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Beyond self-resistance: ABCF ATPase LmrC is a signal-transducing component of an antibiotic-driven signaling cascade hastening the onset of lincomycin biosynthesis

ABSTRACT: In natural environments, antibiotics are an important instrument of inter-species competition. At subinhibitory concentrations, they act as cues or signals inducing antibiotic production: however, our knowledge of well-documented antibiotic-based sensing systems is limited. Here, for the soil actinobacterium Streptomyces lincolnensis we describe a fundamentally new ribosome-mediated signaling cascade that accelerates the onset of lincomycin production in response to an external ribosome-targeting antibiotic to synchronize the antibiotic production within the population. The entire cascade is encoded within the lincomycin biosynthetic gene cluster (BGC) and besides the transcriptional regulator, LmbU it consists of three lincomycin resistance proteins: a lincomycin transporter, LmrA, a 23S rRNA methyltransferase, LmrB, both conferring a high resistance, and an ABCF ATPase LmrC that confers only moderate resistance but is indispensable for the antibiotic-induced signal transduction. Specifically, the antibiotic sensing occurs via a ribosome-mediated attenuation, which activates LmrC production in response to lincosamide, streptogramin A, or pleuromutilin antibiotics. Then, the ribosome-operating LmrC ATPase activity triggers the transcription of lmbU and consequently the expression of lincomycin BGC. Finally, the production of LmrC is downregulated by LmrA and LmrB which reduces the amount of the ribosome-bound antibiotic and thus fine-tune the cascade. We propose that analogous ABCF-mediated signaling systems are relatively common because many BGCs for ribosome-targeting antibiotics encode an ABCF-protein accompanied by additional resistance protein(s) and transcriptional regulators. Moreover, we revealed that three of eight co-produced ABCF proteins of S. lincolnensis are clindamycin-responsive thus the ABCF-mediated antibiotic signaling might be generally utilized tool of chemical communication.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Streptomyces Lincolnensis

SUBMITTER: Karel Harant

LAB HEAD: Gabriela Balíková Novotná, PhD.

PROVIDER: PXD026093 | Pride | 2021-08-18

REPOSITORIES: Pride

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Project description:Tan2012 - Antibiotic Treatment, Inoculum Effect The efficacy of many antibiotics decreases with increasing bacterial density, a phenomenon called the ‘inoculum effect’ (IE). This study reveals that, for ribosome-targeting antibiotics, IE is due to bistable inhibition of bacterial growth, which reduces the efficacy of certain treatment frequencies. This model is described in the article: The inoculum effect and band-pass bacterial response to periodic antibiotic treatment. Tan C, Phillip Smith R, Srimani JK, Riccione KA, Prasada S, Kuehn M, You L. Mol Syst Biol. 2012 Oct 9; 8:617 Abstract: The inoculum effect (IE) refers to the decreasing efficacy of an antibiotic with increasing bacterial density. It represents a unique strategy of antibiotic tolerance and it can complicate design of effective antibiotic treatment of bacterial infections. To gain insight into this phenomenon, we have analyzed responses of a lab strain of Escherichia coli to antibiotics that target the ribosome. We show that the IE can be explained by bistable inhibition of bacterial growth. A critical requirement for this bistability is sufficiently fast degradation of ribosomes, which can result from antibiotic-induced heat-shock response. Furthermore, antibiotics that elicit the IE can lead to 'band-pass' response of bacterial growth to periodic antibiotic treatment: the treatment efficacy drastically diminishes at intermediate frequencies of treatment. Our proposed mechanism for the IE may be generally applicable to other bacterial species treated with antibiotics targeting the ribosomes. This model is hosted on BioModels Database and identified by: MODEL1208300000 . To cite BioModels Database, please use: BioModels Database: An enhanced, curated and annotated resource for published quantitative kinetic models . To the extent possible under law, all copyright and related or neighbouring rights to this encoded model have been dedicated to the public domain worldwide. Please refer to CC0 Public Domain Dedication for more information.

Project description:Deeper understanding of antibiotic-induced physiological responses is critical to identifying means for enhancing our current antibiotic arsenal. Bactericidal antibiotics with diverse targets have been hypothesized to kill bacteria, in part, by inducing production of damaging reactive species. This notion has been supported by many groups, but recently challenged. Here we robustly test the hypothesis using biochemical, enzymatic and biophysical assays along with genetic and phenotypic experiments. We first used a novel intracellular hydrogen peroxide (H2O2) sensor, together with a chemically diverse panel of fluorescent dyes sensitive to an array of reactive species, to demonstrate that antibiotics broadly induce redox stress. Subsequent gene expression analyses reveal that complex antibiotic-induced oxidative stress responses are distinct from canonical responses generated by supra-physiological levels of H2O2. We next developed a method to dynamically quantify cellular respiration and found that bactericidal antibiotics elevate oxygen consumption, indicating significant alterations to bacterial redox physiology. We further show that catalase or DNA mismatch repair enzyme overexpression, as well as antioxidant pre-treatment limit antibiotic lethality, indicating that reactive oxygen species causatively contribute to antibiotic killing. Critically, the killing efficacy of antibiotics was diminished under strict anaerobic conditions, but could be enhanced by exposure to molecular oxygen or addition of alternative electron acceptors, suggesting that environmental factors play a role in killing cells physiologically primed for death. This work provides direct evidence that bactericidal antibiotics, downstream of their target-specific interactions, induce complex redox alterations that contribute to cellular damage and death, thus supporting an evolving, expanded model of antibiotic lethality. Here, we used microarrays to analyze oxidative stress responses to bactericidal antibiotic treatment in wildtype and mutant E coli WT or mutant E coli cells were grown to OD ~0.3. Untreated cells were harvested at time 0 as controls. Treated cells given the appropriate chemical perturbation and harvested 1 hour post-treatment. All experiments were performed in technical triplicate.

			Action	DRS
	1.raw	Raw
	10.CID.ITMS.peak.apl	Other
	10.HCD.FTMS.peak.apl	Other
	10.HCD.ITMS.peak.apl	Other
	10.raw	Raw

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Beyond self-resistance: ABCF ATPase LmrC is a signal-transducing component of an antibiotic-driven signaling cascade hastening the onset of lincomycin biosynthesis

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