Post-transcriptional roles of helix-turn-helix transcription factors in regulation of gene expression in Methicillin-resistant Staphylococcus aureus

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogenic bacterium capable of causing severe and life-threatening infections in humans and animals. The spread of MRSA remains a global healthcare issue. It is one of the leading causes of healthcare- and community-associated infections. Their resistance to different types of antibiotics challenges the clinical management of MRSA infections and is usually associated with prolonged infections and treatment.

The spread and pathogenesis of MRSA rely on the expression of various virulence factors associated with colonisation and biofilm formation, as well as immune evasion, which collectively contribute to their rapid adaption and survival from the hostile host environment. Gene expression regulation plays an essential role during these processes, and it is tightly controlled at the transcriptional, post-transcriptional, translational, and post-translational levels. While the significance of transcription factors in controlling virulence, especially at the transcriptional level, is well-recognised, there is increasing evidence that co- and post-transcriptional regulation by RNA-binding proteins (RBPs) also play critical functions. Nevertheless, there are significant gaps in our understanding of the mechanisms by which RBPs influence the virulence of MRSA.

Our lab recently characterised the MRSA RNA-bound proteome, revealing hundreds of putative RBPs. Surprisingly, nearly half of these proteins contained helix-turn-helix (HTH) domains, a structural feature typically found in DNA-binding proteins and commonly detected in various bacterial transcription factors. The main goal of this thesis was to perform thorough in vivo and in vitro analyses of the RNA-binding properties of these MRSA HTH domain proteins and to gain mechanistic insights into how they interact with RNA. A longer-term aim is to use what we learned from these studies to better understand the biological significance of those RNA-binding properties.

In my research, I focused on characterising the RNA-binding properties of four well-characterised MRSA virulence factors as paradigms for HTH domain proteins, including CcpA, IcaR, Rot and MgrA, because they represent different HTH transcription factor families (LacI, TetR and MarR, respectively).

Using a combination of in vivo and in vitro RNA-binding analysis methods, I demonstrated that these proteins are capable of binding hundreds of different RNAs in MRSA, with a notable preference for binding in 3’ untranslated regions. In most cases, specific RNA binding to these proteins could also be detected in vitro. Delving deeper into CcpA, I was able to determine that this protein binds structured RNA with comparable affinities to double-stranded DNA in vitro. Through site-directed mutagenesis studies, I then demonstrated that CcpA indeed also uses its HTH domain to bind RNA in vitro as it does in vivo. Further analyses suggested that the recognition of RNA by the CcpA HTH domain might involve distinct residues compared to those involved in DNA recognition.

In the final chapter of my thesis, I detail my initial attempts to gain structural insights into how these HTH domain proteins bind RNA, predominantly through X-ray crystallography. Although unsuccessful, these attempts provided a valuable learning experience and laid a solid foundation for optimising crystallisation conditions in future studies.