Studying the riboregulatory role of Hfq in Klebsiella pneumoniae using CLASH (crosslinking, ligation and sequencing of hybrids)

Abstract

In nature, microorganisms are frequently exposed to different types of stresses, such as temperature fluctuations, osmotic pressure, pH variations, nutrient availability changes, attack by host immunity, exposure to free radicals and antibiotics. In response, microorganisms employ various mechanisms to adapt, repair and safeguard themselves. Bacterial adaptation involves reprogramming several molecular pathways that regulate transcription, post-transcriptional regulation (PTR), and translation. Although transcription is an essential regulatory mechanism for gene expression, the post-transcriptional process is critical in fine-tuning the gene expression before translating the mRNAs into proteins. PTR mechanisms involve rewiring the gene expression using small non-coding RNAs (sRNA) and RNA binding proteins (RBPs).

In this study, I investigated Hfq, an RBP in a Gram-negative nosocomial pathogen Klebsiella pneumoniae (Kp), using a high-throughput sequencing technique named CLASH (Crosslinking, ligation, and sequencing of hybrids). I performed CLASH in two Kp strains - Ecl8 and Ecl8ΔramR (RamA overexpressor), and established gene regulation mechanisms between transcription and translation through the involvement of Hfq in PTR (Fig A).

As the transcription of numerous sRNAs is regulated by transcription factors (TF), I explored the link between a TF - “RamA” and its influence on Hfq bound sRNAs in mediating antibiotic resistance (ABR). My analyses between Ecl8 and Ecl8ΔramR provided insights of the transcriptional regulation of 7 sRNAs by RamA, highlighting the intricate and complex regulatory network. I chose MicF and SroC for further analyses. MicF and its target ompF were important in affecting antibiotic susceptibilities of tigecyline, tetracycline, ceftazidime and ciprofloxacin. However, SroC did not alter colistin or polymyxin B resistance in Kp due to the absence of a few downstream interacting targets. These findings imply that not all differentially regulated sRNAs in the presence of RamA produce a direct phenotype.

Analysis of Hfq-CLASH RNA-RNA interactions unravelled numerous canonical and novel sRNA-mRNA interactions important for regulating genes corresponding to virulence and ABR. I investigated some sRNAs such as Spf, RyhB, and OmrA/B, which interact with genes that modulate β-lactam susceptibility. A significant discovery of my thesis is the phenotype obtained due to the interaction between Spf and mrdA (encodes Penicillin Binding Protein 2). Spf deletion mutants exhibited decreased susceptibilities to β-lactam antibiotics like ampicillin, carbenicillin, benzylpenicillin, mecillinam, and penicillin V. Additionally, OmrA/B and RyhB sRNAs were involved in regulating the antibiotic entry of new antibiotic in the β-lactam class - Cefiderocol (CFDC). CFDC is a siderophore-conjugated cephalosporin that utilises a transporter channel named CirA. I demonstrated the role of OmrA/B and RyhB in interacting with cirA and modulating its mRNA expression. However, deletion mutants of OmrA/B and RyhB showed no significant changes in antibiotic susceptibility to CFDC, meaning that other transporters or sRNAs are important for regulating CFDC entry and susceptibility in K. pneumoniae.

In addition to antibiotic susceptibility phenotypes, I also explored the role of sRNAs in oxidative stress response. I studied the role of another sRNA – GcvB, in oxidative stress via its interaction with tpx (thiol peroxidase). The absence of GcvB improves the mutant’s survival rate to hydrogen peroxide exposure, stressing the importance of GcvB in peroxide resistance.

Ultimately, these findings underscore the importance of sRNAs as central players in bacterial stress response networks and pave the way for developing novel therapeutic strategies targeting sRNA-mediated regulatory pathways.