Quorum sensing differentially regulates Pseudomonas aeruginosa type VI secretion locus I and homologous loci II and III, which are required for pathogenesis.

Pseudomonas aeruginosa harbours three type VI secretion (T6S) loci. Although HSI-I has been partially studied, limited knowledge is available on the homologous loci HSI-II and HSI-III. We show that quorum sensing (QS) differentially regulates the expression of genes at all three loci. HSI-I-associated gene expression is suppressed by both the homoserine lactone transcription factor LasR and the 4-hydroxy-2-alkylquinoline (HAQ) transcriptional regulator MvfR. Conversely, both HSI-II and HSI-III loci are positively controlled by LasR and MvfR. PqsE, a key component of the MvfR regulon, is required for the expression of part of HSI-III but not HSI-II, and previously identified inhibitors of HAQ biosynthesis significantly downregulate HSI-II and -III gene expression. Animal and plant infection studies reveal that both HSI-II and -III play important roles in pathogenesis. Furthermore, analysis of a double DeltaHSI-II : : III mutant suggests that these loci functionally compensate for one another in virulence. This study illustrates the contribution of the QS systems to T6S gene regulation and reveals the importance of HSI-II and -III in mediating P. aeruginosa pathogenesis. Moreover, this work provides new insights into the design and development of selective compounds that may restrict human P. aeruginosa and possibly other clinical infections.

A stable isotope dilution assay for the quantification of the Pseudomonas quinolone signal in Pseudomonas aeruginosa cultures.

A stable isotope dilution method was developed to analyse 2-heptyl-3,4-dihydroxyquinoline, also called the Pseudomonas quinolone signal (PQS), directly in Pseudomonas aeruginosa cultures by liquid chromatography coupled to mass spectrometry (LC/MS). PQS, along with the isobaric 2-heptyl-4-hydroxyquinoline N-oxide (HQNO), were quantified in various Pseudomonas liquid cultures using a deuterated PQS analog as internal standard. The kinetic of production of these quinolines in a growing culture of P. aeruginosa PA14 showed that their production starts at the end of the logarithmic growth phase and is maximal at the onset of the stationary growth phase. The concentration of PQS reached a maximum at 13 mg/l and then decreased, while the HQNO concentration reached 18 mg/l and then remained stable. Culture supernatants of P. aeruginosa strains PAO1 and PA14 produced similar concentrations of PQS whereas no PQS or HQNO could be detected in culture supernatants of the P. aeruginosa strain PAK or in the other Pseudomonas species tested, including phytopathogenic pseudomonads.

Common mechanisms for pathogens of plants and animals.

The vast evolutionary gulf between plants and animals--in terms of structure, composition, and many environmental factors--would seem to preclude the possibility that these organisms could act as receptive hosts to the same microorganism. However, some pathogens are capable of establishing themselves and thriving in members of both the plant and animal kingdoms. The identification of functionally conserved virulence mechanisms required to infect hosts of divergent evolutionary origins demonstrates the remarkable conservation in some of the underlying virulence mechanisms of pathogenesis and is changing researchers' thinking about the evolution of microbial pathogenesis.

A quorum sensing-associated virulence gene of Pseudomonas aeruginosa encodes a LysR-like transcription regulator with a unique self-regulatory mechanism.

The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 infects both plants and animals. Previously, using plants to screen directly for P. aeruginosa virulence-attenuated mutants, we identified a locus, pho34B12, relevant in mammalian pathogenesis. Here, nonsense point mutations in the two opposing ORFs identified in the pho34B12 locus revealed that one of them, mvfR (multiple virulence factor Regulator), is able to control all of the phenotypes that mutant phoA34B12 displays. Both genetic and biochemical evidence demonstrate that the mvfR gene encodes a LysR-like transcriptional factor that positively regulates the production of elastase, phospholipase, and of the autoinducers, 3oxo-dodecanoyl homoserine lactone (PAI I) and 2-heptyl-3-hydroxy-4-quinolone (PQS), as well as the expression of the phnAB operon, involved in phenazine biosynthesis. We demonstrate that the MvfR protein is membrane-associated and acts as a transcriptional activator until cells reach stationary phase, when a unique negative feedback mechanism is activated to signal the down-regulation of the MvfR protein. This work reveals an unprecedented virulence mechanism of P. aeruginosa and identifies a unique indispensable player in the P. aeruginosa quorum-sensing cascade.

