Regulatory and Virulence Mechanisms in the Human Bacterial Pathogen the Group A Streptococcus: Dissecting sRNA Function and Mobile Genetic Element Dynamics
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Authors
Baral, Sushila
Issue Date
2025
Type
Dissertation
Language
en_US
Keywords
FasBCA Protein Based Regulators , FasBCAX Regulatory System , FasX Small Regulatory RNA , Group A Streptococcus , Puerperal Sepsis , RD2
Alternative Title
Abstract
The group A Streptococcus (aka GAS, Streptococcus pyogenes) is a Gram-positive bacterium that only infects humans. Normally, this bacterium resides as a commensal organism in the human throat and skin. As a pathogen, it infects a wide variety of anatomical sites in the human body. Infections caused by GAS can be mild (tonsilitis, pharyngitis) as well as severe (puerperal sepsis, toxic shock syndrome and necrotizing fasciitis) with life threatening conditions. In addition, there are various post streptococcal immune sequelae associated with this pathogen like rheumatic fever, rheumatic heart disease, acute glomerulonephritis, etc., which may develop during untreated cases of mild infections or due to repeated infections by the bacterium. As we see from the disease burden data around the globe, GAS is responsible for more than 600 million infections with more than half a million deaths annually. Based on the number of deaths involved, WHO has listed GAS as the ninth most infectious cause of human mortality. Despite the effort from hundreds of years, there is no safe and effective vaccine against GAS. While GAS remains sensitive to penicillin, many published studies have proved the rise in resistance to other antibiotics used in GAS disease treatment. GAS isolates are classified into more than 200 serotypes based on the N terminal hypervariable region of M protein. There is difference in geographical distribution of the GAS serotypes with some serotypes being more prevalent in one region compared to the others. Also, due to genomic variability, different GAS serotypes/strains exhibit different virulence characteristics influencing the disease outcome. The above facts highlight the need to study the molecular mechanisms used by this pathogen to cause disease.
Disease manifestations following GAS infections are influenced by the combined action of various virulence factors whose expression is under the control of several regulatory systems that include two component systems, stand-alone transcriptional regulators and small non-coding regulatory RNAs. Understanding the molecular mechanisms behind the production of various virulence factors will help in the development of vaccine candidates and therapeutic approaches against infections caused by this pathogen. This dissertation includes work that has been done with the central aim of exploring the molecular mechanisms used by GAS to cause disease in humans to better understand the disease-causing ability of GAS.
The work mentioned in the first chapter has helped to expand our knowledge on the least understood aspect of regulatory component of GAS, small regulatory RNAs (sRNAs), by focusing on FasX sRNA. Previous studies from the Sumby lab had uncovered some GAS virulence factor encoding mRNAs as regulatory targets of FasX including those encoding the fibrin clot degrader streptokinase (Ska), the fibronectin binding proteins (PrtF1, PrtF2) and the collagen binding pilus. In addition, these studies also covered the mechanism behind regulation of production of these proteins by FasX. Here, as an additional target regulated by FasX, we have uncovered M-related protein (Mrp) which is a fibrinogen binding protein helping GAS to bind to host fibrinogen and protect itself from the host innate immune system. While the mechanism of Mrp regulation by FasX was not elucidated, we did observe that the regulation occurs at the level of mrp mRNA expression. By positively regulating the production of the GAS spreading factor streptokinase and negatively regulating the expression of adhesion proteins, FasX may act as a regulator to transition GAS from a colonization state to the dissemination state. Mrp is also being investigated as a possible candidate antigen in a GAS vaccine formulation which signifies the importance of this study. During the process of work to expand the FasX regulon, we came through an interesting finding that FasX also has a role promoting the GAS ability to survive in human blood. On further investigation on what imparts this phenotype, we got to know that phagocytic cells are involved. This work is consistent with GAS ability to cause disease by evading the innate defense mechanism mediated by phagocytes like neutrophils. How FasX mediates GAS resistance against phagocytic cell killing and which FasX regulated virulence factors are involved could be the areas for future investigation.
