Binding of Campylobacter jejuni FliW Adjacent to the CsrA RNA-Binding Pockets Modulates CsrA Regulatory Activity.

Marek Bogacz,Zhirui Wang,Jiaqi Li,C Luke Daugherty,Stuart A Thompson,Lynn K H Tran,Jarred S Fiedler,Paul M H Tran,Faiha M El Abbar,Parastoo Azadi,Claudia A Cox,Kate H Blake

doi:10.3389/fmicb.2020.531596

Marek Bogacz, Zhirui Wang + Show 10 more

Open Access

https://doi.org/10.3389/fmicb.2020.531596

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Abstract

Campylobacter jejuni CsrA is an mRNA-binding, post-transcriptional regulator that controls many metabolic- and virulence-related characteristics of this important pathogen. In contrast to E. coli CsrA, whose activity is modulated by binding to small non-coding RNAs (sRNAs), C. jejuni CsrA activity is controlled by binding to the CsrA antagonist FliW. In this study, we identified the FliW binding site on CsrA. Deletion of the C-terminus of C. jejuni CsrA, which is extended relative to sRNA-binding CsrA proteins, abrogated FliW binding. Bacterial two-hybrid experiments were used to assess the interaction of FliW with wild-type CsrA and mutants thereof, in which every amino acid was individually mutated. Two CsrA mutations (V51A and N55A) resulted in a significant decrease in FliW binding. The V51A and N55A mutants also showed a decrease in CsrA-FliW complex formation, as assessed by size-exclusion chromatography and surface plasmon resonance. These residues were highly conserved in bacterial species containing CsrA orthologs whose activities are predicted to be regulated by FliW. The location of FliW binding was immediately adjacent to the two RNA-binding sites of the CsrA homodimer, suggesting the model that FliW binding to CsrA modulates its ability to bind to its mRNA targets either by steric hindrance, electrostatic repulsion, or by altering the overall structure of the RNA-binding sites.

Highlights

Campylobacter jejuni is an important human pathogen, and one of the leading bacterial causes of acute gastroenteritis throughout both developed and developing areas of the world (WHO, 2015)
As the C-terminus of C. jejuni CsrA is longer than the analogous region of CsrA proteins of E. coli and other bacteria, whose activities are regulated by sRNAs rather than by FliW (Fields and Thompson, 2012), and adjacent to the most C-terminal β-strand (β5) that is involved in RNA binding in both E. coli and C. jejuni (Figure 1A; Mercante et al, 2006; El Abbar et al, 2019), we hypothesized that FliW would bind to this region
We previously showed that CsrA is a pleiotropic posttranscriptional regulator in C. jejuni, modulating the expression of numerous proteins including FlaA flagellin (Fields and Thompson, 2008; Fields et al, 2016; Li et al, 2018)

Summary

Introduction

Campylobacter jejuni is an important human pathogen, and one of the leading bacterial causes of acute gastroenteritis throughout both developed and developing areas of the world (WHO, 2015). We previously demonstrated that C. jejuni CsrA regulates multiple processes associated with bacterial virulence, as a csrA mutant had impaired motility and ability to form biofilm, decreased resistance to oxidative stress, reduced epithelial cell binding and mouse colonization, as well as increased host cell invasion (Fields and Thompson, 2008). Some of these phenotypes could be a result of dysregulated translation of flagellin (flaA) mRNA, a known target of C. jejuni CsrA (Dugar et al, 2016; Fields et al, 2016; Radomska et al, 2016)

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