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Molecular Mechanisms Controlling Virulence Factor Gene Expression in the Group A Streptococcus
Cell and Molecular Biology
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The group A Streptococcus (GAS or Streptococcus pyogenes) has a remarkable ability to cause a large variety of human diseases ranging from mild illnesses to serious infections. GAS is the most common cause of bacterial pharyngitis (>600 million infections annually) and a leading cause of superficial skin infections (>100 million infections annually). More severe diseases include necrotizing fasciitis or the flesh-eating syndrome (25 to 50% mortality rate). Also included in the spectrum, are diseases that are toxin-mediated (e.g. toxic shock syndrome) and diseases that are auto-immune in nature and arise post-infection (e.g. acute rheumatic fever or ARF). The consequences of this disease burden are the deaths of >500,000 people per year, globally. Continuing increase in morbidity and mortality caused by GAS, significant health care financial burden, and increasing levels of antibiotic resistance, highlight the need for a more detailed understanding of the molecular strategies used by this pathogen to circumvent the host immune response and cause disease. GAS strains are divided into different serotypes based upon the sequence of the 5’ end of the emm gene. That some GAS serotypes show non-random associations with certain disease manifestations has been known for more than half a century. For example, serotype M28 strains are associated with cases of puerperal sepsis, M3 strains are associated with lethal invasive infections, and M18 strains are associated with outbreaks of ARF. Although these associations are long known, the molecular mechanisms behind them are yet to be fully understood. The work in this dissertation provides new perspectives about the molecular bases of non-random associations of M3 and M28 GAS serotypes with severe invasive infections and puerperal sepsis, respectively. For M3s, this work mainly focuses on characterizing RocA, the regulatory accessory protein of the control of virulence or CovR/S two-component system. The CovR/S system serves mainly as a negative regulator of gene expression, with the membrane-spanning sensor kinase, CovS, and the response regulator, CovR, together regulating the transcription of ∼10% of GAS genes, including almost two dozen that encode immunomodulatory virulence factors. Abundance of active (i.e. phosphorylated [~P]) CovR increases in the presence of a functional RocA. Here, we show that RocA is a pseudokinase that increases the levels of CovR∼P indirectly, possibly by enhancing the CovS-mediated phosphorylation of CovR, with this occurring as a consequence of RocA-CovS interactions via their membrane-spanning domains. Importantly, “hyper-virulent” strain derivatives harboring mutations in covR, covS, or rocA are positively selected for during invasive GAS infections, resulting in the enhanced expression of immunomodulatory virulence factors and subsequently an enhanced ability to inhibit neutrophil-mediated GAS killing. We have identified that there is a positive correlation between the levels of CovR~P (parental> rocA mutant> covS mutant> covR mutant) and GAS survival during upper respiratory tract infections, which is contrary to the previously-identified negative correlation between CovR~P levels and GAS survival during invasive infections. We propose that covR and covS mutant strains are sufficiently attenuated in their ability to cause upper respiratory tract infections, which represent majority of the infections caused by GAS, that they are not maintained within a population. In contrast, the negative impact of rocA mutation on the ability to cause pharyngeal infections is not as severe. This finding may explain the observation that while some GAS serotypes (i.e. M3 and M18) consist exclusively of rocA mutant strains, no serotypes are exclusively covR or covS mutants. For M28s, we show that a pathogenicity island of apparent group B Streptococcus (GBS) origin, RD2, modifies the virulence potential of M28, directly via encoded virulence factors and indirectly via encoded regulatory proteins. RD2 is 36.3 kb long, encodes seven putative virulence factors that are likely to participate in host-pathogen interactions such as cell adhesion, and shares significant homology to two chromosomal regions of multiple GBS isolates. GBS isolates are most commonly associated with neonatal invasive infections and puerperal sepsis. Thus, we hypothesize that acquisition of RD2 from GBS contributes majorly to the decades old association of M28 GAS isolates with cases of puerperal sepsis. In this work, we have shown that RD2 enhances the ability of M28 GAS to adhere to human vaginal epithelial cells, and to colonize the female reproductive tract in a mouse model of infection. In addition, RD2 influences the abundance of mRNAs from more than 100 core chromosomal GAS genes. Thus, our data is consistent with RD2 playing a key role in the association between serotype M28 GAS isolates and puerperal sepsis cases. Overall, this project highlights the importance of regulatory gene mutation and horizontal gene transfer as molecular mechanisms that control virulence factor expression in GAS.