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.     

Killing of Caenorhabditis elegans by Pseudomonas aeruginosa used to model mammalian bacterial pathogenesis

We show that a single clinical isolate of the human opportunistic pathogen Pseudomonas aeruginosa (strain PA14), which previously was shown to be pathogenic in mice and plants, also kills Caenorhabditis elegans. The rate of PA14-mediated killing of C. elegans depends on the composition of the agar medium on which PA14 is grown. When PA14 is grown on minimal medium, killing occurs over the course of several days and is referred to as “slow” killing. When PA14 is grown on high-osmolarity medium, killing occurs over the course of several hours and is referred to as “fast” killing. Several lines of evidence, including the fact that heat-killed bacteria are still capable of fast but not slow killing of C. elegans, indicate that fast and slow killing occur by distinct mechanisms. Slow killing involves an infection-like process and correlates with the accumulation of PA14 within worm intestines. Among 10 PA14 virulence-related mutants that had been shown previously to affect pathogenicity in plants and mice, 6 were less effective in killing C. elegans under both fast- and slow-killing conditions, indicating a high degree of commonalty among the P. aeruginosa factors required for pathogenicity in disparate eukaryotic hosts. Thus, we show that a C. elegans pathogenicity model that is genetically tractable from the perspectives of both host and pathogen can be used to model mammalian bacterial pathogenesis.     

Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence

The opportunistic pathogen Pseudomonas aeruginosa is responsible for systemic infections in immunocompromised individuals and chronic respiratory disease in patients with cystic fibrosis. Cyclic nucleotides are known to play a variety of roles in the regulation of virulence-related factors in pathogenic bacteria. A set of P. aeruginosa genes, encoding proteins that contain putative domains characteristic of diguanylate cyclases (DGCs) and phosphodiesterases (PDEs) that are responsible for the maintenance of cellular levels of the second messenger bis-(3'-5')-cyclic dimeric GMP (c-di-GMP) was identified in the annotated genomes of P. aeruginosa strains PAO1 and PA14. Although the majority of these genes are components of the P. aeruginosa core genome, several are located on presumptive horizontally acquired genomic islands. A comprehensive analysis of P. aeruginosa genes encoding the enzymes of c-di-GMP metabolism (DGC- and PDE-encoding genes) was carried out to analyze the function of c-di-GMP in two disease-related phenomena, cytotoxicity and biofilm formation. Analysis of the phenotypes of DGC and PDE mutants and overexpressing clones revealed that certain virulence-associated traits are controlled by multiple DGCs and PDEs through alterations in c-di-GMP levels. A set of mutants in selected DGC- and PDE-encoding genes exhibited attenuated virulence in a mouse infection model. Given that insertions in different DGC and PDE genes result in distinct phenotypes, it seems likely that the formation or degradation of c-di-GMP by these enzymes is in highly localized and intimately linked to particular targets of c-di-GMP action.     

An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants

Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.     

Social cheating in Pseudomonas aeruginosa quorum sensing

In a process termed quorum sensing, bacteria use diffusible chemical signals to coordinate cell density-dependent gene expression. In the human pathogen Pseudomonas aeruginosa, quorum sensing controls hundreds of genes, many of which encode extracellular virulence factors. Quorum sensing is required for P. aeruginosa virulence in animal models. Curiously, quorum sensing-deficient variants, most of which carry a mutation in the gene encoding the central quorum sensing regulator lasR, are frequently isolated from acute and chronic infections. The mechanism for their emergence is not known. Here we provide experimental evidence suggesting that these lasR mutants are social cheaters that cease production of quorum-controlled factors and take advantage of their production by the group. We detected an emerging subpopulation of lasR mutants after approximately 100 generations of in vitro evolution of the P. aeruginosa wild-type strain under culture conditions that require quorum sensing for growth. Under such conditions, quorum sensing appears to impose a metabolic burden on the proliferating bacterial cell, because quorum-controlled genes not normally induced until cessation of growth were highly expressed early in growth, and a defined lasR mutant showed a growth advantage when cocultured with the parent strain. The emergence of quorum-sensing-deficient variants in certain environments is therefore an indicator of high quorum sensing activity of the bacterial population as a whole. It does not necessarily indicate that quorum sensing is insignificant, as has previously been suggested. Thus, novel antivirulence strategies aimed at disrupting bacterial communication may be particularly effective in such clinical settings.     

