Identification of small molecule compounds that cripple the Uropathogenic E. coli (UPEC) : a chemical genetic approach

Abstract

Uropathogenic Escherichia coli (UPEC) is the most common causative agent for urinary tract infection (UTI). The emerging and spread of antimicrobial-resistant UPEC strains combining with the high incidence of UTI highlight an immediate need for novel therapeutics. Anti-virulence compound is a promising alternative for the treatment of UTI. Instead of killing the pathogen, anti-virulence compounds aim to cripple the UPEC by suppressing its virulence genes, which will intervene in the pathogenesis of UTI and buy time for immune clearance. In this study, the author selected eighteen virulence genes from CFT073, a typical UPEC strain, and cloned their promoters to create a panel of transcriptional reporters. Two reporter strains (CFT073/pGLR1-fim and pGLR1-hly) were used in primary high-throughput screenings to assay 50,240 small molecules from astructurally diverse chemical library. As indicated by the reductions of bioluminescence in reporters, the primary screenings discovered 296 potential inhibitors for fim (type I fimbriae) and hly (hemolysin) expressions. After three rounds of secondary screenings (i.e., tetA reporter, disc diffusion, and western blotting), the compound, 1F7, was identified as a real inhibitor for type I fimbrial production. Surprisingly, 1F7 simultaneously suppressed the transcriptions of fim, pap, and sfa fimbrial operons, as supported by the bioluminescence and qPCR data. Literature reviews on the fimbrial regulations suggested that the Leucine-responsive regulatory protein (Lrp), a shared regulator, could be a bacterial target of 1F7. 1F7-treated CFT073 indeed resembled many phenotypes of its lrpmutant; these affected phenotypes were: i) impaired type I and pap fimbrial productions, ii) reduced bacteria-host adherence, iii) reduced biofilm formation, and iv) attenuated virulence during murine UTI. Gel shift assays (EMSA) confirmed that 1F7 inhibited Lrp-DNA interaction. Nuclear Magnetic Resonance (NMR) experiments further verified the binding of 1F7 on Lrp. Docking software predicted that 1F7 likely touched the DNA binding domain of Lrp or sat at the interfaces between Lrp dimers; however, the actual interacting residues remained elusive. Further results of Dynamic Light Scattering (DLS) and Differential Scanning Fluorimetry (DSF) assays have led to a new model that 1F7 may induce aggregation of the Lrp, destabilize the protein, and disturb its DNA binding function. The author has validated that Lrp is a critical virulence factor for UPEC pathogenicity. Murine infection experiments confirmed that Lrp contributed to UTI and bacteremia caused by CFT073. In vitro assays also suggested a vital role of Lrp in bacteria-host adherence and biofilm formation. The absence of Lrp (achieved by either gene knockout or 1F7 inhibition) significantly reduced fimbrial gene expressions and crippled the CFT073. Therefore, Lrp is a novel and attractive drug target in UPEC.In conclusion, the author employed a chemical genetic approach and identified 1F7 as an inhibitor for multiple fimbrial expressions. Lrp, a global regulator for fimbrial production, might be implicated in the inhibitory effects of 1F7. The physical interaction of 1F7 and Lrp was validated by EMSA, DLS, DSF, and NMR Spectroscopy. As a proof-of-principle, the author has demonstrated that Lrp is a novel druggable target for the treatment of UTI.