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Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa uses quorum sensing to regulate the expression of virulence factors. In static environments, spatial structures, such as biofilms, can increase the expression of these virulence factors. However, in natural settings, biofilms are exposed to physical forces that disrupt spatial structure, which may affect the expression of virulence factors regulated by quorum sensing. We show that periodically disturbing biofilms composed of P. aeruginosa using a physical force reduces the expression of quorum sensing-regulated virulence factors. At an intermediate disturbance frequency, the expression of virulence factors in the las, rhl, and pqs regulons is reduced. Mathematical modeling suggests that perturbation of the pqsR receptor is critical for this reduction. Removing the lasR receptor enhances the reduction in the expression of virulence factors as a result of disturbance. Our results allow identification of environments where virulence is reduced and implicate the lasR receptor as having a buffering role against disturbance.

SUBMITTER: Garcia-Dieguez L

PROVIDER: S-EPMC10225924 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa.

García-Diéguez Laura L Diaz-Tang Gabriela G Marin Meneses Estefania E Cruise Vanessa V Barraza Ivana I Craddock Travis J A TJA Smith Robert P RP

iScience 20230511 6

Pseudomonas aeruginosa uses quorum sensing to regulate the expression of virulence factors. In static environments, spatial structures, such as biofilms, can increase the expression of these virulence factors. However, in natural settings, biofilms are exposed to physical forces that disrupt spatial structure, which may affect the expression of virulence factors regulated by quorum sensing. We show that periodically disturbing biofilms composed of P. aeruginosa using a physical for ...[more]

PMID: 37255658

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Project description:UnlabelledIn Pseudomonas aeruginosa, the transcription factor Anr controls the cellular response to low oxygen or anoxia. Anr activity is high in oxygen-limited environments, including biofilms and populations associated with chronic infections, and Anr is necessary for persistence in a model of pulmonary infection. In this study, we characterized the Anr regulon in biofilm-grown cells at 1% oxygen in the laboratory strain PAO1 and in a quorum sensing (QS)-deficient clinical isolate, J215. As expected, transcripts related to denitrification, arginine fermentation, high-affinity cytochrome oxidases, and CupA fimbriae were lower in the Δanr derivatives. In addition, we observed that transcripts associated with quorum sensing regulation, iron acquisition and storage, type VI secretion, and the catabolism of aromatic compounds were also differentially expressed in the Δanr strains. Prior reports have shown that quorum sensing-defective mutants have higher levels of denitrification, and we found that multiple Anr-regulated processes, including denitrification, were strongly inversely proportional to quorum sensing in both transcriptional and protein-based assays. We also found that in LasR-defective strains but not their LasR-intact counterparts, Anr regulated the production of the 4-hydroxy-2-alkylquinolines, which play roles in quorum sensing and interspecies interactions. These data show that Anr was required for the expression of important metabolic pathways in low-oxygen biofilms, and they reveal an expanded and compensatory role for Anr in the regulation of virulence-related genes in quorum sensing mutants, such as those commonly isolated from infections.ImportancePseudomonas aeruginosa causes acute ocular, soft tissue, and pulmonary infections, as well as chronic infections in the airways of cystic fibrosis patients. P. aeruginosa uses quorum sensing (QS) to regulate virulence, but mutations in the gene encoding the master regulator of QS, lasR, are frequently observed in clinical isolates. We demonstrated that the regulon attributed to Anr, an oxygen-sensitive transcription factor, was more highly expressed in lasR mutants. Furthermore, we show that Anr regulates the production of several different secreted factors in lasR mutants. These data demonstrate the importance of Anr in naturally occurring quorum sensing mutants in the context of chronic infections.

Project description:With the rising development of bacterial resistance the search for new medical treatments beyond conventional antimicrobials has become a key aim of public health research. Possible innovative strategies include the inhibition of bacterial virulence. However, consideration must be given to the evolutionary and environmental consequences of such new interventions. Virulence and cooperative social behaviour of the bacterium Pseudomonas aeruginosa rely on the quorum-sensing (QS) controlled production of extracellular products (public goods). Hence QS is an attractive target for anti-virulence interventions. During colonization, non-cooperating (and hence less virulent) P. aeruginosa QS-mutants, benefiting from public goods provided by wild type isolates, naturally increase in frequency providing a relative protection from invasive infection. We hypothesized that inhibition of QS-mediated gene expression removes this growth advantage and selection of less virulent QS-mutants, and maintains the predominance of more virulent QS-wild type bacteria. We addressed this possibility in a placebo-controlled trial investigating the anti-QS properties of azithromycin, a macrolide antibiotic devoid of bactericidal activity on P. aeruginosa, but interfering with QS, in intubated patients colonized by P. aeruginosa. In the absence of azithromycin, non-cooperating (and hence less virulent) lasR (QS)-mutants increased in frequency over time. Azithromycin significantly reduced QS-gene expression measured directly in tracheal aspirates. Concomitantly the advantage of lasR-mutants was lost and virulent wild-type isolates predominated during azithromycin treatment. We confirmed these results in vitro with fitness and invasion experiments. Azithromycin reduced growth rate of the wild-type, but not of the lasR-mutant. Furthermore, the lasR-mutant efficiently invaded wild-type populations in the absence, but not in the presence of azithromycin. These in vivo and in vitro results demonstrate that anti-virulence interventions based on QS-blockade diminish natural selection towards reduced virulence and therefore may increase the prevalence of more virulent genotypes in the Hospital environment. More generally, the impact of intervention on the evolution of virulence of pathogenic bacteria should be assessed.

Project description:Pseudomonas aeruginosa is a common opportunistic pathogen with growing resistance and presents heightened treatment challenges. Quorum sensing (QS) is a cell-to-cell communication system that contributes to the production of a variety of virulence factors and is also related to biofilm formation of P. aeruginosa. Compared to traditional antibiotics which kill bacteria directly, the anti-virulence strategy by targeting QS is a promising strategy for combating pseudomonal infections. In this study, the QS inhibition potential of the compounds derived from the Traditional Chinese Medicines was evaluated by using in silico, in vitro, and in vivo analyses. The results showed that psoralen, a natural furocoumarin compound derived from Psoralea corylifolia L., was capable of simultaneously inhibiting the three main QS regulators, LasR, RhlR, and PqsR of P. aeruginosa. Psoralen had no bactericidal activity but could widely inhibit the production of extracellular proteases, pyocyanin, and biofilm, and the cell motilities of the model and clinical P. aeruginosa strains. RNA-sequencing and quantitative PCR analyses further demonstrated that a majority of QS-activated genes in P. aeruginosa were suppressed by psoralen. The supplementation of psoralen could protect Caenorhabditis elegans from P. aeruginosa challenge, especially for the hypervirulent strain PA14. Moreover, psoralen showed synergistic antibacterial effects with polymyxin B, levofloxacin, and kanamycin. In conclusions, this study identifies the anti-QS and antibiofilm effects of psoralen against P. aeruginosa strains and sheds light on the discovery of anti-pseudomonal drugs among Traditional Chinese Medicines. KEY POINTS: • Psoralen derived from Psoralea corylifolia L. inhibits the virulence-related phenotypes of P. aeruginosa. • Psoralen simultaneously targets the three core regulators of P. aeruginosa QS system and inhibits the expression of a large part of downstream genes. • Psoralen protects C. elegans from P. aeruginosa challenge and enhances the susceptibility of P. aeruginosa to antibiotics.

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Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in Pseudomonas aeruginosa.

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Periodically disturbing biofilms reduces expression of quorum sensing-regulated virulence factors in <i>Pseudomonas aeruginosa</i>.

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