The oxygen- and iron-dependent sigma factor pvdS of Pseudomonas aeruginosa is an important virulence factor in experimental infective endocarditis

In Pseudomonas aeruginosa, pvdS, a key oxygen (O2)-dependent locus, regulates expression of a number of virulence genes, including toxA (which encodes exotoxin A production). To define the in vivo role of differing O2 tensions on pseudomonal virulence, 2 knockout mutants, DeltapvdS and DeltatoxA, were compared with their parental strain, PA01, in rabbit aortic and tricuspid endocarditis models (representing aerobic vs. microaerobic conditions in vivo, respectively). In aortic endocarditis, DeltapvdS densities were significantly less than those of PA01 in vegetations, kidneys, and spleen (P<.01). In contrast, in tricuspid endocarditis, there were no significant differences between DeltapvdS and PA01 tissue densities in these same target tissues. The DeltatoxA mutant proliferated within target tissues to the same extent as the parental strain. Thus, pvdS (but not toxA) appears to be required for optimal virulence of P. aeruginosa, particularly in tissues preferentially exposed to high O2 tensions (e.g., aortic vegetations).     

Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing antimicrobial peptide gene expression

Insights into the host factors and mechanisms mediating the primary host responses after pathogen presentation remain limited, due in part to the complexity and genetic intractability of host systems. Here, we employ the model Drosophila melanogaster to dissect and identify early host responses that function in the initiation and progression of Pseudomonas aeruginosa pathogenesis. First, we use immune potentiation and genetic studies to demonstrate that flies mount a heightened defense against the highly virulent P. aeruginosa strain PA14 when first inoculated with strain CF5, which is avirulent in flies; this effect is mediated via the Imd and Toll signaling pathways. Second, we use whole-genome expression profiling to assess and compare the Drosophila early defense responses triggered by the PA14 vs. CF5 strains to identify genes whose expression patterns are different in susceptible vs. resistant host-pathogen interactions, respectively. Our results identify pathogenesis- and defense-specific genes and uncover a previously undescribed mechanism used by P. aeruginosa in the initial stages of its host interaction: suppression of Drosophila defense responses by limiting antimicrobial peptide gene expression. These results provide insights into the genetic factors that mediate or restrict pathogenesis during the early stages of the bacterial-host interaction to advance our understanding of P. aeruginosa-human infections.     

Interstrain transfer of the large pathogenicity island (PAPI-1) of Pseudomonas aeruginosa

The large Pseudomonas aeruginosa pathogenicity island PAPI-1 of strain PA14 is a cluster of 108 genes that encode a number of virulence features. We demonstrate that, in a subpopulation of cells, PAPI-1 can exist in an extrachromosomal circular form after precise excision from its integration site within the 3' terminus of the tRNA(Lys) gene. Circular PAPI-1 can reintegrate into either of the two tRNA(Lys) genes, including the one that was used for integration of small pathogenicity island PAPI-2 in strain PA14. The excision requires PAPI-1-encoded integrase, a member of the tyrosine recombinase family. PAPI-1 Soj contains the conserved domains of proteins that are related to chromosome and plasmid partition. soj plays a role in maintaining PAPI-1 and mutations in soj result in the loss of PAPI-1 from P. aeruginosa. We further demonstrate that, during coculture, the PAPI-1-containing strains are able to transfer it into P. aeruginosa recipient strains that do not harbor this island naturally. After transfer, PAPI-1 integrates into either of the two tRNA(Lys) genes. PAPI-1 encompasses many features of mobile elements, including mobilization and maintenance modules. Together with the virulence determinants, PAPI-1 plays an important role in the evolution of P. aeruginosa, by expanding its natural habitat from soil and water to animal and human infections.     

Regulated proteolysis controls mucoid conversion in Pseudomonas aeruginosa

Overproduction of the exopolysaccharide alginate causes mucoid conversion in Pseudomonas aeruginosa and is a poor prognosticator in cystic fibrosis. The ECF sigma factor AlgU and its cognate anti-sigma factor MucA are two principal regulators of alginate production. Here, we report the identification of three positive regulators of alginate biosynthesis: PA4033 (designated mucE), PA3649 (designated mucP), and algW. MucE, a small protein (9.5 kDa), was identified as part of a global mariner transposon screen for new regulators of alginate production. A transposon located in its promoter caused the overexpression of MucE and mucoid conversion in P. aeruginosa strains PAO1 and PA14. Accumulation of MucE in the envelope resulted in increased AlgU activity and reduced MucA levels. Three critical amino acid residues at the C terminus of MucE (WVF) were required for mucoid conversion via two predicted proteases AlgW (DegS) and MucP (RseP/YaeL). Moreover, as in Escherichia coli, the PDZ domain of AlgW was required for signal transduction. These results suggest that AlgU is regulated similarly to E. coli sigma(E) except that the amino acid triad signals from MucE and other envelope proteins that activate AlgW are slightly different from those activating DegS.     

Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa

Identification of host factors that interact with pathogens is crucial to an understanding of infectious disease, but direct screening for host mutations to aid in this task is not feasible in mammals. The nematode Caenorhabditis elegans is a genetically tractable alternative for investigating the pathogenic bacterium Pseudomonas aeruginosa. A P. aeruginosa toxin, produced at high cell density under control of the quorum-sensing regulators LasR and RhlR, rapidly and lethally paralyzes C. elegans. Loss-of-function mutations in C. elegans egl-9, a gene required for normal egg laying, confer strong resistance to the paralysis. Thus, activation of EGL-9 or of a pathway that includes it may lead to the paralysis. The molecular identity of egl-9 was determined by transformation rescue and DNA sequencing. A mammalian homologue of EGL-9 is expressed in tissues in which exposure to P. aeruginosa could have clinical effects.     

A simple alfalfa seedling infection model for Pseudomonas aeruginosa strains associated with cystic fibrosis shows AlgT (sigma-22) and RhlR contribute to pathogenesis

A sensitive plant infection model was developed to identify virulence factors in nontypeable, alginate overproducing (mucoid) Pseudomonas aeruginosa strains isolated from cystic fibrosis (CF) patients with chronic pulmonary disease. Nontypeable strains with defects in lipopolysaccharide O-side chains are common to CF and often exhibit low virulence in animal models of infection. However, 1,000 such bacteria were enough to show disease symptoms in the alfalfa infection. A typical mucoid CF isolate, FRD1, and its isogenic mutants were tested for alfalfa seedling infection. Although defects in the global regulators Vfr, RpoS, PvdS, or LasR had no discernable effect on virulence, a defect in RhlR reduced the infection frequency by >50%. A defect in alginate biosynthesis resulted in plant disease with >3-fold more bacteria per plant, suggesting that alginate overproduction attenuated bacterial growth in planta. FRD1 derivatives lacking AlgT, a sigma factor required for alginate production, were reduced >50% in the frequency of infection. Thus, AlgT apparently regulates factors in FRD1, besides alginate, important for pathogenesis. In contrast, in a non-CF strain, PAO1, an algT mutation did not affect its virulence on alfalfa. Conversely, PAO1 virulence was reduced in a mucA mutant that overproduced alginate. These observations suggested that mucoid conversion in CF may be driven by a selection for organisms with attenuated virulence or growth in the lung, which promotes a chronic infection. These studies also demonstrated that the wounded alfalfa seedling infection model is a useful tool to identify factors contributing to the persistence of P. aeruginosa in CF.     

The broad host range pathogen Pseudomonas aeruginosa strain PA14 carries two pathogenicity islands harboring plant and animal virulence genes

The ubiquitous bacterium Pseudomonas aeruginosa is the quintessential opportunistic pathogen. Certain isolates infect a broad range of host organisms, from plants to humans. The pathogenic promiscuity of particular variants may reflect an increased virulence gene repertoire beyond the core P. aeruginosa genome. We have identified and characterized two P. aeruginosa pathogenicity islands (PAPI-1 and PAPI-2) in the genome of PA14, a highly virulent clinical isolate. The 108-kb PAPI-1 and 11-kb PAPI-2, which are absent from the less virulent reference strain PAO1, exhibit highly modular structures, revealing their complex derivations from a wide array of bacterial species and mobile elements. Most of the genes within these islands that are homologous to known genes occur in other human and plant bacterial pathogens. For example, PAPI-1 carries a complete gene cluster predicted to encode a type IV group B pilus, a well known adhesin absent from strain PAO1. However, >80% of the PAPI-1 DNA sequence is unique, and 75 of its 115 predicted ORF products are unrelated to any known proteins or functional domains. Significantly, many PAPI-1 ORFs also occur in several P. aeruginosa cystic fibrosis isolates. Twenty-three PAPI ORFs were mutated, and 19 were found to be necessary for full plant or animal virulence, with 11 required for both. The large set of "extra" virulence functions encoded by both PAPIs may contribute to the increased promiscuity of highly virulent P. aeruginosa strains, by directing additional pathogenic functions.     

