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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.

Clostridium difficileis 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-(3C. difficileinfection. This synthetic purine shows preferential activity againstC. difficilePolC over those of other organismsin vitroand is effective in an animal model ofC. difficileinfection. 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 ofC. difficileto 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 inC. difficile.

With the impending crisis of antimicrobial resistance, there is an urgent need to develop novel antimicrobials to combat difficult infections and MDR pathogenic microorganisms. DNA replication is essential for cell viability and is therefore an attractive target for antimicrobials. Although several antimicrobials targeting DNA replication proteins have been developed to date, gyrase/topoisomerase inhibitors are the only class widely used in the clinic. Given the numerous essential proteins in the bacterial replisome that may serve as a potential target for inhibitors and the relative paucity of suitable compounds, it is evident that antimicrobials targeting the replisome are underdeveloped so far. In this review, we report on the diversity of antimicrobial compounds targeting DNA replication and highlight some of the challenges in developing new drugs that target this process.