Redox-regulation in Gram-negative Bacteria

项目总结

PROJECT SUMMARY Bacteria frequently encounter a variety of harmful stressors including reactive oxygen and chlorine species (RO/CS). One particularly potent antimicrobial oxidant is hypochlorous acid (HOCl), which is generated during neutrophil-mediated phagocytosis and by enzymes of the mucosal epithelia to control bacterial colonization. Bacteria have evolved strategies to counteract and reduce the harmful effects of RO/CS, e.g. by upregulating the expression of stress-specific genes. RO/CS-induced changes in gene expression are typically mediated by posttranslational modifications of redox-sensitive amino acid side chains in transcriptional regulators, which affect their ability to activate/repress the expression of the corresponding stress-protective target genes. Our preliminary data show that uropathogenic E. coli (UPEC) respond to sublethal HOCl-stress with the upregulation of an operon harboring three uncharacterized UPEC-specific genes. We identified one of them as a HOCl- sensitive transcriptional repressor that reversibly loses its repressor activity during HOCl-stress. Its inactivation results in the de-repression of the two downstream targets contributing to the increased HOCl resistance of UPEC strains compared to commensal and enteropathogenic E. coli. Moreover, our preliminary data show that sublethal HOCl concentrations cause the induction of numerous biofilm genes and stimulate biofilm formation. The overall goal of this project is to comprehensively understand the cause and effects of RO/CS stress and to characterize specific bacterial defense strategies to RO/CS used to mitigate their damage. Our working hypothesis is that RO/CS specifically and reversibly modify surface-exposed cysteine residues in proteins, which become the key factor in redox signaling by affecting gene expression and/or bacterial physiology. In Aim 1, we will use phenotypic and biochemical strategies to assess the mechanism by which the transcriptional repressor is inactivated in vitro and in vivo. Moreover, we will elucidate the function of one of its downstream targets, which appears to contribute substantially to UPEC’s increased HOCl resistance and identify additional HOCl resistance genes by TnSeq. To characterize the benefits of HOCl-mediated biofilm stimulation, we will analyze the composition of CFT073 biofilms before and after exposure to sublethal HOCl and test whether biofilm cells become more resistant to subsequent HOCl stress or common antibiotics used to treat UTIs (Aim 2). In addition, we will investigate how HOCl stress acts to trigger the increase in biofilm formation in CFT073 by determining the role that the diguanylate cyclase YdeH plays in this process. In Aim 3, we will combine transcriptomic and phenotypic analyses to identify novel regulons contributing to UPEC’s resistance to the surface antimicrobial AgXX, which has previously been shown to generate RO/CS. These studies will provide us with fundamentally new insights into the role that RO/CS play in UPEC. Identifying, characterizing and targeting UPEC-specific defense systems has the potential to increase the body’s own capacity to fight UTIs.