Dataset Information

HigB of Pseudomonas aeruginosa Enhances Killing of Phagocytes by Up-Regulating the Type III Secretion System in Ciprofloxacin Induced Persister Cells.

ABSTRACT: Bacterial persister cells are dormant and highly tolerant to lethal antibiotics, which are believed to be the major cause of recurring and chronic infections. Activation of toxins of bacterial toxin-antitoxin systems inhibits bacterial growth and plays an important role in persister formation. However, little is known about the overall gene expression profile upon toxin activation. More importantly, how the dormant bacterial persisters evade host immune clearance remains poorly understood. Here we demonstrate that a Pseudomonas aeruginosa toxin-antitoxin system HigB-HigA is required for the ciprofloxacin induced persister formation. Transcriptome analysis of a higA::Tn mutant revealed up regulation of type III secretion systems (T3SS) genes. Overexpression of HigB increased the expression of T3SS genes as well as bacterial cytotoxicity. We further demonstrate that wild type bacteria that survived ciprofloxacin treatment contain higher levels of T3SS proteins and display increased cytotoxicity to macrophage compared to vegetative bacterial cells. These results suggest that P. aeruginosa accumulates T3SS proteins during persister formation, which can protect the persister cells from host clearance by efficiently killing host immune cells.

SUBMITTER: Li M

PROVIDER: S-EPMC5064212 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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HigB of <i>Pseudomonas aeruginosa</i> Enhances Killing of Phagocytes by Up-Regulating the Type III Secretion System in Ciprofloxacin Induced Persister Cells.

Li Mei M Long Yuqing Y Liu Ying Y Liu Yang Y Chen Ronghao R Shi Jing J Zhang Lu L Jin Yongxin Y Yang Liang L Bai Fang F Jin Shouguang S Cheng Zhihui Z Wu Weihui W

Frontiers in cellular and infection microbiology 20161014

Bacterial persister cells are dormant and highly tolerant to lethal antibiotics, which are believed to be the major cause of recurring and chronic infections. Activation of toxins of bacterial toxin-antitoxin systems inhibits bacterial growth and plays an important role in persister formation. However, little is known about the overall gene expression profile upon toxin activation. More importantly, how the dormant bacterial persisters evade host immune clearance remains poorly understood. Here ...[more]

PMID: 27790409

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Project description:Biofilms are commonly recalcitrant to antibiotics, through incompletely elucidated mechanisms such as tolerance and persistence. We aimed at investigating how a Pseudomonas aeruginosa biofilm escapes ciprofloxacin treatment. P. aeruginosa PA14 in vitro mature biofilms were challenged with supra-MIC ciprofloxacin concentrations. Cell viability was quantified by fluorescein diacetate assay. Population dynamics were determined by counts of surviving culturable cells. Biofilms were analyzed using confocal laser scanning microscopy (CLSM), and the expression of genes involved in stringent response, toxin-antitoxin HigB/HigA, and type 3 secretion system (T3SS) was quantified by RT-qPCR in untreated and treated biofilms. Ciprofloxacin exposure resulted in an initial reduction of bacterial counts following a biphasic time-kill curve. After 24 h of treatment, the overall cell activity and the density of culturable cells significantly decreased as compared to untreated biofilm. No resistant mutant was isolated among the <1% surviving cells. Phenotypic adaptation toward persistence appeared to start after only 1 h of antibiotic exposure, by an overexpression of the genes involved in stringent response and in the toxin-antitoxin system, whereas the expression of genes encoding for the T3SS remained unchanged. After 4 h of ciprofloxacin exposure, stringent response genes returned to their basal level of expression. After a prolonged ciprofloxacin exposure, a deep alteration in the matrix structure that became thinner and lost mushroom-like aggregates was observed, in relation with reduced biovolumes of exopolysaccharides and extracellular DNA. These results support that ciprofloxacin might first induce the bacterial killing of most bacterial cells, but simultaneously activate stringent response mechanisms contributing to the switch of a subpopulation toward a persister phenotype. Once the persister phenotype is expressed, and despite an unexpected alteration of the biofilm matrix, ciprofloxacin fails to eradicate biofilm.

Project description:OUTLINE:In chronic lung infections by Pseudomonas aeruginosa (PA) the bacteria thrive in biofilm structures protected from the immune system of the host and from antibiotic treatment. Increasing evidence suggests that the susceptibility of the bacteria to antibiotic treatment can be significantly enhanced by hyperbaric oxygen treatment. The aim of this study is to simulate the effect of ciprofloxacin treatment in a PAO1 biofilm model with aggregates in agarose when combined with hyperbaric oxygen treatment. This is achieved in a reaction-diffusion model that describes the combined effect of ciprofloxacin diffusion, oxygen diffusion and depletion, bacterial growth and killing, and adaptation of the bacteria to ciprofloxacin. In the model, the oxygen diffusion and depletion use a set of parameters derived from experimental results presented in this work. The part of the model describing ciprofloxacin killing uses parameter values from the literature in combination with our estimates (Jacobs, et al., 2016; Grillon, et al., 2016). Micro-respirometry experiments were conducted to determine the oxygen consumption in the P. aeruginosa strain PAO1. The parameters were validated against existing data from an HBOT experiment by Kolpen et al. (2017). The complete oxygen model comprises a reaction-diffusion equation describing the oxygen consumption by using a Michaelis-Menten reaction term. The oxygen model performed well in predicting oxygen concentrations in both time and depth into the biofilm. At 2.8 bar pure oxygen pressure, HBOT increases the penetration depth of oxygen into the biofilm almost by a of factor 4 in agreement with the scaling that follows from the stationary balance between the consumption term and diffusion term. CONCLUSION:In the full reaction-diffusion model we see that hyperbaric oxygen treatment significantly increases the killing by ciprofloxacin in a PAO1 biofilm in alignment with the experimental results from Kolpen et al. (Kolpen, et al., 2017; Kolpen, et al. 2016). The enhanced killing, in turn, lowers the oxygen consumption in the outer layers of the biofilm, and leads to even deeper penetration of oxygen into the biofilm.

