Biology subjects

Boekhoud, I. M.

Publications and source records attributed to Boekhoud, I. M..

3 recordsLinked to original sources

bioRxiv2020

Heme is crucial for medium-dependent metronidazole resistance in clinical isolates of C. difficile

Until recently, metronidazole was the first-line treatment for Clostridioides difficile infection and it is still commonly used. Though resistance has been reported due to the plasmid pCD-METRO, this does not explain all cases. Here, we investigate resistance to metronidazole in a collection of clinical isolates of C. difficile. We find that nearly all isolates demonstrate a heme-dependent increase in the minimal inhibitory concentration for metronidazole, which in some cases leads to isolates being qualified as resistant (MIC > 2 mg/L). Moreover, whole genome sequence analysis reveals a single nucleotide polymorphism in the heme responsive gene hsmA, which defines a metronidazole resistant lineage of PCR ribotype 010 / multilocus sequence type 15 isolates that also includes pCD-METRO containing strains. Together our data demonstrate that heme is crucial for medium-dependent metronidazole resistance in C. difficile.

microbiology↗

bioRxiv2020

Redefining the Clostridioides difficile σB regulon: σB activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

In many gram-positive bacteria the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B ({sigma}B). In C. difficile {sigma}B has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we used an anti-{sigma}B antibody to demonstrate time-limited overproduction of {sigma}B in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced {sigma}B gene expression. Inducible {sigma}B overproduction uncouples {sigma}B expression from its native regulatory network and allowed for the refinement of the previously proposed {sigma}B regulon. At least 32% the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile {sigma}B was demonstrated through in vitro run-off transcription of specific target genes (cd0350, cd3614, cd3605, cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to {sigma}B-dependent gene activation. ImportanceSigma B is the alternative sigma factor governing stress response in many gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of {sigma}B by evaluating the transcriptional effects of {sigma}B overproduction, provide biochemical evidence of direct transcriptional activation by {sigma}B, and show that {sigma}B-dependent genes can be activated by antimicrobials. Together our data suggest that {sigma}B is a key player in dealing with toxic radicals.

microbiology↗

bioRxiv2018

Genome location dictates the transcriptional response to sub-inhibitory concentrations of PolC-inhibitors in Clostridium difficile

Clostridium difficile is a potentially lethal gut pathogen that causes nosocomial and community acquired infections. Limited treatment options and reports of reduced susceptibility to current treatment emphasize the necessity for novel antimicrobials. The DNA-polymerase of gram-positive organisms is an attractive target for the development of antimicrobials. ACX-362E (N2-(3<,4-Dichlorobenzyl)-7-(2-[1-morpholinyl]ethyl)guanine; MorE-DCBG) is a DNA polymerase inhibitor in pre-clinical development as a novel therapeutic against C. difficile infection. This synthetic purine shows preferential activity against C. difficile PolC over those of other organisms in vitro and is effective in an animal model of C. difficile infection. In this study we have determined its efficacy against a large collection of clinical isolates. At concentrations below the minimal inhibitory concentration, the presumed slowing (or stalling) of replication forks due to ACX-362E leads to a growth defect. We have determined the transcriptional response of C. difficile to replication inhibition and observed an overrepresentation of up-regulated genes near the origin of replication in the presence of PolC-inhibitors, but not when cells were subjected to sub-inhibitory concentrations of other antibiotics. This phenomenon can be explained by a gene dosage shift, as we observed a concomitant increase in the ratio between origin-proximal versus terminus-proximal gene copy number upon exposure to PolC-inhibitors. Moreover, we show that certain genes differentially regulated under PolC-inhibition are controlled by the origin-proximal general stress response regulator sigma factor B. Together, these data suggest that genome location both directly and indirectly determines the transcriptional response to replication inhibition in C. difficile.

microbiology↗