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Quorum quenching activity of Bacillus cereus isolate 30b confers antipathogenic effects in Pseudomonas aeruginosa.

ABSTRACT: Background: Quorum quenching, the interference of a Quorum sensing (QS) system that contributes to the pathogenesis through triggering the production of various virulence determinants, is among the newly suggested antivirulence strategies. Purpose: This study aimed at screening of N-Acyl homoserine lactonase activity from local bacterial isolate, and investigating its effect on Pseudomonas aeruginosa (P. aeruginosa) virulence and biofilm formation. Materials and methods: Soil bacteria were screened for aiiA gene coding for lactonase enzyme by Polymerase Chain reaction and sequencing of aiiA gene homologs. Lactonase activity and spectrum were assessed in the cell-free lysate by well diffusion assay using Agrobacterium tumafaciens KYC55. A bacterial isolate showing the highest N-acyl-homoserine lactones degradation percentage was identified by gene amplification and sequencing of the 16S rRNA gene and its aiiA gene homolog. High performance liquid chromatography was used to confirm N-acyl-homoserine lactone degradation. The effect of cell-free lysate on the biofilm formation ability and cytotoxicity of P. aeruginosa PAO1 and P. aeruginosa clinical isolates from different clinical sources were assessed by static microtiter plate and viability assay, respectively Results: Lactonase gene and activity were identified in three Bacillus spp. isolates. They showed broad catalytic activities against tested N-acyl-homoserine lactones. However, The lactonase activity in the cell- free lysate of isolate 30b showed the highest significant degradation percentage on all tested signals; N-butanoyl-L-homoserine lactone (71%), N-hexanoyl-l-homoserine lactone (100%), N-decanoyl-homoserine lactone (100%), N-(3-oxohexanoyl)-L-homoserine lactone (37.5%), N-(oxodecanoyl)-L-homoserine lactone (100%), and N-(3-oxododecanoyl)-L-homoserine lactone (100%). Alignment of the amino acid sequences of AiiA protein of isolate 30b showed 96% identity with Bacillus cereus (B. cereus) homologous lactonases in the GenBank database, and the isolate was designated as B. cereus isolate 30b. Cell-free lysate of B. cereus isolate 30b reduced biofilm formation significantly in 93% of P. aeruginosa isolates. The highest mean percentage of reduction in the biofilm was 86%. Moreover, the viability percentage of human lung carcinoma A549 cells infected by P. aeruginosa and treated with cell-free lysate of B. cereus isolate 30b increased up to 15%. Conclusion: The results of this study highlight the potential of lactonases as a promising strategy to combat Pseudomonas aeruginosa virulence.

SUBMITTER: Raafat MM

PROVIDER: S-EPMC6559722 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Quorum quenching activity of Bacillus cereus isolate 30b confers antipathogenic effects in Pseudomonas aeruginosa.

Raafat Marwa M MM Ali-Tammam Marwa M Ali Amal E AE

Infection and drug resistance 20190607

Background: Quorum quenching, the interference of a Quorum sensing (QS) system that contributes to the pathogenesis through triggering the production of various virulence determinants, is among the newly suggested antivirulence strategies. Purpose: This study aimed at screening of N-Acyl homoserine lactonase activity from local bacterial isolate, and investigating its effect on Pseudomonas aeruginosa (P. aeruginosa) virulence and biofilm formation. Materials and me ...[more]

PMID: 31239733

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Project description:Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine antibacterials with agents that decrease pathogen virulence via the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen Pseudomonas aeruginosa. Here we describe the characteristics of heterologously expressed YtnP lactonase from Bacillus paralicheniformis ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL–1 concentration reduced the amount of P. aeruginosa-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of P. aeruginosa PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in P. aeruginosa PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL–1 improved the survival of P. aeruginosa PAO1-infected zebrafish (Danio rerio), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of P. aeruginosa PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL–1 YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated P. aeruginosa infections.

Project description:Pseudomonas aeruginosa uses quorum sensing (QS) to modulate the expression of several virulence factors that enable it to establish severe infections. The QS system in P. aeruginosa is complex, intricate and is dominated by two main N-acyl-homoserine lactone circuits, LasRI and RhlRI. These two QS systems work in a hierarchical fashion with LasRI at the top, directly regulating RhlRI. Together these QS circuits regulate several virulence associated genes, metabolites, and enzymes in P. aeruginosa. Paradoxically, LasR mutants are frequently isolated from chronic P. aeruginosa infections, typically among cystic fibrosis (CF) patients. This suggests P. aeruginosa can undergo significant evolutionary pathoadaptation to persist in long term chronic infections. In contrast, mutations in the RhlRI system are less common. Here, we have isolated a clinical strain of P. aeruginosa from a CF patient that has deleted the transcriptional regulator RhlR entirely. Whole genome sequencing shows the rhlR locus is deleted in PA80 alongside a few non-synonymous mutations in virulence factors including protease lasA and rhamnolipid rhlA, rhlB, rhlC. Importantly we did not observe any mutations in the LasRI QS system. PA80 does not appear to have an accumulation of mutations typically associated with several hallmark pathoadaptive genes (i.e., mexT, mucA, algR, rpoN, exsS, ampR). Whole genome comparisons show that P. aeruginosa strain PA80 is closely related to the hypervirulent Liverpool epidemic strain (LES) LESB58. PA80 also contains several genomic islands (GI's) encoding virulence and/or resistance determinants homologous to LESB58. To further understand the effect of these mutations in PA80 QS regulatory and virulence associated genes, we compared transcriptional expression of genes and phenotypic effects with isogenic mutants in the genetic reference strain PAO1. In PAO1, we show that deletion of rhlR has a much more significant impact on the expression of a wide range of virulence associated factors rather than deletion of lasR. In PA80, no QS regulatory genes were expressed, which we attribute to the inactivation of the RhlRI QS system by deletion of rhlR and mutation of rhlI. This study demonstrates that inactivation of the LasRI system does not impact RhlRI regulated virulence factors. PA80 has bypassed the common pathoadaptive mutations observed in LasR by targeting the RhlRI system. This suggests that RhlRI is a significant target for the long-term persistence of P. aeruginosa in chronic CF patients. This raises important questions in targeting QS systems for therapeutic interventions.

Dataset Information

Quorum quenching activity of Bacillus cereus isolate 30b confers antipathogenic effects in Pseudomonas aeruginosa.

Publications

Quorum quenching activity of <i>Bacillus cereus</i> isolate 30b confers antipathogenic effects in <i>Pseudomonas aeruginosa</i>.

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