Clostridioides difficile Senses and Hijacks Host Heme for Incorporation into an Oxidative Stress Defense System

Michel

Corver

et al. 2020

mSphere

In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and 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 σB-dependent gene activation. IMPORTANCE Sigma 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 σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.

Section: Discussionsupporting

confidence: 68%

Redefining the Clostridioides difficile σ ^B Regulon: σ ^B Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

Michel

Corver

et al. 2020

mSphere

“…It has been suggested that antimicrobials at toxic concentrations can influence metabolism and respiration (42,43), potentially resulting in the formation of bactericidal concentrations of radical species (44)(45)(46). A strong connection between σ B and oxidative (and/or nitrosative) stress in C. difficile (Table 2) and other bacteria (7,18,19), as well as a recently described radical scavenging strategy that increases tolerance to antimicrobials (47) are consistent with such a model. However, additional research is necessary to determine exactly how these processes occur and are influenced by antimicrobials in anaerobic organisms under anoxic conditions.…”

Section: Discussionsupporting

confidence: 66%

Redefining theClostridioides difficileσ^Bregulon: σ^Bactivates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

Michel

Corver

et al. 2020

Preprint

In many gram-positive bacteria the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile σB has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allowed for the refinement of the previously proposed σB 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 σB 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 σB-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 σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together our data suggest that σB is a key player in dealing with toxic radicals.

“…Knippel et al has demonstrated that reduced metronidazole susceptibility upon heme supplementation in R20291 was largely mediated by the hsmRA operon, leading to the question whether presence/absence or sequence variants of these genes underlie heme-dependent resistance in other RTs. 29 Based on the present dataset, however, we were unable to identify specific sequence variants of this operon (or in the hatRT operon also involved in heme detoxification) that could explain why the vast majority of strains are less susceptible to metronidazole upon heme supplementation. The genes appear to be (near-)universally conserved amongst different C. difficile types (data not shown) and the same signature is found in strains that do or do not respond to heme supplementation, and those that do or do not qualify as resistant (Table 1, Table 2, Supplemental Table 1).…”

Section: Discussionmentioning

confidence: 60%

“…10 At present, there is no structural information on the HsmA protein, though homology between the protein and heme-containing cytochromes has been noted. 29 HsmA has been postulated to act through sequestration of heme, but the effect of the altered Cterminal sequence on the affinity for heme remains to be elucidated.…”

Section: Elegant Work Bymentioning

confidence: 99%

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

Sidorov

Nooij

et al. 2020

Preprint

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.