The pvc operon regulates the expression of the Pseudomonas aeruginosa fimbrial chaperone/usher pathway (cup) genes.

The Pseudomonas aeruginosa fimbrial structures encoded by the cup gene clusters (cupB and cupC) contribute to its attachment to abiotic surfaces and biofilm formation. The P. aeruginosa pvcABCD gene cluster encodes enzymes that synthesize a novel isonitrile functionalized cumarin, paerucumarin. Paerucumarin has already been characterized chemically, but this is the first report elucidating its role in bacterial biology. We examined the relationship between the pvc operon and the cup gene clusters in the P. aeruginosa strain MPAO1. Mutations within the pvc genes compromised biofilm development and significantly reduced the expression of cupB1-6 and cupC1-3, as well as different genes of the cupB/cupC two-component regulatory systems, roc1/roc2. Adjacent to pvc is the transcriptional regulator ptxR. A ptxR mutation in MPAO1 significantly reduced the expression of the pvc genes, the cupB/cupC genes, and the roc1/roc2 genes. Overexpression of the intact chromosomally-encoded pvc operon by a ptxR plasmid significantly enhanced cupB2, cupC2, rocS1, and rocS2 expression and biofilm development. Exogenously added paerucumarin significantly increased the expression of cupB2, cupC2, rocS1 and rocS2 in the pvcA mutant. Our results suggest that pvc influences P. aeruginosa biofilm development through the cup gene clusters in a pathway that involves paerucumarin, PtxR, and different cup regulators.

Role of Vfr in regulating exotoxin A production by Pseudomonas aeruginosa.

Pseudomonas aeruginosa exotoxin A (ETA) production depends on the virulence-factor regulator Vfr. Recent evidence indicates that the P. aeruginosa iron-starvation sigma factor PvdS also enhances ETA production through the ETA-regulatory gene regA. Mutants defective in vfr, regA and pvdS, plasmids that overexpress these genes individually and lacZ transcriptional/translational fusion plasmids were utilized to examine the relationship between vfr, regA and pvdS in regulating P. aeruginosa ETA production. ETA concentration and regA expression were reduced significantly in PAODeltavfr, but pvdS expression was not affected. Overexpression of Vfr produced a limited increase in ETA production in PAODeltapvdS, but not PAODeltaregA. Additionally, overexpression of either RegA or PvdS did not enhance ETA production in PAODeltavfr. RT-PCR analysis showed that iron did not affect the accumulation of vfr mRNA in PAO1. These results suggest that: (i) Vfr enhances toxA expression in PAO1 both directly and indirectly through regA, but not through pvdS; (ii) vfr expression is not regulated by iron; and (iii) both Vfr and PvdS cooperate in the presence of RegA to achieve a maximum level of toxA expression.

Regulation of Pseudomonas aeruginosa ptxR by Vfr.

Pseudomonas aeruginosa PtxR enhances the expression of the exotoxin A gene toxA. The expression of ptxR itself, which occurs from two promoters (P1 and P2), is not completely understood. We have recently demonstrated that the ptxR upstream region contains potential binding sites for multiple regulators, including the virulence factor regulator Vfr. In this study, we identified within the ptxR upstream region, a 25 bp sequence to which Vfr specifically binds. The sequence is located 20-44 (32.5) bp 5' of the ptxR P2 promoter, and overlaps a potential binding site for the iron-starvation sigma factor PvdS. We also show that, throughout the growth cycle, deletion of vfr reduces ptxR expression from the P2 promoter in the P. aeruginosa strain PAO1 by four- to eightfold, but does not affect ptxR expression from P1. Further, loss of Vfr eliminates the PtxR-induced enhancement in the synthesis of exotoxin A and the metalloproteinase LasB. Our results suggest that Vfr modulates toxA and lasB expression in PAO1 through PtxR. A model defining the relationships between these different genes is presented.

