Genome-wide identification of Pseudomonas aeruginosa virulence-related genes using a Caenorhabditis elegans infection model.

Pseudomonas aeruginosa strain PA14 is an opportunistic human pathogen capable of infecting a wide range of organisms including the nematode Caenorhabditis elegans. We used a non-redundant transposon mutant library consisting of 5,850 clones corresponding to 75% of the total and approximately 80% of the non-essential PA14 ORFs to carry out a genome-wide screen for attenuation of PA14 virulence in C. elegans. We defined a functionally diverse 180 mutant set (representing 170 unique genes) necessary for normal levels of virulence that included both known and novel virulence factors. Seven previously uncharacterized virulence genes (ABC transporters PchH and PchI, aminopeptidase PepP, ATPase/molecular chaperone ClpA, cold shock domain protein PA0456, putative enoyl-CoA hydratase/isomerase PA0745, and putative transcriptional regulator PA14_27700) were characterized with respect to pigment production and motility and all but one of these mutants exhibited pleiotropic defects in addition to their avirulent phenotype. We examined the collection of genes required for normal levels of PA14 virulence with respect to occurrence in P. aeruginosa strain-specific genomic regions, location on putative and known genomic islands, and phylogenetic distribution across prokaryotes. Genes predominantly contributing to virulence in C. elegans showed neither a bias for strain-specific regions of the P. aeruginosa genome nor for putatively horizontally transferred genomic islands. Instead, within the collection of virulence-related PA14 genes, there was an overrepresentation of genes with a broad phylogenetic distribution that also occur with high frequency in many prokaryotic clades, suggesting that in aggregate the genes required for PA14 virulence in C. elegans are biased towards evolutionarily conserved genes.

Comparing insertion libraries in two Pseudomonas aeruginosa strains to assess gene essentiality.

Putative essential genes can be identified by comparing orthologs not disrupted in multiple near-saturated transposon insertion mutation libraries in related strains of the same bacterial species. Methods for identifying all orthologs between two bacterial strains and putative essential orthologs are described. In addition, protocols detailing near-saturation transposon insertion mutagenesis of bacteria are presented, including (1) conjugation-mediated mutagenesis, (2) automated colony picking and liquid handling of mutant cultures, and (3) arbitrary polymerase chain reaction amplification and sequencing of genomic DNA adjacent to transposon insertion sites.

BifA, a cyclic-Di-GMP phosphodiesterase, inversely regulates biofilm formation and swarming motility by Pseudomonas aeruginosa PA14.

The intracellular signaling molecule, cyclic-di-GMP (c-di-GMP), has been shown to influence bacterial behaviors, including motility and biofilm formation. We report the identification and characterization of PA4367, a gene involved in regulating surface-associated behaviors in Pseudomonas aeruginosa. The PA4367 gene encodes a protein with an EAL domain, associated with c-di-GMP phosphodiesterase activity, as well as a GGDEF domain, which is associated with a c-di-GMP-synthesizing diguanylate cyclase activity. Deletion of the PA4367 gene results in a severe defect in swarming motility and a hyperbiofilm phenotype; thus, we designate this gene bifA, for biofilm formation. We show that BifA localizes to the inner membrane and, in biochemical studies, that purified BifA protein exhibits phosphodiesterase activity in vitro but no detectable diguanylate cyclase activity. Furthermore, mutational analyses of the conserved EAL and GGDEF residues of BifA suggest that both domains are important for the observed phosphodiesterase activity. Consistent with these data, the DeltabifA mutant exhibits increased cellular pools of c-di-GMP relative to the wild type and increased synthesis of a polysaccharide produced by the pel locus. This increased polysaccharide production is required for the enhanced biofilm formed by the DeltabifA mutant but does not contribute to the observed swarming defect. The DeltabifA mutation also results in decreased flagellar reversals. Based on epistasis studies with the previously described sadB gene, we propose that BifA functions upstream of SadB in the control of biofilm formation and swarming.

Genomic analysis reveals that Pseudomonas aeruginosa virulence is combinatorial.

BACKGROUND: Pseudomonas aeruginosa is a ubiquitous environmental bacterium and an important opportunistic human pathogen. Generally, the acquisition of genes in the form of pathogenicity islands distinguishes pathogenic isolates from nonpathogens. We therefore sequenced a highly virulent strain of P. aeruginosa, PA14, and compared it with a previously sequenced (and less pathogenic) strain, PAO1, to identify novel virulence genes. RESULTS: The PA14 and PAO1 genomes are remarkably similar, although PA14 has a slightly larger genome (6.5 megabses [Mb]) than does PAO1 (6.3 Mb). We identified 58 PA14 gene clusters that are absent in PAO1 to determine which of these genes, if any, contribute to its enhanced virulence in a Caenorhabditis elegans pathogenicity model. First, we tested 18 additional diverse strains in the C. elegans model and observed a wide range of pathogenic potential; however, genotyping these strains using a custom microarray showed that the presence of PA14 genes that are absent in PAO1 did not correlate with the virulence of these strains. Second, we utilized a full-genome nonredundant mutant library of PA14 to identify five genes (absent in PAO1) required for C. elegans killing. Surprisingly, although these five genes are present in many other P. aeruginosa strains, they do not correlate with virulence in C. elegans. CONCLUSION: Genes required for pathogenicity in one strain of P. aeruginosa are neither required for nor predictive of virulence in other strains. We therefore propose that virulence in this organism is both multifactorial and combinatorial, the result of a pool of pathogenicity-related genes that interact in various combinations in different genetic backgrounds.

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.

