Project description:Oxidative stress caused by exposure to reactive oxygen species (ROS), is a major challenge for aerobic and especially anaerobic organisms. Bacteria coordinate the response to oxidative stress through the LysR-type transcriptional regulator (LTTR) OxyR. Extensive studies have focused on the classical Escherichia coli system to shed light on the mode of action of defensive weapons against oxidative stress. The underlying mechanism is mediated via the formation of redox-dependent disulfide bond between two conserved cysteines of OxyR, thus activating transcription of members of the OxyR regulon. However, only fragmentary information on the regulation and function of OxyR has been gleaned through genetic and biochemical analyses in the important opportunistic pathogen P. aeruginosa. In this report, we used a comprehensive transcriptional profiling analysis to delineate the OxyR regulon under three conditions (KingM-bM-^@M-^Ys A medium [Pseudomonas medium or PM], Luria Broth (LB), and LB when oxyR is overexpressed), to investigate its roles in different cellular aspects that are independent of the classical oxidative stress response. Interestingly, when grown in LB, OxyR was found to regulating many genes involved in the process of inter-cellular communication known as quorum sensing (QS). In contrast, when grown in PM, OxyR regulate the expression of a newly identified CSS (cell-surface signaling) system in an OxyR-dependent fashion. In addition, the results from oxyR overexpression further confirmed that OxyR was linked to regulation of QS and Type 3 Secretion (T3SS) in addition to the regulation of antioxidative genes. Taken together, our results show that, apart from its dominant role in defense against oxidative stress in P. aeruginosa, OxyR acts as a global regulator that provides a link between the regulation of oxidative stress response, QS and virulence. 15 samples, representing 5 different biological conditions, including 3 biological replicates for each condition
Project description:Oxidative stress caused by exposure to reactive oxygen species (ROS), is a major challenge for aerobic and especially anaerobic organisms. Bacteria coordinate the response to oxidative stress through the LysR-type transcriptional regulator (LTTR) OxyR. Extensive studies have focused on the classical Escherichia coli system to shed light on the mode of action of defensive weapons against oxidative stress. The underlying mechanism is mediated via the formation of redox-dependent disulfide bond between two conserved cysteines of OxyR, thus activating transcription of members of the OxyR regulon. However, only fragmentary information on the regulation and function of OxyR has been gleaned through genetic and biochemical analyses in the important opportunistic pathogen P. aeruginosa. In this report, we used a comprehensive transcriptional profiling analysis to delineate the OxyR regulon under three conditions (King’s A medium [Pseudomonas medium or PM], Luria Broth (LB), and LB when oxyR is overexpressed), to investigate its roles in different cellular aspects that are independent of the classical oxidative stress response. Interestingly, when grown in LB, OxyR was found to regulating many genes involved in the process of inter-cellular communication known as quorum sensing (QS). In contrast, when grown in PM, OxyR regulate the expression of a newly identified CSS (cell-surface signaling) system in an OxyR-dependent fashion. In addition, the results from oxyR overexpression further confirmed that OxyR was linked to regulation of QS and Type 3 Secretion (T3SS) in addition to the regulation of antioxidative genes. Taken together, our results show that, apart from its dominant role in defense against oxidative stress in P. aeruginosa, OxyR acts as a global regulator that provides a link between the regulation of oxidative stress response, QS and virulence.
Project description:In this study, we present a novel transcriptional regulator, PA1226, which modulates biofilm formation and virulence in P. aeruginosa. Mutation in this regulator abolished the ability of P. aeruginosa to produce biofilm in vitro, without any effect on the planktonic growth. This regulator is essential to the in vivo fitness and pathogenesis in both Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA1226 regulates many essential virulence genes/pathways, including alginate, pili, and LPS biosynthesis.
Project description:P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. We identified bacterial metabolite - phenylacetic acid (PAA) which acts as an inhibitory molecule counteracting its pathogenic infection. Microarray and genetic analyses were conducted to investigate the inhibitory mechanism of the identified inhibitor PAA on bacterial virulence. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of type 3 secretion systems (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Our findings present a new insight to the puzzle of high-cell-density-modulated virulence attenuation in P. aeruginosa and the regulatory mechanisms of T3SS which is associated with bacterial acute infection.
Project description:Anticipating the risk for infectious disease during space exploration and habitation is a critical factor to ensure safety, health and performance of the crewmembers. As a ubiquitous environmental organism that is occasionally part of the human flora, Pseudomonas aeruginosa could pose a health hazard for the immuno-compromised astronauts. In order to gain insights in the behavior of P. aeruginosa in spaceflight conditions, two spaceflight-analogue culture systems, i.e. the rotating wall vessel (RWV) and the random position machine (RPM), were used. Microarray analysis of P. aeruginosa PAO1 grown in the low shear modeled microgravity (LSMMG) environment of the RWV compared to the normal gravity control (NG), revealed a regulatory role for AlgU (RpoE). Specifically, P. aeruginosa cultured in LSMMG exhibited increased alginate production and up-regulation of AlgU-controlled transcripts, including those encoding stress-related proteins. This study also shows the involvement of Hfq in the LSMMG response, consistent with its previously identified role in the Salmonella LSMMG- and spaceflight response. Furthermore, cultivation in LSMMG increased heat- and oxidative stress resistance and caused a decrease in the culture oxygen transfer rate. Interestingly, the global transcriptional response of P. aeruginosa grown in the RPM was similar to that in NG. The possible role of differences in fluid mixing between the RWV and RPM is discussed, with the overall collective data favoring the RWV as the optimal model to study the LSMMG-response of suspended cells. This study represents a first step towards the identification of specific virulence mechanisms of P. aeruginosa activated in response to spaceflight-analogue conditions, and could direct future research regarding the risk assessment and prevention of Pseudomonas infections for the crew in flight and the general public.
