Identification of a novel regulator of the quorum-sensing systems in Pseudomonas aeruginosa

Quorum sensing is a global gene-regulatory mechanism in bacteria that enables individual bacterial cells to communicate and coordinate their population behaviors. Quorum sensing is central to the pathogenesis of many bacterial pathogens including Pseudomonas aeruginosa and therefore has been exploited as a target for developing novel antipathogenic drugs. In P. aeruginosa , three intertwined quorum-sensing systems, las, rhl , and the 2-alkyl-4(1 H )-quinolone system, which includes the Pseudomonas quinolone signal (PQS), control virulence factor production, and pathogenesis processes. Previously, we obtained a mutant with diminished expression of the phzA1B1C1D1E1F1G1 operon that is involved in the production of virulence factor phenazine compounds. In this study, the mutant was further characterized, and evidence indicating that the disrupted gene PA1196 in the mutant is a potential regulator of the rhl and PQS systems is presented. PA1196 positively controls the expression of the rhl and PQS systems and affects bacterial motility and multiple virulence factor expression via the quorum-sensing systems. This adds an important new player in the complex quorum-sensing network in P. aeruginosa .     

The global regulator Crc modulates metabolism,susceptibility to antibiotics and virulence in Pseudomonas aeruginosa

The capacity of a bacterial pathogen to produce a disease in a treated host depends on the former's virulence and resistance to antibiotics. Several scattered pieces of evidence suggest that these two characteristics can be influenced by bacterial metabolism. This potential relationship is particularly important upon infection of a host, a situation that demands bacteria adapt their physiology to their new environment, making use of newly available nutrients. To explore the potential cross‐talk between bacterial metabolism, antibiotic resistance and virulence, a Pseudomonas aeruginosa model was used. This species is an important opportunistic pathogen intrinsically resistant to many antibiotics. The role of Crc, a global regulator that controls the metabolism of carbon sources and catabolite repression in Pseudomonas, was analysed to determine its contribution to the intrinsic antibiotic resistance and virulence of P. aeruginosa. Using proteomic analyses, high‐throughput metabolic tests and functional assays, the present work shows the virulence and antibiotic resistance of this pathogen to be linked to its physiology, and to be under the control (directly or indirectly) of Crc. A P. aeruginosa strain lacking the Crc regulator showed defects in type III secretion, motility, expression of quorum sensing‐regulated virulence factors, and was less virulent in a Dictyostelium discoideum model. In addition, this mutant strain was more susceptible to beta‐lactams, aminoglycosides, fosfomycin and rifampin. Crc might therefore be a good target in the search for new antibiotics.     

The sensor kinase CbrA is a global regulator that modulates metabolism, virulence, and antibiotic resistance in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen that possesses a large arsenal of virulence factors enabling the pathogen to cause serious infections in immunocompromised patients, burn victims, and cystic fibrosis patients. CbrA is a sensor kinase that has previously been implied to play a role with its cognate response regulator CbrB in the metabolic regulation of carbon and nitrogen utilization in P. aeruginosa. Here it is demonstrated that CbrA and CbrB play an important role in various virulence and virulence-related processes of the bacteria, including swarming, biofilm formation, cytotoxicity, and antibiotic resistance. The cbrA deletion mutant was completely unable to swarm while exhibiting an increase in biofilm formation, supporting the inverse regulation of swarming and biofilm formation in P. aeruginosa. The cbrA mutant also exhibited increased cytotoxicity to human lung epithelial cells as early as 4 and 6 h postinfection. Furthermore, the cbrA mutant demonstrated increased resistance toward a variety of clinically important antibiotics, including polymyxin B, ciprofloxacin, and tobramycin. Microarray analysis revealed that under swarming conditions, CbrA regulated the expression of many genes, including phoPQ, pmrAB, arnBCADTEF, dnaK, and pvdQ, consistent with the antibiotic resistance and swarming impairment phenotypes of the cbrA mutant. Phenotypic and real-time quantitative PCR (RT-qPCR) analyses of a PA14 cbrB mutant suggested that CbrA may be modulating swarming, biofilm formation, and cytotoxicity via CbrB and that the CrcZ small RNA is likely downstream of this two-component regulator. However, as CbrB did not have a resistance phenotype, CbrA likely modulates antibiotic resistance in a manner independent of CbrB.     

