Characterization and Attenuation of Regulatory Network of Vibrio vulnificus rtxA Encoding a MARTX Toxin
Year: 2020
Type: dissertation
Abstract: Bacterial pathogens have evolved the ability to survive and develop diseases in several different environments within the host. The ability requires the production of various virulence factors whose expressions are coordinately controlled by regulatory networks in response to environmental changes. The opportunistic human pathogen Vibrio vulnificus can cause food-borne diseases from gastroenteritis to life-threatening septicemia. Among a wide array of virulence factors produced by V. vulnificus, a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin RtxA encoded by the rtxA gene plays an essential role in the virulence of the pathogen. It has been previously reported that the expression of rtxA is negatively and positively regulated by direct binding of H-NS and HlyU to the rtxA promoter, PrtxA, respectively. In the present study, I have further examined additional regulatory proteins as well as environmental signals involved in the rtxA expression and identified a small-molecule inhibitor that attenuates the virulence of V. vulnificus. As a result, a leucine-responsive regulatory protein (Lrp) was found as a positive regulator of rtxA. Electrophoretic mobility shift and DNase I protection assays revealed that Lrp activates the rtxA expression by binding directly and specifically to PrtxA. Notably, DNase I cleavage of the PrtxA regulatory region showed phased hypersensitivity, suggesting that Lrp probably induces the DNA bending in PrtxA. Lrp activates rtxA in an independent manner with H-NS and HlyU, and leucine inhibits the binding of Lrp to PrtxA and thus decreases the Lrp-mediated activation. Moreover, a cyclic AMP receptor protein (CRP) acts as a negative regulator of the rtxA transcription, and exogenous glucose relieves the CRP-mediated repression. Interestingly, biochemical and mutational analyses demonstrated that CRP binds directly and specifically to the upstream regions of PrtxA, which presumably changes the DNA conformation in PrtxA and represses rtxA. Furthermore, CRP represses the expressions of lrp and hlyU by directly binding to their upstream regions, forming coherent feedforward loops with Lrp and HlyU. Taken together, a regulatory network comprising CRP, Lrp, H-NS, and HlyU coordinates the expression of rtxA in response to changes in host environmental signals such as leucine and glucose. This collaborative regulation will contribute to the precise expression of rtxA during the pathogenesis of V. vulnificus. \nMy next concern was about new approaches called anti-virulence strategies that target virulence of bacterial pathogens in an attempt to control the virulence of V. vulnificus. Anti-virulence strategies have the advantage of less selective pressure for inducing resistance than conventional strategies that target viability of the pathogens. Therefore, I performed a high-throughput screening of the small-molecule library containing 8,385 compounds to inhibit HlyU, a transcriptional activator essential for the expression of V. vulnificus rtxA. A small molecule [N-(4-oxo-4H-thieno[3,4-c]chromen-3-yl)-3-phenylprop-2-ynamide] was identified as an inhibitor of the HlyU activity and named CM14. CM14 reduces HlyU-dependent virulence gene expression in V. vulnificus, but does not suppress the bacterial growth or cause host cell death. Treatment of CM14 decreases hemolysis of human erythrocytes and impedes host cell rounding and lysis caused by V. vulnificus. Remarkably, co-administration of CM14 improves the survival of mice infected with V. vulnificus by alleviating hepatic and renal dysfunction and systemic inflammation. As revealed by biochemical, mass spectrometric, and mutational analyses, CM14 covalently modifies the Cys30 residue of HlyU to prevent the protein from binding to the target DNA. Based on these results, a possible molecular mechanism is proposed for the covalent modification of HlyU by CM14. Because HlyU is a conserved transcriptional activator of virulence genes in Vibrio species, CM14 is also capable of reducing the expressions of multiple virulence genes in other Vibrio species and attenuating their virulence-related phenotypes. The combined results suggest that this small-molecule could be an anti-virulence agent against HlyU-harboring pathogenic Vibrio species with a low possibility of developing resistance.
Author: Ji‐Won Lee
Language: English
Subfield: Microbiology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W3101234674