The Fatty Acid Synthesis Protein Enoyl-ACP Reductase II (FabK) is a Target for Narrow-Spectrum Antibacterials for Clostridium difficile Infection

Abstract

Clostridium difficile infection (CDI) is an antibiotic-induced microbiota shift disease of the large bowel. While there is a need for narrow-spectrum CDI antibiotics, it is unclear which cellular proteins are appropriate drug targets to specifically inhibit C. difficile. We evaluated the enoyl-acyl carrier protein reductase II (FabK) that catalyzes the final step of bacterial fatty acid biosynthesis. Bioinformatics showed C. difficile uses FabK as its sole enoyl-ACP reductase, unlike several major microbiota species. The essentiality of fabK for C. difficile growth was confirmed by failure to delete this gene using Clostron mutagenesis and by growth inhibition upon gene silencing with CRISPR-interference antisense to fabK transcription or by blocking protein translation. Inhibition of C. difficile's FASII pathway could not be circumvented by supply of exogenous fatty acids, either during fabK's gene silencing or upon inhibition of the enzyme with a phenylimidazolederived inhibitor 1. The inability for fatty acids to bypass FASII inhibition is likely due to the function of transcriptional repressor FapR. Inhibition of FabK also inhibited spore formation, reflecting the enzyme's role in de novo fatty acid biosynthesis for the formation of spore membrane lipids. Compound 1 did not inhibit growth of key microbiota species. These findings suggest that C. difficile FabK is a druggable target for discovering narrow-spectrum anti-C. difficile drugs to treat CDI but avoid collateral damage to the gut microbiota.

Chromosomal Resistance to Metronidazole inClostridioides difficilecan be Mediated By Epistasis Between Iron Homeostasis and Oxidoreductases

“…To assess the roles played by nifJ, xdhA1 and iscR in metronidazole resistance, we silenced these genes using a xylose-inducible CRISPR-interference vector. In this approach, fusion of antisense nucleotides to the guide RNA of Cas9 blocks gene transcription (37) and is a more facile approach than gene deletions in C. difficile . As a positive control, an antisense to feoB1 was included to measure the effect of gene silencing on growth in metronidazole.…”

“…The xylose-inducible vector pXWpxyl- dcas9 was used to silence the transcription of feoB1, nifJ, iscR and xdhA1 , as previously described (37). The sgRNA ( Table S5 ) targeting the abovementioned genes were synthesized and cloned into the vector’s PmeI site by Genscript Biotech (New Jersey).…”

A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile

“…A further advantage of CRISPRi is that it can be used to explore the consequences of inactivating essential genes, as illustrated here for ftsZ (depletion phenotype, filamentation) and pbp-0712 (depletion phenotype, a complex mixture of lysis and aberrant morphologies). In fact, after we submitted this study to the review process, Marreddy et al used CRISPRi to demonstrate that the fatty acid biosynthesis gene fabK is essential in C. difficile (45). Finally, CRISPRi could be used to tune the expression of a gene of interest to suboptimal levels, with potential applications in drug discovery screens for synthetic phenotypes (23,46,47).…”

Expanding the Clostridioides difficile Genetics Toolbox

“…Impact of the research. The utility of the CRISPRi system in C. difficile was also recently shown by Marreddy et al, who developed a similar xylose-inducible CRISPRi system to show that the fatty acid biosynthesis gene fabK is essential in C. difficile (24). The CRISPRi system developed by the Hurdle group was not necessarily optimized for C. difficile, which may explain why their antisense RNA approach for knocking down fabK expression was more effective at repressing gene expression than their CRISPRi system.…”