Differential roles of the Pseudomonas aeruginosa PA14 rpoN gene in pathogenicity in plants, nematodes, insects, and mice.

We cloned the rpoN (ntrA, glnF) gene encoding the alternate sigma factor sigma(54) from the opportunistic multihost pathogen Pseudomonas aeruginosa strain PA14. A marker exchange protocol was used to construct the PA14 rpoN insertional mutation rpoN::Gen(r). PA14 rpoN::Gen(r) synthesized reduced levels of pyocyanin and displayed a variety of phenotypes typical of rpoN mutants, including a lack of motility and the failure to grow on nitrate, glutamate, or histidine as the sole nitrogen source. Compared to wild-type PA14, rpoN::Gen(r) was ca. 100-fold less virulent in a mouse thermal injury model and was significantly impaired in its ability to kill the nematode Caenorhabditis elegans. In an Arabidopsis thaliana leaf infectivity assay, although rpoN::Gen(r) exhibited significantly reduced attachment to trichomes, stomata, and the epidermal cell surface, did not attach perpendicularly to or perforate mesophyll cell walls, and proliferated less rapidly in Arabidopsis leaves, it nevertheless elicited similar disease symptoms to wild-type P. aeruginosa PA14 at later stages of infection. rpoN::Gen(r) was not impaired in virulence in a Galleria mellonella (greater wax moth) pathogenicity model. These data indicate that rpoN does not regulate the expression of any genes that encode virulence factors universally required for P. aeruginosa pathogenicity in diverse hosts.

The roles of mucD and alginate in the virulence of Pseudomonas aeruginosa in plants, nematodes and mice.

We are exploiting the broad host range of the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 to elucidate the molecular basis of bacterial virulence in plants, nematodes, insects and mice. In this report, we characterize the role that two PA14 gene products, MucD and AlgD, play in virulence. MucD is orthologous to the Escherichia coli periplasmic protease and chaperone DegP. DegP homologues are known virulence factors that play a protective role in stress responses in various species. AlgD is an enzyme involved in the biosynthesis of the exopolysaccharide alginate, which is hyperinduced in mucD mutants. A PA14 mucD mutant was significantly impaired in its ability to cause disease in Arabidopsis thaliana and mice and to kill the nematode Caenorhabditis elegans. Moreover, MucD was found to be required for the production of an extracellular toxin involved in C. elegans killing. In contrast, a PA14 algD mutant was not impaired in virulence in plants, nematodes or mice. A mucDalgD double mutant had the same phenotype as the mucD single mutant in the plant and nematode pathogenesis models. However, the mucDalgD double mutant was synergistically reduced in virulence in mice, suggesting that alginate can partially compensate for the loss of MucD function in mouse pathogenesis.

Pathogenesis of the human opportunistic pathogen Pseudomonas aeruginosa PA14 in Arabidopsis.

The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 is a multihost pathogen that can infect Arabidopsis. We found that PA14 pathogenesis in Arabidopsis involves the following steps: attachment to the leaf surface, congregation of bacteria at and invasion through stomata or wounds, colonization of intercellular spaces, and concomitant disruption of plant cell wall and membrane structures, basipetal movement along the vascular parenchyma, and maceration and rotting of the petiole and central bud. Distinctive features of P. aeruginosa pathogenesis are that the surface of mesophyll cell walls adopt an unusual convoluted or undulated appearance, that PA14 cells orient themselves perpendicularly to the outer surface of mesophyll cell walls, and that PA14 cells make circular perforations, approximately equal to the diameter of P. aeruginosa, in mesophyll cell walls. Taken together, our data show that P. aeruginosa strain PA14 is a facultative pathogen of Arabidopsis that is capable of causing local and systemic infection, which can result in the death of the infected plant.

Elucidating the molecular mechanisms of bacterial virulence using non-mammalian hosts.

Several strains of the human opportunistic pathogen Pseudomonas aeruginosa infect plants, nematodes and insects. Our laboratory has developed a multihost pathogenesis system based on the P. aeruginosa clinical isolate PA14, in which non-mammalian hosts are used to screen directly for virulence-attenuated mutants. The majority of PA14 mutants isolated using non-mammalian hosts also displayed reduced virulence in a burned mouse model. Surprisingly, only a few host-specific virulence factors were identified, and many of the P. aeruginosa mutants were attenuated in virulence in all the hosts. These studies illustrate the extensive conservation in the virulence mechanisms used by P. aeruginosa to infect evolutionarily diverged hosts, and validate the multihost method of screening for virulence factors relevant to mammalian pathogenesis. Through the use of genetically tractable hosts, the multihost pathogenesis model also provides tools for elucidating host responses and dissecting the fundamental molecular interactions that underlie bacterial pathogenesis.