Integration of Caenorhabditis elegans MAPK pathways mediating immunity and stress resistance by MEK-1 MAPK kinase and VHP-1 MAPK phosphatase.

The p38 and JNK classes of mitogen-activated protein kinases (MAPKs) have evolutionarily conserved roles in the control of cellular responses to microbial and abiotic stresses. The mechanisms by which crosstalk between distinct p38 and c-Jun N-terminal kinase (JNK) MAPK pathways occurs with resultant integration of signaling information have been difficult to establish, particularly in the context of whole organism physiology. In Caenorhabditis elegans a PMK-1 p38 MAPK pathway is required for resistance to bacterial infection, and a KGB-1 JNK-like MAPK pathway has recently been shown to mediate resistance to heavy metal stress. Here, we show that two components of the KGB-1 pathway, MEK-1 MAPK kinase (MAPKK), a homolog of mammalian MKK7, and VHP-1 MAPK phosphatase (MKP), a homolog of mammalian MKP7, also regulate pathogen resistance through the modulation of PMK-1 activity. The regulation of p38 and JNK-like MAPK pathways mediating immunity and heavy metal stress by common MAPKK and MKP signaling components suggests pivotal roles for MEK-1 and VHP-1 in the integration of diverse stress signals contributing to pathogen resistance in C. elegans. In addition, these data point to mechanisms in multicellular organisms by which signals transduced by distinct MAPK pathways may be subject to physiological integration at the level of regulation of MAPK activity by MAPKKs and MKPs.

Casein kinase Iepsilon plays a functional role in the transforming growth factor-beta signaling pathway.

The transforming growth factor-beta (TGF-beta) signaling pathway is known to be involved in a wide range of biological events, including development, cellular differentiation, apoptosis, and oncogenesis. The TGF-beta signal is mediated by ligand binding to the type II receptor, leading to the recruitment and activation of the type I receptor, and subsequent activation of a family of intracellular signal transducing proteins called Smads. Here we report a regulatory role for casein kinase Iepsilon (CKIepsilon) in the TGF-beta signaling cascade. We find that CKIepsilon binds to all Smads and the cytoplasmic domains of the type I and type II receptors both in vitro and in vivo. The interaction of CKIepsilon with the type I and type II receptors is independent of TGF-beta stimulation, whereas the CKIepsilon/Smad interaction is transiently disrupted by ligand treatment. Additionally, CKIepsilon is able to phosphorylate the receptor-activated Smads (Smads 1-3 and 5) and the type II receptor in vitro. Transcriptional reporter assays reveal that transient overexpression of wild type CKIepsilon dramatically reduces basal reporter activity but enhances TGF-beta-stimulated transcription. Furthermore, overexpression of a kinase-dead mutant of CKIepsilon inhibits both basal and ligand-induced transcription, whereas inhibition of endogenous CKI catalytic activity with IC261 blocks only TGF-beta-stimulated reporter activity. Finally, knocking down CKIepsilon protein levels results in a significant increase in basal and TGF-beta-induced transcription. These results suggest that CKIepsilon plays a ligand-dependent, differential, and dual regulatory role within the TGF-beta signaling pathway.

Requirement for a conserved Toll/interleukin-1 resistance domain protein in the Caenorhabditis elegans immune response.

The p38 mitogen-activated protein kinase pathway regulates innate immune responses in evolutionarily diverse species. We have previously shown that the Caenorhabditis elegans p38 mitogen-activated protein kinase, PMK-1, functions in an innate immune response pathway that mediates resistance to a variety of microbial pathogens. Here, we show that tir-1, a gene encoding a highly conserved Toll/IL-1 resistance (TIR) domain protein, is also required for C. elegans resistance to microbial pathogens. RNA interference inactivation of tir-1 resulted in enhanced susceptibility to killing by pathogens and correspondingly diminished PMK-1 phosphorylation. Unlike all known TIR-domain adapter proteins, overexpression of the human TIR-1 homologue, SARM, in mammalian cells was not sufficient to induce expression of NF-kappaB or IRF3-dependent reporter genes that are activated by Toll-like receptor signaling. These data reveal the involvement of a previously uncharacterized, evolutionarily conserved TIR domain protein in innate immunity that is functionally distinct from other known TIR domain signaling adapters.

Transforming growth factor beta-mediated transcriptional repression of c-myc is dependent on direct binding of Smad3 to a novel repressive Smad binding element.

Smad proteins are the most well-characterized intracellular effectors of the transforming growth factor beta (TGF-beta) signal. The ability of the Smads to act as transcriptional activators via TGF-beta-induced recruitment to Smad binding elements (SBE) within the promoters of TGF-beta target genes has been firmly established. However, the elucidation of the molecular mechanisms involved in TGF-beta-mediated transcriptional repression are only recently being uncovered. The proto-oncogene c-myc is repressed by TGF-beta, and this repression is required for the manifestation of the TGF-beta cytostatic program in specific cell types. We have shown that Smad3 is required for both TGF-beta-induced repression of c-myc and subsequent growth arrest in keratinocytes. The transcriptional repression of c-myc is dependent on direct Smad3 binding to a novel Smad binding site, termed a repressive Smad binding element (RSBE), within the TGF-beta inhibitory element (TIE) of the c-myc promoter. The c-myc TIE is a composite element, comprised of an overlapping RSBE and a consensus E2F site, that is capable of binding at least Smad3, Smad4, E2F-4, and p107. The RSBE is distinct from the previously defined SBE and may partially dictate, in conjunction with the promoter context of the overlapping E2F site, whether the Smad3-containing complex actively represses, as opposed to transactivates, the c-myc promoter.

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.