Genome Location Dictates the Transcriptional Response to PolC Inhibition in Clostridium difficile

Abstract

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 preclinical 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 MIC, 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 upregulated genes near the origin of replication in the presence of PolC inhibitors, but not when cells were subjected to subinhibitory 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 and 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.

Redefining theClostridioides difficileσBregulon: σBactivates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

“…Although the promoters of cd3412 (uvrB) and cd3614 were reported to have a σ B consensus sequence and are differentially expressed upon σ B overexpression (19,35), our results clearly demonstrate that they can also be expressed in a σ B -independent manner ( Figure 5B). This is most notable for Pcd3412, which is still activated by metronidazole in the absence of σ B , in line with results obtained with ibezapolstat in a different study (20). Both metronidazole and ibezapolstat treatment can cause DNA damage, and DNA-damage dependent induction of cd3412 therefore likely depends on a sigB-independent pathway.…”

“…Therefore, we evaluated the effects of different antimicrobial compounds and the radical producer H2O2 as a positive control (19), using the Pcd0350 reporter construct, as this promoter demonstrated the clearest σ B dependent activation in the presence of metronidazole ( Figure 5B). We tested the cell wall biosynthesis inhibitor vancomycin, the protein synthesis inhibitor lincomycin and the DNA polymerase inhibitor ibezapolstat (formerly known as ACX-362E) (35). We observed clear activation of Pcd0350 in the presence of all added stressors, but not for a negative control containing water ( Figure 5C).…”

“…(encoding a hypothetical protein involved in oxidative stress) were selected on the basis of our transcriptome analyses (Dataset S1), previous reporter gene assays (20) and/or the presence of a putative σ B recognition site (11). The gene cd0872 (maltose Oacetyltransferase) was not differentially expressed in our transcriptome data and was thus included as a negative control.…”

“…The resulting DNA fragment was digested and ligated into NdeI-xhoI digested pET21b(-) vector, generating expression vector pIB14. Plasmids pIB27, pIB68, pIB69 and pIB74 have been described previously (20). The CD0872 promoter area was amplified using primers oIB-14 and oIB15 and the Pcd0872 luciferase-reporter plasmid was created by restriction-ligation using restriction enzymes KpnI and SacI in digested pAP24 backbone, generating plasmid pIB21.…”

“…However, the underlying molecular mechanism remains unknown (19). Finally, indirect activation of σ B -dependent genes as the result of a gene-dosage shift has been demonstrated for C. difficile exposed to DNA-polymerase inhibitors such as the Phase I drug ibezapolstat/ACX-362E (20).…”