The immunopathogenesis of group A streptococcal skin disease
Embargoed until: 2019-04-12
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Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-‐positive bacterial pathogen exhibiting human host exclusivity. GAS is responsible for a wide array of non-‐ invasive suppurative infections of the throat and skin, such as pharyngitis and pyoderma respectively. GAS is also capable of causing severe invasive diseases and post-‐streptococcal complications, each with high rates of morbidity and mortality. Epidemiological studies have demonstrated a high prevalence of pyoderma in tropical regions such as in northern Australia. Streptococcal skin infections are thought to be a significant risk factor for the development of rheumatic fever in Indigenous Australians within these tropical regions. The skin is also a common portal of entry for invasive GAS disease. The ability of GAS to cause these invasive diseases relies upon the timely expression of specific virulence factors enabling the bacteria to evade host immune responses. Several important virulence factors are under the transcriptional control of the CovR/S operon. GAS strains possessing a mutation within their CovR/S regulatory system are particularly adept at evading host immunity resulting in hypervirulence and an increased capacity for invasive disease. CovR/S wild-‐type (WT) and mutant (MT) GAS pairs were identified from a panel of clinical and laboratory isolates. Each CovR/S MT assessed possessed a different mutation within their CovR or CovS gene. It was observed that mice immunised with the M protein-‐derived vaccine candidate J8-‐DT/Alum were protected from local and systemic infections by the CovR/S WT strains; however, protection was significantly compromised for all mice infected with the CovR/S MT strains. Analysis of gene expression via RT-‐PCR revealed that each of the CovR/S MT isolates were up-‐regulating specific virulence factors, namely streptolysin O (SLO), SpyCEP, and the hyaluronic acid capsule, that enhanced their immune evasion capabilities. The increased gene expression by CovR/S MT strains observed using in vitro assays with increased red blood cell lysis, increased IL-‐8 chemokine degradation, and a greater production of hyaluronic acid. The virulence of these CovR/S mutant strains was then tested in the context of the redesigned J8-‐DT vaccine (J8-‐DT combined with an inactive 20-‐mer fragment from SpyCEP, ‘S2’). The J8 CombiVax (comprising J8 and S2 with four lysine residues, ‘K4S2’) afforded significantly better protection compared to J8-‐DT, as each of the CovR/S MT strains were unable to cause a systemic infection in a murine model of pyoderma in the immunised cohorts. The combination of two minimal epitopes provided a synergistic effect through the opsonic J8-‐specific antibodies and the S2-‐ specific antibodies neutralizing SpyCEP, and thus preserving IL-‐8-‐mediated neutrophil chemotaxis. Further investigation into the altered virulence profile of CovR/S mutants underscored SLO as an essential virulence factor for the pathogenesis of these hypervirulent strains. Utilising the CovR/S mutant 5448 (5448 MT) as a representative M1T1 GAS isolate, we generated several additional CovR/S mutants lacking SLO (ΔSLO) to investigate the contribution of this toxin to GAS virulence. The up-‐regulation of SLO by 5448 MT resulted in increased SLO-‐mediated hemolysis, decreased dendritic cell (DC) viability post-‐infection, and an increased production of pro-‐inflammatory cytokines TNF and MCP-‐1 under in vitro conditions. Further to this, it was observed that when SLO was absent from the isolate, the viability of infected DCs improved whilst inflammatory cytokine production decreased. This was despite the observation that infecting isolates still exhibited the characteristic CovR/S mutant virulence factor up-‐regulation of SpyCEP and the hyaluronic acid capsule. Moreover, histological analyses showed that DC presence was restored in murine skin post-‐infection with 5448 MT if SLO was absent from the strain. The presence of SLO correlated with systemic infection and severe pathology at the site of infection in a murine model of pyoderma. Conversely, the absence of SLO significantly attenuated the virulence of 5448 MT in vivo. J8 CombiVax immunisation was effective against all CovR/S mutant infections and provided significant systemic protection. Neutrophils have been shown to be critical in controlling GAS infection, and also for the protective efficacy of J8 CombiVax. Therefore, we sought to investigate the main cellular source of CXCL2, the primary murine neutrophil chemoattractant (a homologue of human IL-‐8) and target of SpyCEP-‐mediated proteolysis. We used a clinical isolate (NS88.2 MT) sourced from the Northern Territory of Australia that possessed a natural CovS mutation, in parallel with the genetically repaired isogenic CovR/S wild-‐type strain (NS88.2 Rep). Following cutaneous infection in a murine model, we observed hypervirulence of the NS88.2 MT strain and differential interactions with neutrophils between the NS88.2 MT and NS88.2 Rep strains. The NS88.2 Rep strain was observed via immunofluorescence analysis interacting with neutrophils in skin sections in vivo and also being killed by human neutrophils in vitro. Conversely, NS88.2 MT appeared to inhibit neutrophil ingress in vivo and proliferated in the presence of human neutrophils in vitro. RT-‐PCR revealed that NS88.2 MT significantly up-‐regulated its SpyCEP expression compared to NS88.2 Rep providing a likely causative factor for the observed differential neutrophil interactions. To specifically target the up-‐regulated expression of SpyCEP, mice were immunised with K4S2-‐DT/Alum. Whilst there was no significant protection against NS88.2 Rep or MT infection with K4S2 alone, spleen samples of immunised mice showed evidence of germinal centre formation post-‐infection, particularly in the context of NS88.2 MT, which was consistent with immunological boosting of K4S2-‐specific B cells by MT infection. Investigation into the cellular response of infection using intracellular staining (ICS) revealed that neutrophils were the primary source of CXCL2 in the skin. This in turn, enabled further recruitment of neutrophils to the site of the infection. This was highlighted by the significant increase in neutrophil abundance in K4S2-‐immunised cohorts compared to the non-‐immunised cohorts, suggesting that protection against SpyCEP-‐mediated cleavage through K4S2 antibodies in vivo contributes to the adaptive immune response against GAS skin infections. ICS also revealed that CXCL2+ve neutrophils were more abundant in the skin of CovR/S WT-‐infected mice compared to the skin of mice infected with the CovR/S MT NS88.2. Similarly, significantly higher levels of CXCL2 protein were found in the skin of mice infected with the CovR/S WT compared to those infected with the CovR/S MT by day 3 post-‐infection. The degradation of CXCL2 in vitro by NS88.2 Rep or NS88.2 MT supernatant was effectively inhibited by the addition of K4S2 anti-‐sera. Overall, the studies within this thesis highlight the hypervirulent nature of CovR/S mutant GAS and some of the mechanisms by which each strain can evade the host immune system. Gaining a greater understanding of host-‐pathogen interactions during GAS infections has enabled improved vaccine design strategies, such as the generation of vaccine candidate J8 CombiVax from J8-‐DT/Alum. J8, being a highly conserved cryptic epitope, in synergy with a 20-‐mer epitope from SpyCEP, provides protective coverage against GAS by neutralizing two of their most important virulence factors.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
Institute for Glycomics
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