Abstract
Antibiotics with novel and/or multiple targets are highly desirable in the face of the steady rise of clinical antibiotic resistance. We have screened and identified small molecules, typified by the compound TPI1609-10 (aka SM10), with antibiotic activity against both gram-positive and gram-negative bacteria. SM10 was screened in vitro to bind branched Holliday junction intermediates of homologous recombination and tyrosine recombinase-mediated recombination; thus, the cellular targets of the small molecules were expected to include the RuvABC Holliday junction resolvasome and the XerCD complex involved in proper segregation of replicated chromosomes to daughter cells. SM10 indeed induces DNA damage and filamentation in E. coli. However, SM10 also induces envelope stress and causes increased production of intracellular reactive oxygen species. In addition, SM10 has similar effects to endogenously-induced envelope stress via overproducing outer membrane proteins (OmpC and OmpF), which also induces the SOS response, chromosome fragmentation, and production of reactive oxygen species. The synergy between SM10, and cerulenin, a fatty acid synthesis inhibitor, together with the SM10 hypersensitivity of cpx and rpoE mutants, further support that SM10's mode of action damages membrane damage. The lethality of SM10 treatment and of OmpC overproduction are observed in both aerobically- and anaerobically-grown cells, and is accompanied by substantial DNA damage even anaerobically. Thus, only some DNA damage is due to reactive oxygen. We propose that membrane depolarization and the potential reduction in intracellular pH, leading to abasic site formation, cause a substantial amount of the DNA damage associated with both SM10 treatment and endogenous envelope stress. While it is difficult to completely exclude effects related to envelope damage as the sources of DNA damage, trapping intermediates associated with DNA repair and chromosome segregation pathways remains very likely. Thus SM10 may have distinct but synergistic modes of action.
Highlights
IntroductionBacteria may face many environmental challenges in the form of toxic chemicals (naturally occurring or man-made) or physical conditions (suboptimal pH, desiccation, UV or other irradiation, etc.)
During their lifetime, bacteria may face many environmental challenges in the form of toxic chemicals or physical conditions
To further characterize the antimicrobial mechanism of SM10, we studied its effect on the viability of E. coli MG1655 and showed that it inhibits growth severely at 20–30 mg/ ml, and is bactericidal at 30 mg/ml based on a reduction in viability by more than 99.9% compared to starting cell counts (Figure 1B)
Summary
Bacteria may face many environmental challenges in the form of toxic chemicals (naturally occurring or man-made) or physical conditions (suboptimal pH, desiccation, UV or other irradiation, etc.). Envelope stress guards the integrity of the cell’s membranes and of the cell itself, and is mediated through the sigma factor sE [6,7,8,9] and the two component signal transduction systems CpxA/ R [6,10,11,12] and BaeR/S [13,14,15], which respond to both unique and overlapping signals. These three cross–regulate factors (e.g., proteases and chaperones) that protect and restore the integrity of the bacterial envelope [7,10,16]. While the envelope stress response was discovered as the means to repair damage due to over-expression of major bacterial porins [17], more recently it has been implicated during bacterial growth in the presence of antibiotics [18,19,20,21]
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