Plants and animals share functionally common bacterial virulence factors.

By exploiting the ability of Pseudomonas aeruginosa to infect a variety of vertebrate and nonvertebrate hosts, we have developed model systems that use plants and nematodes as adjuncts to mammalian models to help elucidate the molecular basis of P. aeruginosa pathogenesis. Our studies reveal a remarkable degree of conservation in the virulence mechanisms used by P. aeruginosa to infect hosts of divergent evolutionary origins.

Positive correlation between virulence of Pseudomonas aeruginosa mutants in mice and insects.

Strain PA14, a human clinical isolate of Pseudomonas aeruginosa, is pathogenic in mice and insects (Galleria mellonella). Analysis of 32 different PA14 mutants in these two hosts showed a novel positive correlation in the virulence patterns. Thus, G. mellonella is a good model system for identifying mammalian virulence factors of P. aeruginosa.

Pseudomonas aeruginosa killing of Caenorhabditis elegans used to identify P. aeruginosa virulence factors.

We reported recently that the human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans and that many P. aeruginosa virulence factors (genes) required for maximum virulence in mouse pathogenicity are also required for maximum killing of C. elegans. Here we report that among eight P. aeruginosa PA14 TnphoA mutants isolated that exhibited reduced killing of C. elegans, at least five also exhibited reduced virulence in mice. Three of the TnphoA mutants corresponded to the known virulence-related genes lasR, gacA, and lemA. Three of the mutants corresponded to known genes (aefA from Escherichia coli, pstP from Azotobacter vinelandii, and mtrR from Neisseria gonorrhoeae) that had not been shown previously to play a role in pathogenesis, and two of the mutants contained TnphoA inserted into novel sequences. These data indicate that the killing of C. elegans by P. aeruginosa can be exploited to identify novel P. aeruginosa virulence factors important for mammalian pathogenesis.

Molecular mechanisms of bacterial virulence elucidated using a Pseudomonas aeruginosa-Caenorhabditis elegans pathogenesis model.

The human opportunistic pathogen Pseudomonas aeruginosa strain PA14 kills Caenorhabditis elegans. Using systematic mutagenesis of PA14 to identify mutants that fail to kill C. elegans and a C. elegans mutant that lacks P-glycoproteins, we identified phenazines, secreted P. aeruginosa pigments, as one of the mediators of killing. Analysis of C. elegans mutants with altered responses to oxidative stress suggests that phenazines exert their toxic effects on C. elegans through the generation of reactive oxygen species. Finally, we show that phenazines and other P. aeruginosa factors required for C. elegans killing are also required for pathogenesis in plants and mice, illustrating that this model tackles the dual challenges of identifying bacterial virulence factors as well as host responses to them.

Modulation of expression of the ToxR regulon in Vibrio cholerae by a member of the two-component family of response regulators.

The ToxRS system in Vibrio cholerae plays a central role in the modulation of virulence gene expression in response to environmental stimuli. An integration of multiple signalling inputs mediated by ToxR, -S, and -T controls virulence gene expression leading to cholera toxin (CT) production. Recently, we identified a new virulence locus, varA (virulence associated regulator), in classical V. cholerae O1 that positively controls transcription of tcpA, the major subunit of the toxin-coregulated pilus (TCP) and the production of CT, two key factors in cholera pathogenesis. The varA locus is a homolog of gacA (originally described for the soil organism Pseudomonas fluorescens), which encodes a conserved global regulator belonging to the family of two-component signal transducing molecules. GacA homologs in a number of diverse gram-negative pathogenic bacterial species have been implicated in controlling the production of diverse virulence factors. varA mutants showed reduced levels of tcpA message and TcpA protein, lacked visible signs of autoagglutination (a phenotype associated with functional TCP), produced decreased levels of CT, and were attenuated in colonizing infant mice. Transcription of varA appears to be independent of ToxR, and overexpression of the regulators tcpPH and toxT from plasmids in the varA mutant restored wild-type levels of CT production and the ability to autoagglutinate. varA represents an additional modulating factor in the coordinate expression of virulence factors in V. cholerae.