Human probiotic bacteria attenuate Pseudomonas aeruginosa biofilm and virulence by quorum-sensing inhibition

Abstract

This work investigated chloroform extracts from culture supernatants of two human probiotic bacteria, Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730 for the production of virulence factors and quorum sensing (QS) interference against three Pseudomonas aeruginosa strains. Both extracts inhibited biofilm biomass (up to 50%), biofilm metabolic activity (up to 39%), the production of the enzyme elastase (up to 63%) and pyocyanin (up to 77%), and decreased QS, without presenting any antibacterial acgivity. In addition, the chloroform extracts of both strains disrupted preformed biofilms of the three strains of P. aeruginosa analyzed (up to 40%). GC-MS analysis revealed that the major compounds detected in the bioactive extracts were four diketopiperazines. This study suggests that the metabolites of L. casei and L. acidophilus could be a promising alternative to combat the pathogenicity of P. aeruginosa.     

A new quorum-sensing inhibitor attenuates virulence and decreases antibiotic resistance in Pseudomonas aeruginosa

Quorum sensing (QS) has been a novel target for the treatment of infectious diseases. Here structural analogs of Pseudomonas aeruginosa autoinducer N-acyl homoserine lactone (AHL) were investigated for QS inhibitor (QSI) activity and a novel QSI was discovered, N-decanoyl-L-homoserine benzyl ester (C2). Virulence assays showed that C2 down-regulated total protease and elastase activities, as well as the production of rhamnolipid, that are controlled by QS in P. aeruginosa wild-type strain PAO1 without affecting growth. C2 was also shown to inhibit swarming motility of PAO1. Using a microdilution checkerboard method, we identified synergistic interactions between C2 and several antibiotics, tobramycin, gentamycin, cefepime, and meropenem. Data from real-time RT-PCR suggested that C2 inhibited the expression of lasR (29.67%), lasI (21.57%), rhlR (28.20%), and rhlI (29.03%).     

The quorum-sensing negative regulator RsaL of Pseudomonas aeruginosa binds to the lasI promoter

A mutation in the rsaL gene of Pseudomonas aeruginosa produces dramatically higher amounts of N-acyl homoserine lactone with respect to the wild type, highlighting the key role of this negative regulator in controlling quorum sensing (QS) in this opportunistic pathogen. The DNA binding site of the RsaL protein on the rsaL-lasI bidirectional promoter partially overlaps the binding site of the LasR protein, consistent with the hypothesis that RsaL and LasR could be in binding competition on this promoter. This is the first direct demonstration that RsaL acts as a QS negative regulator by binding to the lasI promoter.     

P. aeruginosa quorum-sensing systems and virulence

Quorum sensing is an important mechanism for the regulation of genes in many Gram-negative and Gram-positive bacteria. In the opportunistic pathogen Pseudomonas aeruginosa, the absence of one or more components of the quorum-sensing system results in a significant reduction in virulence. Recent advances in the past year have demonstrated that the quorum-sensing signal molecule 3O-C(12)-HSL is also a potent stimulator of multiple eukaryotic cells and thus may alter the host response during P. aeruginosa infections. Therefore, via the regulation of multiple factors and the production of 3O-C(12)-HSL, quorum-sensing systems have a significant effect on the virulence of the bacteria and also on how the host responds to P. aeruginosa infections.     

PchR-box recognition by the AraC-type regulator PchR of Pseudomonas aeruginosa requires the siderophore pyochelin as an effector

Under iron limitation, the opportunistic human pathogen Pseudomonas aeruginosa produces the siderophore pyochelin. When secreted into the extracellular environment, pyochelin complexes ferric ions and delivers them, via the outer membrane receptor FptA, to the bacterial cytoplasm. Extracellular pyochelin also acts as a signalling molecule, inducing the expression of pyochelin biosynthesis and uptake genes by a mechanism involving the AraC-type regulator PchR. We have identified a 32 bp conserved sequence element (PchR-box) in promoter regions of pyochelin-controlled genes and we show that the PchR-box in the pchR-pchDCBA intergenic region is essential for the induction of the pyochelin biosynthetic operon pchDCBA and the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin and iron. PchR-box mutations that interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. We conclude that pyochelin, probably in its iron-loaded state, is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation suggests that the siderophore can enter the cytoplasm.