The roles of a universally conserved DNA-and RNA-binding domain in controlling MRSA virulence and antibiotic resistance

Antimicrobial medicines have saved millions of lives since the introduction of penicillin in the 1940s. However, their overuse has resulted in the appearance of multidrug-resistant bacteria at a rate that has outpaced the discovery of new antibiotics. The rapid spread of highly virulent and multi-drug-resistant S. aureus strains (such as methicillin-resistant S. aureus (MRSA)) is causing major healthcare problems worldwide as S. aureus skin and respiratory infections can be life-threatening and are becoming increasingly more challenging to treat. MRSA uses several clever tactics to increase its resistance to host immune systems and antibiotic therapies. These include attaching to and killing host cells to extract essential nutrients while evading intracellular immune response and forming biofilm structures that protect the bacterial cells from host immune response and antibiotics. To accomplish this, MRSA must quickly produce new proteins to execute these tasks. Like all organisms, MRSA makes temporary copies of its genes, called messenger RNA (mRNA) molecules. This requires the activity of the transcription machinery, the RNA Polymerase, and other proteins, called transcription factors, that help determine for which genes mRNA copies are generated. The mRNAs can subsequently be read (translated) by another important machinery, called the ribosome, to create proteins.
Besides transcription factors, RNA-binding proteins (RBPs) also play vital roles in helping MRSA survive the hostile host environment. By binding to mRNAs, RBPs control how efficiently ribosomes translate the temporary mRNA copies. RBPs can also aid in removing mRNAs that are no longer needed.
Although the importance of RBPs for bacteria is well established, we know remarkably little about how these proteins contribute to S. aureus survival during host infection. To address this, we performed pioneering experiments that uncovered many new RBPs in S. aureus. To our surprise, this dataset contained many proteins belonging to a group of transcription factors called Helix-Turn-Helix proteins (HTH). Interestingly, several of these HTH proteins have well-established functions in antibiotic resistance and host immune evasion. Using methodologies from various scientific disciplines, this research programme aims to determine how HTH proteins can recognise distinct DNA and RNA molecules and how important this newly discovered RNA-binding function is for MRSA survival in the host. Finally, using innovative drug discovery techniques, we aim to identify small molecules that control the activity of a select number of HTH proteins. A longer-term goal is to determine whether these small molecules can be repurposed for battling bacterial infections and whether the RNA-binding activities of HTH proteins can be exploited for developing new therapeutics.

Methicillin-resistant Staphylococcus aureus (MRSA) poses a significant threat to healthcare worldwide as infections are becoming increasingly difficult to treat. MRSA is an effective pathogen because it expresses numerous virulence factors, including cytolytic toxins, that enable the bacterium to survive hostile host environments. Transcription factors dictate the temporal expression of virulence factors during infection. However, it is increasingly evident that post-transcriptional regulation by RNA-binding proteins (RBPs) makes major contributions to regulation and virulence but remains an understudied target for therapy. Unexpectedly, we recently found that many MRSA Helix-Turn-Helix domain transcription factors (HTH-TFs) also globally bind RNA in vivo. Some HTH-TFs have well-established transcriptional roles in regulating MRSA infectivity and antibiotic resistance. Moreover, the transcription activity of numerous HTH-TFs can be controlled by small molecules, making them promising targets for developing novel antimicrobials.
Our data indicate that the regulatory impact of HTH-TFs is much more profound than anticipated, underscoring the need for a thorough characterisation of these proteins. We hypothesise that HTH-TFs post-transcriptionally control a substantial fraction of antibiotic resistance and virulence factors by altering the stability/translation of RNA substrates. RNA may also compete with HTH-TF transcriptional activities. By integrating state-of-the-art biochemical, structural, and phenotypic approaches, we will obtain detailed mechanistic insights into (i) how key HTH-TFs bind both DNA and RNA molecules, (ii) how small molecule effectors control this activity, and (iii) how these RNA-binding activities impact gene expression and bacterial survival during host infection. A detailed understanding of MRSA HTH-TF function and how its nucleic acid binding is regulated may underpin future therapeutic approaches.