Use of model plant hosts to identify Pseudomonas aeruginosa virulence factors.

We used plants as an in vivo pathogenesis model for the identification of virulence factors of the human opportunistic pathogen Pseudomonas aeruginosa. Nine of nine TnphoA mutant derivatives of P. aeruginosa strain UCBPP-PA14 that were identified in a plant leaf assay for less pathogenic mutants also exhibited significantly reduced pathogenicity in a burned mouse pathogenicity model, suggesting that P. aeruginosa utilizes common strategies to infect both hosts. Seven of these nine mutants contain TnphoA insertions in previously unknown genes. These results demonstrate that an alternative nonvertebrate host of a human bacterial pathogen can be used in an in vivo high throughput screen to identify novel bacterial virulence factors involved in mammalian pathogenesis.

Common virulence factors for bacterial pathogenicity in plants and animals.

A Pseudomonas aeruginosa strain (UCBPP-PA14) is infectious both in an Arabidopsis thaliana leaf infiltration model and in a mouse full-thickness skin burn model. UCBPP-PA14 exhibits ecotype specificity for Arabidopsis, causing a range of symptoms from none to severe in four different ecotypes. In the mouse model, UCBPP-PA14 is as lethal as other well-studied P. aeruginosa strains. Mutations in the UCBPP-PA14 toxA, plcS, and gacA genes resulted in a significant reduction in pathogenicity in both hosts, indicating that these genes encode virulence factors required for the full expression of pathogenicity in both plants and animals.

Plant and environmental sensory signals control the expression of hrp genes in Pseudomonas syringae pv. phaseolicola.

The hrp genes of Pseudomonas syringae pv. phaseolicola control the development of primary disease symptoms in bean plants and the elicitation of the hypersensitive response in resistant plants. We examined the expression of the seven operons located in the 22-kb hrp cluster (L. G. Rahme, M. N. Mindrinos, and N. J. Panopoulos, J. Bacteriol. 173:575-586, 1991) in planta and in vitro under different physiological and nutritional conditions by using chromosomally located hrp::inaZ reporter fusions. We show that (i) a plant signal(s) is specifically required for the induction of the seven hrp operons, during both compatible and incompatible interactions; (ii) hrpL and hrpRS are regulated by different mechanisms in planta and in vitro; and (iii) expression of individual hrp loci is differentially affected by pH, osmotic strength, and type of carbon source: hrpAB, hrpC, and hrpD were downregulated similarly by osmolarity, pH, and certain carbon sources; hrpE expression was affected strongly by pH and carbon substrate and slightly by osmolarity; and hrpF was not substantially affected by any of these factors. These findings suggest complex signaling mechanisms taking place during plant-pathogen interactions.

Genetic and transcriptional organization of the hrp cluster of Pseudomonas syringae pv. phaseolicola.

The hrp cluster of Pseudomonas syringae pv. phaseolicola encodes functions that are essential for pathogenicity on bean plants and for the elicitation of the hypersensitive response on resistant plants. The cluster was saturated with insertions of transposon Tn3-spice that served both as a mutagen and as a sensitive reporter of the expression of the target regions. The mutations covered a 17.5-kb segment in strain NPS3121, in which seven hrp::Tn5 insertions had been previously mapped, and regions outside this segment. The cluster is organized into seven distinct complementation groups (hrpL, hrpAB, hrpC, hrpD, hrpE, hrpF, and hrpSR) on the basis of the analysis of over 100 Tn3-spice insertions in plasmids and 43 similar insertions in the chromosome; it spans nearly 22 kb and is chromosomally located. The transcriptional orientation of all genes in the cluster was established by measuring the level of ice nucleation activity of complemented merodiploids carrying chromosomal hrp::inaZ fusions after inoculation in Red Kidney bean leaves. Although all seven loci were actively expressed in Red Kidney bean leaves, none of them was substantially expressed when the bacteria were grown in King B broth medium. Mutations in all loci, except those in hrpC, greatly reduced the ability of the bacteria to multiply in bean leaves. Mutations in the hrpC locus, although preventing the bacteria from eliciting a hypersensitive reaction on tobacco, allowed the bacteria to produce delayed and attenuated symptoms in Red Kidney bean leaves and to multiply to a level 10(2)- to 10(3)-fold lower than that of the wild-type strain. This is the first comprehensive report of the genetic and transcriptional organization of the hrp gene cluster in a phytopathogenic bacterium.