Oral Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Characterisation of a master regulator of Uropathogenic Escherichia coli virulence (104307)

Yvette 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. Australian Infectious Diseases Research Centre, The University of Queensland, Brisbane, QLD, Australia
  3. Institute for Molecular Bioscience, The University of Queensland, Saint Lucia, Queensland, Australia

Uropathogenic Escherichia coli (UPEC) is the most common cause of urinary tract infections. UPEC has many virulence factors contributing to its pathogenesis, including the capsule and LPS that confer protection from host innate immunity, and adhesins such as fimbriae and Ag43 that enhance colonisation and biofilm formation. Many of these virulence determinants are regulated by the transcriptional regulator RfaH. Here, we investigated the function of RfaH as a master regulator of UPEC virulence.

The global impact of RfaH on UPEC transcription was examined using RNAseq. Bioinformatic analyses were performed by designing a HMM RfaH binding motif matrix and screening complete E. coli genomes. A novel genome-wide screening method using magnetic bead separation with transposon directed insertion-site sequencing (TraDIS) was developed to investigate how cell-surface polysaccharides interfere with adhesin function. 


Transcriptomic mapping of the multidrug-resistant UPEC strain S65EC revealed that the RfaH regulon is extremely complex, comprising virulence genes encoding cell-surface polysaccharides (such as the capsule, LPS and O-antigen), a repressor of flagella, and factors associated with stress adaptation. Bioinformatic analyses uncovered novel targets of RfaH – an uncharacterised operon and a Type VI Secretion System. Using TraDIS, we showed that RfaH-mediated control of surface polysaccharides modulates the function of shorter adherence factors indirectly by either shielding or unmasking their exposure on the cell surface. We propose a model whereby RfaH works in tandem with other response regulators of its direct targets to achieve this effect, thus revealing an extended network of genes indirectly controlled by RfaH in multiple key cellular processes including adhesion, biofilm formation and adaptation to stress.


In summary, this study characterised how RfaH functions as a master regulator of UPEC virulence beyond transcriptional enhancement of long operons encoding virulence factors. The complex RfaH regulon defined in this work demonstrates its critical role as a global regulator of UPEC pathogenesis.