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A switch in the poly(dC)/RmlB complex regulates bacterial persister formation.

ABSTRACT: Bacterial persisters are phenotypic variants that tolerate exposure to lethal antibiotics. These dormant cells are responsible for chronic and recurrent infections. Multiple mechanisms have been linked to persister formation. Here, we report that a complex, consisting of an extracellular poly(dC) and its membrane-associated binding protein RmlB, appears to be associated with persistence of the opportunistic pathogen Pseudomonas aeruginosa. Environmental stimuli triggers a switch in the complex physiological state (from poly(dC)/RmlB to P-poly(dC)/RmlB or RmlB). In response to the switch, bacteria decrease proton motive force and intracellular ATP levels, forming dormant cells. This alteration in complex status is linked to a (p)ppGpp-controlled signaling pathway that includes inorganic polyphosphate, Lon protease, exonuclease VII (XseA/XseB), and the type III secretion system. The persistence might be also an adaptive response to the lethal action of the dTDP-L-rhamnose pathway shutdown, which occurs due to switching of poly(dC)/RmlB.

SUBMITTER: Chen X

PROVIDER: S-EPMC6318315 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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A switch in the poly(dC)/RmlB complex regulates bacterial persister formation.

Chen Xu X Li Gen G Liao Xuewei X Fang Jie J Li Bo B Yu Shanshan S Sun Mingming M Wu Jun J Zhang Lihao L Hu Yi Y Jiao Jiaguo J Liu Ting T Xu Li L Chen Xiaoyun X Liu Manqiang M Li Huixin H Hu Feng F Sun Kouhong K

Nature communications 20190103 1

Bacterial persisters are phenotypic variants that tolerate exposure to lethal antibiotics. These dormant cells are responsible for chronic and recurrent infections. Multiple mechanisms have been linked to persister formation. Here, we report that a complex, consisting of an extracellular poly(dC) and its membrane-associated binding protein RmlB, appears to be associated with persistence of the opportunistic pathogen Pseudomonas aeruginosa. Environmental stimuli triggers a switch in the complex p ...[more]

PMID: 30604752

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Project description:Persisters represent a small subpopulation of cells within a bacterial culture that are tolerant to killing by antibiotics. Persisters have been linked to recalcitrant infections caused by numerous bacterial pathogens, including Pseudomonas aeruginosa A classic example is the incurable infection of the airways for patients with cystic fibrosis. The genetic mediators of persister formation for P. aeruginosa are poorly understood. We generated a high-density transposon insertion library of P. aeruginosa PAO1 and determined the relative frequency of each insertion following fluoroquinolone treatment using transposon sequencing (Tn-seq). Of the 4,411 disrupted genes included in the screen, 137 had a ≥10-fold impact on survival. The gene disruption that resulted in the lowest survival rate was disruption of carB, which codes for the large subunit of carbamoyl phosphate synthetase (CPSase). CPSase is a metabolic enzyme that is involved in pyrimidine and arginine synthesis. Disruption of carB resulted in survival rates that were reduced by up to 2,500-fold following antibiotic treatment, and this phenotype was abolished by the addition of uracil, highlighting the importance of de novo pyrimidine biosynthesis for persister formation. Disruption of carB resulted in intracellular ATP accumulation, and lowering ATP levels using arsenate restored the antibiotic tolerance profile of the mutant to levels similar to those seen with the wild type. A decrease in ATP would lead to reduced antibiotic target activity and increased survival.IMPORTANCE Antibiotic treatment of P. aeruginosa residing in the lung of cystic fibrosis patients is ineffective. Treatment failure is attributed in part to antibiotic-tolerant phenotypic variants known as persister cells. Understanding how these cells emerge will likely inform future therapeutic strategies. In the current study, we identified carB, which codes for the large subunit of carbamoyl-phosphate synthetase, as a persister gene that contributes to multidrug tolerance in P. aeruginosa Disruption of carB resulted in a metabolic perturbation that increased cellular ATP and reduced persister formation. Conversely, lowering ATP in the mutant restored antibiotic tolerance. Our data support the hypothesis that a drop in intracellular ATP is a general mechanism of persister formation in bacteria.

Project description:Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%-11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B--an antibiotic that is believed to act on non-dividing cells as well--failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy.

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A switch in the poly(dC)/RmlB complex regulates bacterial persister formation.

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A switch in the poly(dC)/RmlB complex regulates bacterial persister formation.

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