silD mutants exchanged markers with the emm14 mutant, silB mutant did not. Thus, we identified a novel locus, which controls GAS spreading into deeper tissues and could be involved in DNA transfer.

A mini-Tn5 transposon derivative, mini-Tn5cyaA', has been constructed. It contains a promoter-less and ribosome binding site-deficient reporter gene, encoding the catalytic domain of Bordetella pertussis adenylate cyclase toxin (CyaA'). We used this system to mutagenize B. bronchiseptica and we developed a screen for identification of mutants containing cyaA' translational fusions. This system was used to identify B. bronchiseptica genes that encode surface-exposed and secreted proteins.

The M protein of group A streptococcus (GAS) is considered to be a major virulence factor because it renders GAS resistant to phagocytosis and allows bacterial growth in human blood. There are more than 80 known serotypes of M proteins, and protective opsonic antibodies produced during disease in humans are serotype specific. M proteins also mediate bacterial adherence to epithelial cells of skin and pharynx. GAS strains vary in the genomic organization of the mga regulon, which contains the genes encoding M and M-like proteins and other virulence factors. This diversity of organization makes it difficult to assess virulence of M proteins of different serotypes, unless they can be expressed in an isogenic background. Here, we express M proteins of different serotypes in the M protein- and protein F1-deficient GAS strain, SAM2, which also lacks M-like proteins. Genes encoding M proteins of different serotypes (emmXs) have been integrated into the SAM2 chromosome in frame with the emm6.1 promoter and its mga regulon, resulting in similar levels of emmX expression. Although SAM2 exhibits a very low level of adherence to and invasion of HEp-2 and HaCaT cells, a SAM2-derived strain expressing M6 protein adheres to and invades both cell types. In contrast, the isogenic strain expressing M18 protein adheres to both cell types, but invades with a very low efficiency. A strain expressing M3 protein adheres to both types of cells, but its invasion of HEp-2 cells is serum dependent. A GAS strain expressing M6 protein does not compete with the isogenic strain expressing M18 protein for adherence to or invasion of HaCaT cells. We conclude that M proteins of different serotypes recognize different repertoires of receptors on the surfaces of eukaryotic cells.

Adenylate cyclase toxin (CyaA) of Bordetella pertussis belongs to the RTX family of toxins. These toxins are characterized by a series of glycine- and aspartaterich nonapeptide repeats located at the C-terminal half of the toxin molecules. For activity, RTX toxins require Ca2+, which is bound through the repeat region. Here, we identified a stretch of 15 amino acids (block A) that is located C-terminally to the repeat and is essential for the toxic activity of CyaA. Block A is required for the insertion of CyaA into the plasma membranes of host cells. Mixing of a short polypeptide composed of block A and eight Ca2+ binding repeats with a mutant of CyaA lacking block A restores toxic activity fully. This in vitro interpolypeptide complementation is achieved only when block A is present together with the Ca2+ binding repeats on the same polypeptide. Neither a short polypeptide composed of block A only nor a polypeptide consisting of eight Ca2+ binding repeats, or a mixture of these two polypeptides, complement toxic activity. It is suggested that functional complementation occurs because of binding between the Ca2+ binding repeats of the short C-terminal polypeptide and the Ca2+ binding repeats of the CyaA mutant lacking block A.

Enteropathogenic Escherichia coli (EPEC) induces formation of actin pedestals in infected host cells. Agents that inhibit the activity of Rho, Rac, and Cdc42, including Clostridium difficile toxin B (ToxB), compactin, and dominant negative Rho, Rac, and Cdc42, did not inhibit formation of actin pedestals. In contrast, treatment of HeLa cells with ToxB inhibited EPEC invasion. Thus, Rho, Rac, and Cdc42 are not required for assembly of actin pedestals; however, they may be involved in EPEC uptake by HeLa cells.

Group A streptococci were recently shown to be capable of invading human epithelial cell monolayers. Cell invasion might be an important virulence trait of streptococci that enable the pathogen to gain entry into deeper tissues after initial binding to host cells. Nothing is known concerning the nature of streptococcal components that mediate invasion. Using isogenic mutants of strain JRS4 that are defective in the expression of either M6 protein or protein F1, or both proteins, it was demonstrated that both adhesins are required for efficient invasion. Further more, expression of protein F1 on the surface of a non-invasive strain rendered the latter invasive, suggesting that protein F1 is directly involved in the invasion process.

