Doctor of Philosophy
Illawarra Health and Medical Research Institute
Tsatsaronis, James Anthony, Characterisation of group A streptococcal innate immune resistance and host response mechanisms, Doctor of Philosophy thesis, Illawarra Health and Medical Research Institute, University of Wollongong, 2014. http://ro.uow.edu.au/theses/4125
Group A Streptococcus (GAS, Streptococcus pyogenes) is a Gram-positive bacterial pathogen responsible for life-threatening, invasive human diseases, including necrotising fasciitis and streptococcal toxic shock syndrome. In the last 30 years, a global increase in the rates of severe and fatal forms of GAS infection has been noted. Coinciding with this epidemiological trend has been a pandemic spread of particularly hypervirulent GAS, notably a clone of the M1T1 serotype. Studies investigating this serotype highlight the unique genetic makeup of M1T1 GAS and its interactions with the host innate immune response as key elements coordinating the virulence of this serotype. Phage-mediated horizontal gene transfer and selective pressure exerted by polymorphonuclear leukocytes (PMNs) for mutations in the control of virulence regulator operon (covRS) bestow M1T1 GAS with enhanced pathogenic capacity. However, isolates of virulent GAS also occur in genetic backgrounds lacking phageencoded factors, such as sda1, and other M1T1-specific core genomic elements necessary for covRS mutation. In this study, high-throughput next-generation sequencing was utilised to generate a draft genome sequence of NS88.2, an emm98.1 GAS isolate exhibiting a hypervirulent and PMN resistant phenotype. The multi-locus sequence type (MLST) was determined from the NS88.2 draft genome sequence, which was compared to fully sequenced GAS strains by whole genome BLAST alignment. NS88.2 was found to have a MLST sequence type of 205, and regions of nucleotide sequence divergence between NS88.2 and other GAS included the multiple gene regulator (MGA) and fibronectin, collagen and T-antigen (FCT) loci. The NS88.2 MGA locus showed low sequence similarity to GAS of other emm-types, and the FCT locus corresponded to an FCT type 3. A ~30 kb region containing putative prophagelike elements constituted the majority of NS88.2 novel sequence data, however interrogation of these elements did not reveal novel genes with obvious roles in modulating PMN responses. Bioinformatic prediction of genes encoding cell surface and secreted proteins was conducted, and identified 10 putative cell surface proteins and 190 putative proteins with secretion signal peptides. The addition of this genome draft to public databases will facilitate other studies of GAS genome biology, and further in-depth analyses of the NS88.2 isolate described here.
The phage-encoded extracellular DNase streptodornase 1 (Sda1) has previously been shown as essential for M1T1 GAS acquisition of covRS mutations. Mutations of covRS in M1T1 GAS bestow hypervirulent and PMN resistant qualities. These phenotypes are also observed in the NS88.2 isolate, which encodes a truncated, non-functional covS gene, but not the sda1 gene. In this study, NS88.2 and derivative strains with intact covS (NS88.2rep) or reverse complemented inactive covS (NS88.2covS) were examined to determine potential novel mechanisms by which these strains interact with the innate immune response. Phenotypic comparison of NS88.2, NS88.2rep and NS88.2covS to M1T1 GAS demonstrated that similar to M1T1 GAS, mutation of covS impairs the ability of NS88.2 and NS88.2covS to adhere to HEp-2 cells in vitro, murine skin in vivo, and to biofilm production relative to NS88.2rep. The ability of NS88.2 strains to degrade neutrophil extracellular traps (NETs) was determined, and in congruence with previously described genome data describing the absence of sda1, these strains showed significantly reduced ability to degrade NETs over virulent M1T1 GAS. In extension to the NS88.2 genomic sequence draft, proteomic and transcriptomic approaches utilising 2D-PAGE and transcriptional microarray analyses were undertaken. Proteomics revealed that covS-mutation lead to increased amounts of streptococcal collagen-like protein A (SclA), general stress protein 24 (Gls24) and other known or potential virulence factors in culture supernatants. Analysis of the NS88.2 transcriptome demonstrated that sclA was upregulated by covS mutation, and that gls24 was highly upregulated in response to incubation in whole blood. NS88.2 isogenic deletion mutants of sclA and gls24 exhibited significantly reduced ability to proliferate in whole blood, and reduced ability to resist killing by purified PMNs. This study describes genomic, transcriptomic and proteomic characterisation of a non-M1T1 GAS isolate, and identification of a novel role for two GAS virulence determinants in modulating interactions with PMNs.
Infection of PMNs with M1T1 GAS rapidly triggers an apoptotic cell death program, however the impact of differentially PMN-resistant GAS strains on PMN cell death responses is unclear. In this study, the interactions of PMNs with PMN-resistant (NS88.2) and PMN-sensitive (NS88.2rep) GAS were characterised to determine the effect on PMN cell death modality. In vitro infection of purified PMNs demonstrated that PMNs phagocytosed significantly less virulent NS88.2 than avirulent NS88.2rep and subsequently generated less reactive oxygen species. PMNs that had phagocytosed NS88.2rep also underwent a higher degree of mitochondrial membrane depolarisation than NS88.2. Scanning electron microscopy of NS88.2rep infected PMNs indicated morphological signs of apoptotic cell death, which was supported by biochemical evidence of nuclear DNA fragmentation and caspase-3 activation. Conversely, apoptotic markers were absent in NS88.2 infected PMNs, which displayed ultrastructural markers of oncotic cell death and loss of plasma membrane integrity. Intradermal infection of C57BL/6 mice determined that phagocytosis of NS88.2 was impaired in vivo, which contributed to the survival and virulence of this strain. Immunohistological analysis of murine dermal tissue showed that infection by NS88.2rep lead to migration of murine PMNs, and cells associated with infection stained strongly for apoptotic markers associated with a pro-resolution of inflammation phenotype. Murine dermal tissue infected with NS88.2 also exhibited murine PMN influx, however was absent for apoptotic markers and exhibited adverse histopathologies. This study indicates that the manner of PMN cell death program induced by GAS infection may enhance GAS virulence and affect disease pathologies. The work described here illustrates interactions between the human innate immune response and GAS in a non-M1T1 genetic background. These findings emphasise heterologous mechanisms by which GAS of different serotypes resist killing by PMNs, and how PMN cell death responses are shaped by virulent and avirulent GAS. A deepened understanding of both host and GAS cellular and molecular interactions during infection holds high potential to identify novel targets that can be exploited for the development of improved diagnostics and therapeutics for GAS diseases.