Regulation of the Pseudomonas aeruginosa toxA, regA and ptxR genes by the iron-starvation sigma factor PvdS under reduced levels of oxygen.

The level of environmental oxygen (EO) within various Pseudomonas aeruginosa infection sites is low (microaerobic), and this can affect the production of different virulence factors. Expression of the toxA gene, encoding exotoxin A (ETA), is regulated by regA, ptxR and pvdS. Moreover, the iron-starvation sigma factor PvdS directs the transcription of pyoverdine siderophore genes (e.g. pvdD). DNA-protein binding analysis using recombinant PvdS showed that the PvdS-RNA polymerase holoenzyme complex specifically bound the toxA, regA and ptxR promoter regions. All three promoters contain a PvdS-binding site, the iron-starvation box. To determine the relationship between these different genes and PvdS, we conducted a comparative analysis of toxA, regA, ptxR and pvdD transcription throughout the growth cycle of wild-type P. aeruginosa and its pvdS mutant in iron-deficient medium under aerobic-shaking (A-sh) and microaerobic-static (M-st) conditions. Under both EO conditions, optimal toxA, regA and pvdD expression and pyoverdine production required PvdS, while ptxR expression was moderately dependent on PvdS only under A-sh conditions. Expression of regA, pvdD and pyoverdine production in wild-type P. aeruginosa was significantly lower under M-st in comparison with A-sh conditions, while the opposite was observed for toxA and ptxR. Although low, the level of toxA expression and ETA production in the pvdS mutant were higher under M-st than under A-sh conditions. Transcription of pvdS and PvdS expression were also reduced by low EO. We propose that the regulation of toxA expression under aerobic conditions primarily involves PvdS, while an additional EO-responsive regulator(s) besides PvdS is required under low EO levels. Thus, PvdS may control the transcription of the ptxR, regA and toxA genes, and respond to EO by acting at different levels of the toxA regulatory cascade.

Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa.

The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.

PtxR modulates the expression of QS-controlled virulence factors in the Pseudomonas aeruginosa strain PAO1.

The production of several virulence factors by Pseudomonas aeruginosa is regulated through the hierarchical cell-density dependent quorum sensing (QS) systems las and rhl. A third component of the QS hierarchy, the Pseudomonas quinolone signal PQS, also controls the expression of several genes. We previously described P. aeruginosa PtxR as a transcriptional activator of the exotoxin A gene toxA. Here, we provide evidence that PtxR regulates the production of other virulence factors. Mutation of ptxR in PAO1 increased pyocyanin production. This increase was reduced in the presence of a ptxR plasmid. Throughout the growth cycle, PtxR reduced the expression of the pyocyanin operon phzA1-G1 but not phzA2-G2. As pyocyanin production is stringently controlled by QS, we examined the effect of PtxR on QS-related genes in PAO1. PtxR also reduced the expression of the PQS synthesis operon pqsABCDE. ptxR mutation increased the expression of the rhamnolipid synthesis gene rhlA but decreased lasB expression. The expression of the RhlI synthase gene rhlI and the production of the C(4)-HSL autoinducer were increased in the ptxR mutant, while the expression of the LasI synthase gene lasI and the production of 3OC(12)-HSL were reduced. These results suggest that PtxR negatively regulates the expression of the rhamnolipid and pyocyanin genes through rhlI and the pqsABCDE operon while it positively regulates the expression of lasB through lasI.

mvaT mutation modifies the expression of the Pseudomonas aeruginosa multidrug efflux operon mexEF-oprN.

Pseudomonas aeruginosa carries several multidrug efflux operons, including mexEF-oprN, that contribute to its resistance to multiple antibiotics. mvaT affects the expression of several P. aeruginosa genes. In this study, we show that the mvaT mutant PAODeltamvaT is more resistant than its parent PAO1 strain to chloramphenicol and norfloxacin but more sensitive to imipenem; yet both were less resistant to chloramphenicol, norfloxacin, and imipenem than 'typical'nfxC-type mutants. Neither strain carries the deletion described for nfxC-type mutants in mexT, the mexEF-oprN regulatory gene. Expression of mexEF-oprN is increased by five- to sixfold in PAODeltamvaT, while the expression of oprD is reduced by approximately twofold. mvaT mutation had no effect on the expression of other multidrug resistance operons, although it increased the expression of several ATP-binding cassette transporter genes. We show that mvaT mutation does not affect mexEF-oprN expression through mexT or mexS. We also explored several other potential mechanisms.