In the second chapter we have characterized a four-component regulatory system, FasBCAX, of GAS. The Fas system is a combined protein and small regulatory RNA based system. As the small regulatory RNA of this system is none other than FasX, the first motivation to characterize this previously uncharacterized system arose from the previous work on the FasX sRNA. The surprising part is that the protein components, FasBCA, encoded by the fasBCA genes located upstream of fasX, by an unknown mechanism increase FasX abundance/expression by ~100-fold. These protein components deviate slightly from typical bacterial two-component systems (TCs) by consisting of two sensor kinase like proteins (FasB and FasC) and one response regulator like protein (FasA). In this chapter, the major focus has been to characterize FasBCA proteins at the molecular level by delineating domains of FasBCA important for the regulation of FasX expression. We have also worked on the mechanistic approach on what kind of signal may activate the system and how the proteins may interact among one another to influence FasX abundance. After delineating the domains of FasBCA important for FasX regulation, along with the finding that a protein component in plasma activates FasX sRNA, we are able to come up with a model of how this Fas system works. Upon sensing an extracellular signal by FasC, through its transmembrane domain, FasC and FasB heterodimerize in the bacterial cell membrane, then the ATPase catalytic domain of FasB catalyzes ATP into ADP and free phosphate which is used to phosphorylate FasC at amino acid H246. The phosphate from FasCH246 gets transferred to the cytoplasmically located response regulator FasA at amino acid D60. After getting phosphorylated, FasA is activated, then binds the fasX promoter thus enhancing FasX transcription. This is the first data to show that two sensor kinase-like proteins in bacteria interact by forming a heterodimer. Though there is more to be done to further understand the Fas system in depth, our study has illuminated significant light on how the Fas system works. Future work includes co-immunoprecipitation to confirm FasB and FasC interaction, demonstration of FasB kinase activity, and study on proteins present in plasma or THY on how they activate the Fas system. Delineation of the domains of FasB, FasC and FasA may help in the design of therapeutic approaches targeting FasBCA to inhibit their interaction or activity and hence may reduce GAS disease burden by decreasing FasX abundance.
The third chapter explores physical and genetic barriers in the gain of mobile genetic element RD2. Epidemiological studies have established that almost all isolates of serotype M28 GAS, but not isolates of other serotypes, contain RD2 and are non-randomly associated with the cases of puerperal sepsis, a life-threatening infection that may occur in women following childbirth. There remains an important question yet to be answered: Why is RD2 limited only to certain GAS populations like M28? Based on high conjugation frequency of intra-serotype conjugation of RD2 from M28 GAS isolates containing RD2 to M28 isolates lacking RD2 (constructed in lab), but low inter-serotype conjugation frequency from M28 to other GAS serotypes like M1, M49 and M59, we proposed that the capsule, which is present in M1, M49 and M59, but is naturally absent from M28 isolates serves as a barrier to the gain of RD2. Our data show that the presence and absence of capsule has no direct role in influencing the intra- or inter serotype conjugation frequency. We questioned if capsule may indirectly influence the distribution of RD2 in the GAS population by inhibiting the activity of RD2-encoded factors. While the presence of capsule was found to inhibit RD2 mediated adherence to two different vaginal epithelial cell lines, the presence of capsule had no effect on RD2 mediated colonization in mouse model of vaginal colonization. Thus, our study suggested that capsule in not a barrier in the acquisition of RD2. Finally, by using hsdR mutant and complemented mutant GAS strains, we found that a Type I restriction- modification (R-M) system serves as a genetic barrier to the gain and transfer of RD2 across GAS serotypes. We further tested RD2s ability to modify the GAS transcriptome following growth in human plasma, an ex vivo model of infection and found that the presence of RD2 leads to differential expression of large numbers of virulence genes involved in host adherence, dissemination and adaptation to the host during invasive infections, consistent with RD2 associating with cases of puerperal sepsis.
In summary, our work has expanded our insights into the molecular mechanisms used by GAS to cause disease in humans, focusing on the FasX small regulatory RNA, the FasBCA proteins and the RD2 mobile genetic element. The data generated from this dissertation work may pave the way for the creation of therapeutic approaches against GAS infection.