Reducing the Level of Undecaprenyl Pyrophosphate Synthase Has Complex Effects on Susceptibility to Cell Wall Antibiotics

Abstract

Undecaprenyl pyrophosphate synthase (UppS) catalyzes the formation of the C 55 lipid carrier (UPP) that is essential for bacterial peptidoglycan biosynthesis. We selected here a vancomycin (VAN)-resistant derivative of Bacillus subtilis W168 that contains a single-point mutation in the ribosome-binding site of the uppS gene designated uppS1. Genetic reconstruction experiments demonstrate that the uppS1 allele is sufficient to confer low-level VAN resistance and causes reduced UppS translation. The decreased level of UppS renders B. subtilis slightly more susceptible to many late-acting cell wall antibiotics, including ␤-lactams, but significantly more resistant to fosfomycin and D-cycloserine, antibiotics that interfere with the very early steps of cell wall synthesis. We further show that the uppS1 allele leads to slightly elevated expression of the M regulon, possibly helping to compensate for the stress caused by a decrease in UPP levels. Notably, the uppS1 mutation increases resistance to VAN, fosfomycin, and D-cycloserine in wild-type cells, but this effect is greatly reduced or eliminated in a sigM mutant background. Our findings suggest that, although UppS is an attractive antibacterial target, incomplete inhibition of UppS function may lead to increased resistance to some cell wall-active antibiotics.

MurJ and a novel lipid II flippase are required for cell wall biogenesis inBacillus subtilis

“…However, recent work has suggested that available undecaprenylphosphate might serve as the signal that is sensed by the anti-σ M factors YhdK/L to maintain inhibition of σ M (22,23). Our results are consistent with this idea.…”

“…amj is regulated by the stress-response sigma factor σ M . σ M is held inactive by the two integral membrane proteins YhdL and YhdK, and inhibition is relieved in response to an unknown signal that is thought to be generated by cell wall stress (21)(22)(23). Expression profiling experiments indicate that σ M -controlled genes are transcribed at low levels under normal growth conditions and are induced twoto fivefold in response to acute exposure to cell wall synthesis inhibitors (19,24).…”

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

“…Specifically, the M regulon is selectively induced by UPP-Pase depletion, with comparatively weak effects noted for other ECF factor stress responses. Induction of the M regulon is consistent with the inclusion of the BcrC UPP-Pase as part of this regulon (19), as well as the previously noted induction in cells treated with bacitracin (48) or containing mutations that decrease UPP synthesis (17). In contrast with M , the W regulon, most strongly activated by membrane stress conditions (50), was downregulated in the depleted cells compared to its expression in the control.…”

“…The depletion strain was not significantly more sensitive to several other tested stress conditions, including compounds that elicit membrane stress (nisin, SDS, and Triton X-100). The reason for the increased sensitivity to some but not other peptidoglycan synthesis inhibitors is presently unclear, but interpreting such effects can be complex, as discussed previously (17).…”

“…Recent in silico, in vitro, and in vivo approaches have identified inhibitors of UppS (13)(14)(15), including a method that used clustered regularly interspersed short palindromic repeat (CRISPR) interference (CRISPRi) to identify drug targets (16). We demonstrated previously that a ribosome-binding-site (RBS) mutation that decreased the expression of UppS led to vancomycin resistance and activation of the M -dependent cell envelope stress response (17). Compounds that inhibit the recycling of UPP may also serve as effective antibiotics.…”