Project description:Factor for inversion stimulation (Fis) is a versatile DNA binding protein that plays an important role in coordinating bacterial global gene expression in response to growth phases and environmental stresses. Previously, we demonstrated that Fis regulates the type III secretion system (T3SS) in Pseudomonas aeruginosa In this study, we explored the role of Fis in the antibiotic resistance of P. aeruginosa and found that mutation of the fis gene increases the bacterial susceptibility to ciprofloxacin. We further demonstrated that genes related to pyocin biosynthesis are upregulated in the fis mutant. The pyocins are produced in response to genotoxic agents, including ciprofloxacin, and the release of pyocins results in lysis of the producer cell. Thus, pyocin biosynthesis genes sensitize P. aeruginosa to ciprofloxacin. We found that PrtN, the positive regulator of the pyocin biosynthesis genes, is upregulated in the fis mutant. Genetic experiments and electrophoretic mobility shift assays revealed that Fis directly binds to the promoter region of prtN and represses its expression. Therefore, our results revealed novel Fis-mediated regulation on pyocin production and bacterial resistance to ciprofloxacin in P. aeruginosaIMPORTANCEPseudomonas aeruginosa is an important opportunistic pathogenic bacterium that causes various acute and chronic infections in human, especially in patients with compromised immunity, cystic fibrosis (CF), and/or severe burn wounds. About 60% of cystic fibrosis patients have a chronic respiratory infection caused by P. aeruginosa The bacterium is intrinsically highly resistant to antibiotics, which greatly increases difficulties in clinical treatment. Therefore, it is critical to understand the mechanisms and the regulatory pathways that are involved in antibiotic resistance. In this study, we elucidated a novel regulatory pathway that controls the bacterial resistance to fluoroquinolone antibiotics, which enhances our understanding of how P. aeruginosa responds to ciprofloxacin.

Project description:UnlabelledThe opportunistic pathogen Pseudomonas aeruginosa utilizes an injectisome-type III secretion system (injectisome-T3SS) to elicit cytotoxicity toward epithelial cells and macrophages. Macrophage killing results from the cytotoxic properties of the translocated effector proteins (ExoS, ExoT, ExoU, and ExoY) and inflammasome-mediated induction of pyroptosis. Inflammasome activation can occur following Nlrc4-mediated recognition of cytosolic translocated flagellin (FliC). In the present study, we demonstrate that FliC is a secretion substrate of both the injectisome- and flagellum-associated T3SSs. Molecular analyses indicate that the first 20 amino-terminal residues of FliC are sufficient for secretion by the injectisome-T3SS and that the first 100 residues are sufficient for translocation of FliC into host cells. Although maximal inflammasome activation requires FliC, activation can also occur in the absence of FliC. This prompted us to examine whether other flagellar components might also be translocated into cells to elicit inflammasome activation. Indeed, we find that the flagellar cap (FliD), hook-associated (FlgK and FlgL), hook (FlgE), and rod (FlgE) proteins are secretion substrates of the injectisome-T3SS. None of these proteins, however, result in increased inflammasome activation when they are overexpressed in a fliC mutant and appear to be translocated into host cells. While a role in inflammasome activation has been excluded, these data raise the possibility that flagellar components, which are highly conserved between different bacterial species, trigger other specific host responses from the extracellular milieu or contribute to the pathogenesis of P. aeruginosa.ImportanceThe inflammasome is a host defense mechanism that recognizes invading bacteria and triggers an inflammatory immune response. The opportunistic pathogen P. aeruginosa produces both inflammasome agonists and antagonists. In this study, we demonstrate that overexpression of an agonist suppresses the activity of an antagonist, thereby resulting in inflammasome activation. Since the relative expression levels of agonists and antagonists likely vary between strains, these differences could be important predictors of whether a particular P. aeruginosa strain elicits inflammasome activation.

Dataset Information

HigB of Pseudomonas aeruginosa Enhances Killing of Phagocytes by Up-Regulating the Type III Secretion System in Ciprofloxacin Induced Persister Cells.

Publications

HigB of <i>Pseudomonas aeruginosa</i> Enhances Killing of Phagocytes by Up-Regulating the Type III Secretion System in Ciprofloxacin Induced Persister Cells.

Similar Datasets

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