Effect of static growth and different levels of environmental oxygen on toxA and ptxR expression in the Pseudomonas aeruginosa strain PAO1.

Within certain infection sites, such as the lung of cystic fibrosis patients, Pseudomonas aeruginosa grows statically under either decreased oxygen tension or anaerobic conditions, a situation that is likely to influence the production of virulence factors. The goal of this study was to determine the effect of static growth under microaerobic (decreased oxygen) and anaerobic conditions on the expression of the P. aeruginosa exotoxin A (ETA) gene toxA and its positive regulator ptxR. Using toxA-lacZ and ptxR-lacZ fusion plasmids, the level of toxA and ptxR expression was measured throughout the growth cycle of strain PAO1, which was grown in either iron-deficient or iron-sufficient medium under four different conditions: 20%-SH (aerobic, shaking), 20%-ST (aerobic, static), 10%-ST (microaerobic, static) and 0%-ST (anaerobic, static). In iron-deficient medium, toxA expression was higher under 20%-ST and 10%-ST than under 20%-SH. However, the highest level of toxA expression occurred under 0%-ST. Analysis of ETA protein using sandwich ELISA revealed that at time points between 8 and 24 h of the growth curve, PAO1 produced higher levels of ETA under 0%-ST than under 20%-SH. In iron-sufficient medium, toxA expression was significantly repressed under all conditions. Additional analyses using PAO1 strains that carry lacZ fusions with the toxA regulatory genes regA and pvdS revealed that the expression of regA and pvdS is reduced rather than increased at 0%-ST. ptxR expression under different conditions paralleled that of toxA expression, except that it was repressed by iron under 20 %-SH only. Between 6 and 24 h of growth, and under all conditions, the level of dissolved oxygen (DO) within the PAO1 cultures was sharply reduced. These results suggest that (1) the combined effect of static growth and anaerobic conditions produce a significant increase in toxA and ptxR expression in PAO1; (2) this effect appears to be unique to toxA and ptxR, since the level of regA and pvdS expression was reduced under the same conditions; (3) neither static growth nor anaerobic conditions interfere with the repression of toxA expression by iron, although static growth deregulates ptxR expression with respect to iron; and (4) the enhanced expression of toxA and ptxR is not related to the reduced levels of DO in PAO1 cultures.

Analysis of quorum sensing-deficient clinical isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces multiple virulence factors and causes different types of infections. Previous clinical studies identified P. aeruginosa isolates that lack individual virulence factors. However, the impact of losing several virulence factors simultaneously on the in vivo virulence of P. aeruginosa is not completely understood. The P. aeruginosa cell-to-cell communication system, or quorum sensing (QS), controls the production of several virulence factors. Animal studies using constructed QS mutants indicated that loss of the QS system severely impacts the virulence of P. aeruginosa. In this study, we tried to determine if deficiency within the QS system compromises the ability of P. aeruginosa to establish infections in humans. We have identified five QS-deficient strains through screening 200 isolates from patients with urinary tract, lower respiratory tract and wound infections. These strains lacked LasB and LasA activities and produced either no or very low levels of the autoinducers N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone. PCR analysis revealed that three isolates contained all four QS genes (lasI, lasR, rhlI and rhlR) while two isolates lacked both the lasR and rhlR genes. We also examined the five isolates for other virulence factors. The isolates produced variable levels of exotoxin A and, with one exception, were deficient in pyocyanin production. One isolate produced the type III secretion system (TTSS) effector proteins ExoS and ExoT, two isolates produced ExoT only and two isolates produced no TTSS proteins. The isolates produced weak to moderate biofilms on abiotic surfaces. Analysis of the patients' data revealed that two of the isolates represented a single strain that was isolated twice from the same patient within a 1 month interval. One QS-deficient clinical isolate (CI-1) lacked all tested virulence factors and produced a weak biofilm. These results suggest that naturally occurring QS-deficient strains of P. aeruginosa do occur and are capable of causing infections; and, that besides the known virulence factors, additional factors may contribute to the ability of certain strains such as CI-1 to establish an infection.