Pathogen-host interactions in Pseudomonas aeruginosa pneumonia

Pseudomonas aeruginosa is an important pathogen causing a wide range of acute and chronic infections. P. aeruginosa rarely causes infection in the normal host, but is an efficient opportunistic pathogen causing serious infections in patients who are mechanically ventilated, individuals who are immunocompromised, and patients with malignancies or HIV infection. Among these risk groups, the most vulnerable hosts are neutropenic and patients who are mechanically ventilated. In addition, P. aeruginosa is the most prevalent chronic infection contributing to the pathogenesis of cystic fibrosis. Because of the ubiquitous nature of P. aeruginosa and its ability to develop resistance to antibiotics, it continues to be problematic from a treatment perspective. The pathogenicity of P. aeruginosa is largely caused by multiple bacterial virulence factors and genetic flexibility enabling it to survive in varied environments. Lung injury associated with P. aeruginosa infection results from both the direct destructive effects of the organism on the lung parenchyma and exuberant host immune responses. This article focuses on the major bacterial virulence factors and important aspects of the host immunity that are involved in the pathogenesis of serious P. aeruginosa infection. In addition to antibiotic therapy, strategies directed toward enhancing host defense and/or limiting excessive inflammation could be important to improve outcome in P. aeruginosa lung infections.     

Genetic footprinting with mariner-based transposition in Pseudomonas aeruginosa

The complete DNA sequence of Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major human pathogen. A comparative genomics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in P. aeruginosa. Use of a highly efficient in vivo mariner transposition system in P. aeruginosa facilitated the analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-SceI homing endonuclease in conjunction with in vitro mariner mutagenesis to generate mutants within targeted regions of the P. aeruginosa chromosome for genetic footprinting analyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformable or may lack well developed in vivo transposition systems. We tested this system with three genes in P. aeruginosa that have putative essential homologs in Haemophilus influenzae. We show that one of three H. influenzae essential gene homologs is needed for growth in P. aeruginosa, validating the practicality of this comparative genomics strategy to identify essential genes in P. aeruginosa.     

Alginate is not a significant component of the extracellular polysaccharide matrix of PA14 and PAO1 Pseudomonas aeruginosa biofilms

The bacterium Pseudomonas aeruginosa causes chronic respiratory infections in cystic fibrosis (CF) patients. Such infections are extremely difficult to control because the bacteria exhibit a biofilm-mode of growth, rendering P. aeruginosa resistant to antibiotics and phagocytic cells. During the course of infection, P. aeruginosa usually undergoes a phenotypic switch to a mucoid colony, which is characterized by the overproduction of the exopolysaccharide alginate. Alginate overproduction has been implicated in protecting P. aeruginosa from the harsh environment present in the CF lung, as well as facilitating its persistence as a biofilm by providing an extracellular matrix that promotes adherence. Because of its association with biofilms in CF patients, it has been assumed that alginate is also the primary exopolysaccharide expressed in biofilms of environmental nonmucoid P. aeruginosa. In this study, we examined the chemical nature of the biofilm matrix produced by wild-type and isogenic alginate biosynthetic mutants of P. aeruginosa. The results clearly indicate that alginate biosynthetic genes are not expressed and that alginate is not required during the formation of nonmucoid biofilms in two P. aeruginosa strains, PAO1 and PA14, that have traditionally been used to study biofilms. Because nonmucoid P. aeruginosa strains are the predominant environmental phenotype and are also involved in the initial colonization in CF patients, these studies have implications in understanding the early events of the infectious process in the CF airway.     

Polyphosphate kinase is essential for biofilm development, quorum sensing, and virulence of Pseudomonas aeruginosa

The human opportunistic pathogen Pseudomonas aeruginosa causes a variety of infections in immunocompromised hosts and in individuals with cystic fibrosis. A knockout mutation in the polyphosphate kinase (ppk) gene, encoding PPK responsible for the synthesis of inorganic polyphosphate from ATP, renders P. aeruginosa cells unable to form a thick and differentiated biofilm. The mutant is aberrant in quorum sensing and responses in that production of the quorum-sensing controlled virulence factors elastase and rhamnolipid are severely reduced. In a burned-mouse pathogenesis model, the virulence of the mutant is greatly reduced with severe defects in the colonization of mouse tissues. The conservation of PPK among many bacterial pathogens and its absence in eukaryotes suggest that PPK might be an attractive target for antimicrobial drugs.