Project description:Colistin is an important cationic antimicrobial peptide (CAMP) in the fight against Pseudomonas aeruginosa infection within the cystic fibrosis (CF) lungs. The effects of sub-inhibitory colistin on gene expression in P. aeruginosa were investigated by transcriptome microarray and functional analysis. Analysis revealed an alteration in the expression of 60 genes in total from a variety of pathways. Genes associated with bacterial chronic colonisation and virulence such as response to osmotic stress, motility, and biofilm formation, as well as those associated with LPS modification and quorum sensing are the most highly represented. Most striking among these is the upregulation of the PQS biosynthesis operon including pqsH, pqsE, and the anthranilate biosynthetic genes phnAB. Early activation of this central component of the QS-network may represent a switch to a more robust population, with increased fitness in the competitive environment of the CF-lung.
Project description:While it is known that PqsE can stimulate the ability of RhlR to control some virulence factors, no data are available allowing clear discrimination of the PqsE and RhlR regulons. In this study, a P. aeruginosa PAO1 mutant strain with deletion of multiple QS elements and inducible expression of PqsE and/or RhlR has been generated and validated. RNA-seq performed in this genetic background allowed to unambiguously define the regulons controlled by PqsE and RhlR when produced alone or in combination Data produced in this study demonstrate that PqsE mainly impacts on the P. aeruginosa transcriptome via an RhlR-dependent pathway, and splits the RhlR-regulon in PqsE-dependent and PqsE-independent sub-regulons.
Project description:The ParS/ParR two component regulatory system plays important roles for multidrug resistance in Pseudomonas aeruginosa. In this study we report RNA-seq analyses of the transcriptomes of P. aeruginosa PAO1 wild type and par mutants growing in a minimal medium containing 2% casamino acids. This has allowed the quantification of PAO1 transcriptome, and further defines the regulon that is dependent on the ParS/ParR system for expression. Our RNA-seq analysis produced the first estimates of absolute transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished the expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to affecting drug resistance genes, transcripts of quorum sensing genes (rhlIR and pqsABCDE-phnAB), were significantly up-regulated in both parS and parR mutants. Consistent with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of par genes also lead to overproduction of phenazines and increased swarming motility, consistent with the up-regulation of quorum sensing genes. Our results established a link among ParS/ParR, MexEF-OprN and quorum sensing in Pseudomonas aeruginosa. Based on these results, we propose a model to illustrate the relationship among these regulatory systems in P. aeruginosa.
Project description:P. aeruginosa is known to cause acute cytotoxicity against various human and animal cells and tissues. We identified bacterial metabolite - phenylacetic acid (PAA) which acts as an inhibitory molecule counteracting its pathogenic infection. Microarray and genetic analyses were conducted to investigate the inhibitory mechanism of the identified inhibitor PAA on bacterial virulence. Microarray analysis revealed that treatment of P. aeruginosa with PAA down-regulated the transcriptional expression of type 3 secretion systems (T3SS) genes and related regulatory genes including rsmA and vfr, which were confirmed by transcriptional and translational analysis. Our findings present a new insight to the puzzle of high-cell-density-modulated virulence attenuation in P. aeruginosa and the regulatory mechanisms of T3SS which is associated with bacterial acute infection. Overnight PAO1 culture were diluted 1:200 to fresh LB medium supplemented with nitriloacetic acid (NTA) with or without addition of 1 mM of phenylacetic acid (PAA). The growth was continued with shaking at 37M-BM-0C for 4 h to allow OD600 reaching about 1.5 and the cells were used for RNA extraction and hybridization on Affymetrix microarrays.
Project description:The ParS/ParR two component regulatory system plays important roles for multidrug resistance in Pseudomonas aeruginosa. In this study we report RNA-seq analyses of the transcriptomes of P. aeruginosa PAO1 wild type and par mutants growing in a minimal medium containing 2% casamino acids. This has allowed the quantification of PAO1 transcriptome, and further defines the regulon that is dependent on the ParS/ParR system for expression. Our RNA-seq analysis produced the first estimates of absolute transcript abundance for the 5570 coding genes in P. aeruginosa PAO1. Comparative transcriptomics of P. aeruginosa PAO1 and par mutants identified a total of 464 genes regulated by ParS and ParR. Results also showed that mutations in the parS/parR system abolished the expression of the mexEF-oprN operon by down-regulating the regulatory gene mexS. In addition to affecting drug resistance genes, transcripts of quorum sensing genes (rhlIR and pqsABCDE-phnAB), were significantly up-regulated in both parS and parR mutants. Consistent with these results, a significant portion of the ParS/ParR regulated genes belonged to the MexEF-OprN and quorum sensing regulons. Deletion of par genes also lead to overproduction of phenazines and increased swarming motility, consistent with the up-regulation of quorum sensing genes. Our results established a link among ParS/ParR, MexEF-OprN and quorum sensing in Pseudomonas aeruginosa. Based on these results, we propose a model to illustrate the relationship among these regulatory systems in P. aeruginosa. A total of 9 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas aeruginosa PAO1 wild type strain (3 replicates); Pseudomonas aeruginosa parS mutant (3 replicates); Pseudomonas aeruginosa parR mutant (3 replicates).