An Analysis of Serotype-Specific Virulence in the Group A Streptococcus
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Authors
Roshika, Roshika
Issue Date
2023
Type
Dissertation
Language
Keywords
Group A Streptococcus , M28 , M3 , Pilus , Puerperal Sepsis , RD2
Alternative Title
Abstract
Group A Streptococcus (GAS or Streptococcus pyogenes) is a Gram-positive coccus responsible for a wide range of human diseases, from mild infections such as pharyngitis/strep throat to invasive infections such as puerperal sepsis and necrotizing fasciitis. GAS results in nearly 500,000 annual fatalities, with invasive infections and acute rheumatic fever/rheumatic heart disease (ARF/RHD) being the primary causes of death. The burden of invasive GAS diseases is high, with ~650,000 cases and ~150,000 deaths annually. GAS strains are divided into more than 200 emm-types/M-types/serotypes based on the molecular typing of the sequence of the hypervariable N-terminal region of the primary cell surface protein called the M-protein. Over the past several decades, epidemiological studies have established associations between specific serotypes (M-types) and certain GAS infections like serotype M28 with puerperal sepsis, serotype M1 with the resurgence in severe invasive GAS infections observed since the mid-1980s, and serotype M3 with the lethal invasive infections. Nevertheless, the molecular mechanisms behind these associations are yet to be fully understood. Fundamental questions include: Does the presence or absence of certain genes in specific serotypes affect disease outcomes? Does serotype-specific gene expression play a role? This dissertation explores three main areas: i) The impact of the region of difference 2 (RD2), a genomic island, acquisition on the virulence of serotypes other than M28. ii) The potential role of the capsule as a barrier to RD2 acquisition in other serotypes. iii) The molecular basis for the low nra expression in M3 GAS serotype. The aforementioned areas are expected to shed new light on how serotypic gene expression and gene content lead to varying diseases/virulence outcomes.For the RD2 gain-of-function study, we show that the virulence-altering ability of RD2 is not restricted to M28 GAS isolates but also applies to isolates of other GAS serotypes. RD2, a mobile genetic element, is present in all M28 GAS isolates but absent in most of the other GAS serotypes. M28 GAS isolates are non-randomly associated with severe/lethal cases of puerperal sepsis. RD2 is of apparent group B Streptococcus (GBS) origin and is 36.3 kb long, encoding seven putative extracellular proteins that are possibly involved in establishing host-pathogen interactions, such as adhesion and colonization of the host. RD2 shares significant homology with two chromosomal regions of GBS. Notably, GBS isolates are associated with invasive neonatal infections and puerperal sepsis. Hence, we hypothesized that RD2 is the factor that non-randomly associates M28 GAS isolates with cases of puerperal sepsis. Previous studies have associated RD2 with enhanced adherence and colonization abilities in M28 GAS isolates, directly or indirectly, through encoded virulence factors or regulatory proteins. In this work, we have shown that RD2 enhances the adherence and colonization abilities of GAS isolates and the blood survival ability of isolates in a strain/serotype-specific manner. RD2 also influences the GAS transcriptome in a strain/serotype-specific manner, suggesting that this strain/serotype-specific virulence modulation by RD2 may determine GAS isolates' ability to establish infection in the host. Hence, this could be why RD2 is not ubiquitous in GAS serotypes. For RD2 and the barrier to its acquisition study, we study capsule as a factor that may be a potential barrier to the acquisition of RD2 by other GAS serotypes in nature. Our research shows that the presence and absence of the capsule do not affect the intra- or inter-serotype conjugation frequency. On investigating the role of capsule on RD2-mediated phenotypes using derivatives of M1 and M28 strains, we observed that while the presence of capsule does influence the RD2-mediated adherence ability of GAS isolates, there is no significant difference in the colonization ability of strains with RD2 in the presence and absence of a capsule. However, RD2-containing strains showed a significantly higher colonization ability than RD2-lacking strains. Therefore, our current study suggests that the capsule does not act as a barrier to RD2 acquisition, at least in the case of M1 isolates. For M3s, our work uncovers the molecular basis of a lower abundance of Nra, a positive regulator of pilus expression in the M3 GAS serotype, compared to the M1 GAS serotype. Pili are hair-like structures on bacterial cell surfaces that help organisms adhere, colonize, and establish host infection. The CovR/S system is a two-component regulatory system that negatively regulates virulence gene expression. CovS is a sensor kinase, and CovR is the response regulator, jointly regulating the transcription of approximately 10% of GAS genes. Our previous studies showed that nra gene expression and pili production are lower in M3s compared to M49s and M1s (Note that M1 has RofA, and not Nra, as a positive regulator of pilus gene expression). In this work, we conclude that the lower nra expression and pili abundance in M3s are a cumulative result of a point mutation in the nra gene and the repression of pilus gene expression by the CovR/S system. In addition, we also show that RofA and Nra are not interchangeable in function. Therefore, our study shows that the repression of pilus genes by CovR/S and expression of these genes occur in a strain- or type-specific manner. In summary, our work provides insight into strain- and serotype-specific variation in regulating virulence genes, particularly those related to adherence, colonization, and infection establishment in group A Streptococcus. This research contributes to developing prophylactic measures for puerperal sepsis and enhances our understanding of the variation in pilus expression, a potential vaccine candidate, across GAS serotypes.