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The small molecule nitazoxanide selectively disrupts BAM-mediated folding of the outer membrane usher protein.

ABSTRACT: Bacterial pathogens assemble adhesive surface structures termed pili or fimbriae to initiate and sustain infection of host tissues. Uropathogenic Escherichia coli, the primary causative agent of urinary tract infections, expresses type 1 and P pili required for colonization of the bladder and kidney, respectively. These pili are assembled by the conserved chaperone-usher (CU) pathway, in which a periplasmic chaperone works together with an outer membrane (OM) usher protein to build and secrete the pilus fiber. Previously, we found that the small molecule and antiparasitic drug nitazoxanide (NTZ) inhibits CU pathway-mediated pilus biogenesis in E. coli by specifically interfering with proper maturation of the usher protein in the OM. The usher is folded and inserted into the OM by the ?-barrel assembly machine (BAM) complex, which in E. coli comprises five proteins, BamA-E. Here, we show that sensitivity of the usher to NTZ is modulated by BAM expression levels and requires the BamB and BamE lipoproteins. Furthermore, a genetic screen for NTZ-resistant bacterial mutants isolated a mutation in the essential BamD lipoprotein. These findings suggest that NTZ selectively interferes with an usher-specific arm of the BAM complex, revealing new details of the usher folding pathway and BAM complex function. Evaluation of a set of NTZ derivatives identified compounds with increased potency and disclosed that NTZ's nitrothiazole ring is critical for usher inhibition. In summary, our findings indicate highly specific effects of NTZ on the usher folding pathway and have uncovered NTZ analogs that specifically decrease usher levels in the OM.

SUBMITTER: Psonis JJ

PROVIDER: S-EPMC6768635 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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The small molecule nitazoxanide selectively disrupts BAM-mediated folding of the outer membrane usher protein.

Psonis John J JJ Chahales Peter P Henderson Nadine S NS Rigel Nathan W NW Hoffman Paul S PS Thanassi David G DG

The Journal of biological chemistry 20190807 39

Bacterial pathogens assemble adhesive surface structures termed pili or fimbriae to initiate and sustain infection of host tissues. Uropathogenic <i>Escherichia coli</i>, the primary causative agent of urinary tract infections, expresses type 1 and P pili required for colonization of the bladder and kidney, respectively. These pili are assembled by the conserved chaperone-usher (CU) pathway, in which a periplasmic chaperone works together with an outer membrane (OM) usher protein to build and se ...[more]

PMID: 31391254

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Project description:Several antibacterial compounds have recently been discovered that potentially inhibit the activity of BamA, an essential subunit of a heterooligomer (the barrel assembly machinery or BAM) that assembles outer membrane proteins (OMPs) in Gram-negative bacteria, but their mode of action is unclear. To address this issue, we examined the effect of three inhibitors on the biogenesis of a model E. coli OMP (EspP) in vivo. We found that darobactin potently inhibited the interaction of a conserved C-terminal sequence motif (the "β signal") with BamA, but had no effect on assembly if added at a postbinding stage. In contrast, Polyphor peptide 7 and MRL-494 inhibited both binding and at least one later step of assembly. Taken together with previous studies that analyzed the binding of darobactin and Polyphor peptide 7 to BamA in vitro, our results strongly suggest that the two compounds inhibit BAM function by distinct competitive and allosteric mechanisms. In addition to providing insights into the properties of the antibacterial compounds, our results also provide direct experimental evidence that supports a model in which the binding of the β signal to BamA initiates the membrane insertion of OMPs. IMPORTANCE There is a clear need to develop novel broad-spectrum antibiotics to address the global problem of antimicrobial resistance. Multiple compounds that have strong antibacterial activity have recently been described that appear to inhibit the activity of the barrel assembly machinery (BAM), an essential complex that catalyzes the assembly (i.e., folding and membrane insertion) of outer membrane proteins (OMPs) in all Gram-negative bacteria. We analyzed the effects of three of these compounds on OMP biogenesis in vivo and found that they inhibited different stages of the assembly process. Because these compounds have distinct modes of action, they can be used in combination to reduce the emergence of resistant strains. As a corollary, we obtained evidence that these compounds will be valuable tools in future studies on BAM function.

Dataset Information

The small molecule nitazoxanide selectively disrupts BAM-mediated folding of the outer membrane usher protein.

Publications

The small molecule nitazoxanide selectively disrupts BAM-mediated folding of the outer membrane usher protein.

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