Pseudomonas aeruginosa autoinducer enters and functions in mammalian cells.

Quorum sensing (QS) is a cell density-dependent signaling mechanism used by many bacteria to control gene expression. Several recent reports indicate that the signaling molecules (autoinducers) that mediate QS in Pseudomonas aeruginosa may also modulate gene expression in host cells; however, the mechanisms are largely unknown. Here we show that two P. aeruginosa autoinducers, N-3-oxododecanoyl-homoserine lactone and N-butyryl-homoserine lactone, can both enter eukaryotic cells and activate artificial chimeric transcription factors based on their cognate transcriptional activators, LasR and RhlR, respectively. The autoinducers promoted nuclear localization of chimeric proteins containing the full LasR or RhlR coding region, and the LasR-based proteins were capable of activating transcription of a LasR-dependent luciferase gene. Responsiveness to autoinducer required the N-terminal autoinducer-binding domains of LasR and RhlR. Truncated proteins consisting of only the C-terminal helix-turn-helix DNA-binding domains of both proteins attached to a nuclear localization signal efficiently translocated to the nucleus in the absence of autoinducer, and truncated LasR-based proteins functioned as constitutively active transcription factors. Chimeric LasR proteins were only activated by their cognate autoinducer ligand and not by N-butyryl-L-homoserine lactone. These data provide evidence that autoinducer molecules from human pathogens can enter mammalian cells and suggest that autoinducers may influence gene expression in host cells by interacting with and activating as-yet-unidentified endogenous proteins.

Regulation of toxA by PtxR in Pseudomonas aeruginosa PA103.

Exotoxin A (ETA) production in Pseudomonas aeruginosa requires the regulatory locus regAB. Pseudomonas aeruginosa PA103 produces significantly higher levels of ETA than the prototypic strain PAO1 does, partly because of differences in the regAB locus. Other factors that contribute to this variation are not known. We previously described the P. aeruginosa gene ptxR that positively regulates production of ETA through regAB. ETA production was enhanced but still iron regulated in the PAO1 strain PAO1-XR that carries two copies of ptxR on its chromosome. Here we determine whether ptxR regulation of ETA is different in PA103. In contrast to PAO1-XR, ETA activity produced by PA103-2R, a PA103 strain carrying two copies of ptxR, is enhanced tenfold and partially deregulated in the presence of iron. Real-time PCR transcriptional analysis showed that the copy number of toxA mRNA in PA103-2R is significantly higher than in PA103 in both the presence and absence of iron, yet no similar increase in either regAB or ptxR mRNA copy number was detected. The integrated plasmid together with adjoining DNA was retrieved from the PA103-2R chromosome to determine whether integration-induced DNA changes played a role in this phenotype. Introduction of the retrieved plasmid in PA103 produced a phenotype similar to that of PA103-2R. Sequence analysis of the plasmid revealed the loss of 322 bp within the region 3' of ptxR. A plasmid construct carrying a 4-bp insertion in this same region produced in PA103 a phenotype similar to that of PA103-2R. Our results suggest that the effect of ptxR on toxA expression is different in PA103 than in PAO1 and that this variation in PA103-2R does not occur solely through regAB. Changes within the region 3' of ptxR are critical for the production of the unique PA103-2R phenotype, which occurs in trans and requires intact ptxR, but is not caused by ptxR overexpression.