Abstract
Abstract Background Compared to other Firmicutes, enterococci are often tolerant to many antibiotics; combination therapy is used for severe, deep-seated infections. We have previously reported the LiaFSR system, a three-component cell-envelop stress regulatory system, mediates resistance and tolerance to daptomycin (DAP). The main effector, LiaX, harbors distinct N- and C-terminal domains. The N-terminal portion of LiaX is released into the extracellular milieu where it recognizes DAP and other antimicrobial peptides functioning as a sentinel and signal transduction protein to decrease DAP-mediated killing. LiaX likely acts through LiaF, a transmembrane protein necessary for the LiaX effect. We postulated that the LiaX-LiaF relay system may be involved in the β-lactam tolerance shown by E. faecalis (Efs).Figure 1.N-terminal domain of LiaX protects E. faecalis from ampicillin and ceftriaxone. Methods We used wild type (WT) Efs OG117 and derivatives: OG117liaF*11-14 (liaF deletion) and OG117 liaF*11-14::liaF (liaF complemented). Ampicillin (AMP) and ceftriaxone (CRO) broth-microdilution MICs were performed. N-terminal LiaX1-275 was expressed in E. coli and purified. Killing assays (5 replicates) with 0.5 X MICs of AMP and CRO were done, with and without LiaX1-275 (80 nM). Bacteria were diluted (∼1x105 CFU/mL) and used to inoculate test media containing antibiotic alone, antibiotic+LiaX1-275, and no antibiotic. After 2-hours of incubation at 37°C, samples were plated on BHI agar for enumeration. Results The CRO MICs for WT and complemented strains were 64 μg/mL, whereas it was 2-fold higher (128 μg/mL) for OG117liaF*11-14. All strains showed AMP MICs of 1 μg/mL. In the AMP killing assay, OG117 survival increased with the addition of LiaX1-275 (p < .005). The addition of LiaX1-275 failed to increase survival of OG117liaF*11-14 in the presence of AMP compared to WT (p = 0.4). The survival advantage was restored in the complemented strain (p < .0001). CRO showed a similar trend, i.e., increased survival in OG117 (p < .01) and complemented strain (p < .001), but not in OG117liaF*11-14 (p = 0.29). Conclusion We have discovered a novel pathway in Efs for antibiotic tolerance. The LiaF-LiaX signal relay, part of the enterococcal response to antibiotics, boosts organism survival, contributing to tolerance to multiple anti-enterococcal drugs. Disclosures William R. Miller, M.D., Merck: Grant/Research Support|UptoDate: Royalties
Published Version
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