Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2023

Dissecting the role of RfaH in uropathogenic Escherichia coli virulence (92817)

Yvette Yu Ting Ong 1 2 3 , Nhu Nguyen 1 2 3 , Minh Duy Phan 1 2 3 , Mark Schembri 1 2 3
  1. School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia
  2. Institute for Molecular Bioscience, The University of Queensland, Saint Lucia, Queensland, Australia
  3. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia

Introduction

Uropathogenic Escherichia coli (UPEC) are one of the most common causes of urinary tract and bloodstream infections (Flores-Mireles et al., 2015). UPEC possess key virulence determinants such as the capsule and lipopolysaccharide (LPS) that confer protection from host innate immune factors while advancing disease progression (Totsika et al., 2012). Many of these virulence determinants are regulated by the transcriptional elongation factor RfaH (Nagy et al., 2002), which binds to a specific motif known as the JUMPstart sequence located upstream of target virulence genes (Hobbs & Reeves, 1994). RfaH binding enhances RNA polymerase activity and increases the efficiency of long operon transcription and translation (Svetlov & Nudler, 2012).

Methods

The UPEC RfaH regulon was investigated using a combined bioinformatic and molecular (RNA-seq) approach. A HMM motif matrix was built using characterized JUMPstart sequences and used to identify RfaH-regulated genes in 1378 completely sequenced E. coli genomes. The RfaH regulon was experimentally mapped in the multidrug resistant UPEC ST131 strain S65EC at mid-log phase using RNA-seq and validated with qRT-PCR.

Results

Overall, the matrix successfully identified all known JUMPstart sequences and detected a potentially new JUMPstart sequence belonging to an operon comprising 7 genes of unknown function. The RNA-seq analysis and qRT-PCR validation was congruent with the bioinformatic analysis, demonstrating that RfaH was required for the transcription of genes encoding the capsule, LPS, and O-antigen.

Conclusion

RfaH is a key regulator of multiple UPEC cell surface virulence determinants. Deciphering the function of RfaH will improve our understanding of UPEC pathogenesis and may form a framework for the development of urgently needed anti-virulence therapeutics to address the enormous problem of increasing antibiotic resistance.

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