The antiphagocytic effect of M protein has been considered a critical element in virulence of the group A streptococcus. The hyaluronic acid capsule also appears to play an important role: studies of an acapsular mutant derived from the mucoid or highly encapsulated M protein type 18 group A streptococcal strain 282 indicated that loss of capsule expression was associated with decreased resistance to phagocytic killing and with reduced virulence in mice. To study directly the relative contributions to virulence of M protein and the hyaluronic acid capsule in strain 282, we inactivated the gene encoding the M protein (emm18) both in wild-type strain 282 and in its acapsular mutant, strain TX72. Inactivation of emm18 was accomplished by integrational plasmid mutagenesis, using the temperature-sensitive shuttle vector pJRS233 harboring a 5' DNA segment of emm18. As reported previously, wild-type strain 282 was resistant to phagocytic killing in vitro, both in whole human blood and in 10% serum. The capsule mutant TX72 was highly susceptible to phagocytic killing in 10% serum and moderately sensitive in whole blood. The M protein mutant 282KZ was highly susceptible to phagocytic killing in blood but only moderately sensitive in 10% serum. The double mutant TX74 was sensitive to killing in both conditions. In a mouse infection model, the 50% lethal dose was increased by 60- and 80-fold for the capsule and double mutants, respectively, compared with that of strain 282, but only by 6-fold for the M protein mutant. Integration of the strain 282 capsule genes into the chromosome of a nonmucoid M1 strain resulted in high-level capsule production and rendered the transformed strain resistant to phagocytic killing in 10% serum. These results provide further evidence that the hyaluronic acid capsule confers resistance to phagocytosis and enhances group A streptococcal virulence. The results suggest also that assessment of in vitro resistance to phagocytosis in 10% serum rather than in whole blood may be a more accurate reflection of virulence in vivo of group A streptococci.

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)-binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn-binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene (prtF2) from GAS strain 100076 encoding a novel Fn-binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high-affinity Fn binding. prtF2-related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn-binding protein in GAS. Protein F2 is highly homologous to Fn-binding proteins from Streptococcus dysgalactiae and Streptococcus equisimilis, particularly in its carboxy-terminal portion. Two domains are responsible for Fn binding by protein F2. One domains (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non-repeated stretch of approximately 100 amino acids and is located 100 amino acids aminoterminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.

In a previous study we reported the identification of protein F, a fibronectin-binding protein that was essential to the ability of Streptococcus pyogenes JRS4 to adhere to respiratory epithelial cells (E. Hanski and M. Caparon, Proc. Natl. Acad. Sci. USA, 89:6172-6176, 1992). To further evaluate the role of protein F in the adherence of the group A streptococci, we screened other group A streptococcal strains, including six recent clinical isolates, and one strain of Enterococcus faecalis for their capacity to bind fibronectin and for the presence of the gene encoding protein F (prtF). Seven of eight group A streptococcal strains analyzed, including all recent clinical isolates, both bound fibronectin at high affinity and contained DNA sequences that hybridized with a prtF-specific probe. One group A streptococcal isolate and the strain of E. faecalis examined neither contained a prtF-related gene nor bound fibronectin. These two strains also could not efficiently adhere to respiratory epithelial cells. However, upon the introduction of the cloned prtF gene, both of these strains gained the capacity to bind fibronectin and to adhere to respiratory epithelial cells. These results suggest that protein F is an important adhesin, which may have a general role in the virulence of the group A streptococci.

Treatment of intact hepatocytes with islet-activating protein, from Bordatella pertussis, led to a pronounced increase in the ability of glucagon to raise intracellular cyclic AMP concentrations. Islet-activating protein, however, caused no apparent increase in the intracellular concentration of cyclic AMP under basal conditions. These effects were attributed to an enhanced ability of adenylate cyclase, in membranes from hepatocytes treated with islet-activating protein, to be stimulated by glucagon. When forskolin was used to amplify the basal adenylate cyclase activity, elevated GTP concentrations were shown to inhibit adenylate cyclase activity in membranes from control hepatocytes. This inhibitory effect of GTP was abolished if the hepatocytes had been pre-treated with islet activating protein. In isolated liver plasma membranes, islet-activating protein caused the NAD-dependent ribosylation of a Mr-40000 protein, the putative inhibitory guanine nucleotide regulatory protein, Ni. This effect was inhibited if guanosine 5'-[beta-thio]diphosphate rather than GTP was present in the ribosylation incubations. The ability of glucagon to uncouple or desensitize the activity of adenylate cyclase in intact hepatocytes was also blocked by pre-treating hepatocytes with islet-activating protein. Islet-activating protein thus heightens the response of hepatocytes to the stimulatory hormone glucagon. It achieves this by both inhibiting the expression of desensitization and also removing a residual inhibitory input expressed in the presence of glucagon.