Genetic and biochemical analyses of a eukaryotic-like phospholipase D of Pseudomonas aeruginosa suggest horizontal acquisition and a role for persistence in a chronic pulmonary infection model

Phospholipases D (PLDs) are virtually ubiquitous in eukaryotic organisms; however, they are relatively uncommon in prokaryotes. In this report, we demonstrate that the environmentally acquired, opportunistic pathogen Pseudomonas aeruginosa expresses PLD activity. A gene designated pldA was identified in the genomic database of P. aeruginosa PAO1 encoding a protein with significant homology to eukaryotic PLDs, but not to any prokaryotic PLDs. PldA is most homologous to PLDs from mammals and yeast. The pldA gene was cloned and shown to express an approximately 116 kDa protein with calcium-regulated PLD activity that is localized to the periplasm. Interestingly, not all strains of P. aeruginosa carry pldA. When present, pldA is always linked to an open reading frame (ORF), ORF4, and a gene (vgrA1) encoding a protein homologous to Vgr from Escherichia coli. Vgr proteins contain regularly repeated dipeptide motifs (valine-glycine repeats). In E. coli, genes encoding Vgr are associated with multicopy genetic elements designated Rhs (rearrangement hot-spots). P. aeruginosa PAO1 has 10 vgr homologues dispersed throughout its genome, but the copy number of these genetic elements varies considerably in different strains. Neither vgrA1 nor ORF4 is present in strains lacking pldA. Furthermore, sequences flanking vgrA1, pldA and ORF4 in the P. aeruginosa strains examined are highly conserved, suggesting a specific site of insertion. These and other data suggest that vgrA1, pldA and ORF4 constitute an approximately 7 kb mobile genetic element and that pldA was acquired horizontally, perhaps from a eukaryotic organism. Competition studies between a PldA knock-out mutant and the parental wild-type strain indicate that PldA contributes to the ability of P. aeruginosa PAO1 to persist in a chronic pulmonary infection model in rats.     

Identification of Virulence Genes in a Pathogenic Strain of Pseudomonas aeruginosa by Representational Difference Analysis

Pseudomonas aeruginosa is an opportunistic pathogen that may cause severe infections in humans and other vertebrates. In addition, a human clinical isolate of P. aeruginosa, strain PA14, also causes disease in a variety of nonvertebrate hosts, including plants, Caenorhabditis elegans, and the greater wax moth, Galleria mellonella. This has led to the development of a multihost pathogenesis system in which plants, nematodes, and insects have been used as adjuncts to animal models for the identification of P. aeruginosa virulence factors. Another approach to identifying virulence genes in bacteria is to take advantage of the natural differences in pathogenicity between isolates of the same species and to use a subtractive hybridization technique to recover relevant genomic differences. The sequenced strain of P. aeruginosa, strain PAO1, has substantial differences in virulence from strain PA14 in several of the multihost models of pathogenicity, and we have utilized the technique of representational difference analysis (RDA) to directly identify genomic differences between P. aeruginosa strains PA14 and PAO1. We have found that the pilC, pilA, and uvrD genes in strain PA14 differ substantially from their counterparts in strain PAO1. In addition, we have recovered a gene homologous to the ybtQ gene from Yersinia, which is specifically present in strain PA14 but absent in strain PAO1. Mutation of the ybtQ homolog in P. aeruginosa strain PA14 significantly attenuates the virulence of this strain in both G. mellonella and a burned mouse model of sepsis to levels comparable to those seen with PAO1. This suggests that the increased virulence of P. aeruginosa strain PA14 compared to PAO1 may relate to specific genomic differences identifiable by RDA.     

The Pseudomonas aeruginosa devB/SOL homolog, pgl, is a member of the hex regulon and encodes 6-phosphogluconolactonase

A cyclic version of the Entner-Doudoroff pathway is used by Pseudomonas aeruginosa to metabolize carbohydrates. Genes encoding the enzymes that catabolize intracellular glucose to pyruvate and glyceraldehyde 3-phosphate are coordinately regulated, clustered at 39 min on the chromosome, and collectively form the hex regulon. Within the hex cluster is an open reading frame (ORF) with homology to the devB/SOL family of unidentified proteins. This ORF encodes a protein of either 243 or 238 amino acids; it overlaps the 5' end of zwf (encodes glucose-6-phosphate dehydrogenase) and is followed immediately by eda (encodes the Entner-Doudoroff aldolase). The devB/SOL homolog was inactivated in P. aeruginosa PAO1 by recombination with a suicide plasmid containing an interrupted copy of the gene, creating mutant strain PAO8029. PAO8029 grows at 9% of the wild-type rate using mannitol as the carbon source and at 50% of the wild-type rate using gluconate as the carbon source. Cell extracts of PAO8029 were specifically deficient in 6-phosphogluconolactonase (Pgl) activity. The cloned devB/SOL homolog complemented PAO8029 to restore normal growth on mannitol and gluconate and restored Pgl activity. Hence, we have identified this gene as pgl and propose that the devB/SOL family members encode 6-phosphogluconolactonases. Interestingly, three eukaryotic glucose-6-phosphate dehydrogenase (G6PDH) isozymes, from human, rabbit, and Plasmodium falciparum, contain Pgl domains, suggesting that the sequential reactions of G6PDH and Pgl are incorporated in a single protein. 6-Phosphogluconolactonase activity is induced in P. aeruginosa PAO1 by growth on mannitol and repressed by growth on succinate, and it is expressed constitutively in P. aeruginosa PAO8026 (hexR). Taken together, these results establish that Pgl is an essential enzyme of the cyclic Entner-Doudoroff pathway encoded by pgl, a structural gene of the hex regulon.     

Microarray analysis of global gene expression in mucoid Pseudomonas aeruginosa

Pseudomonas aeruginosa is the dominant pathogen causing chronic respiratory infections in cystic fibrosis (CF). After an initial phase characterized by intermittent infections, a chronic colonization is established in CF upon the conversion of P. aeruginosa to the mucoid, exopolysaccharide alginate-overproducing phenotype. The emergence of mucoid P. aeruginosa in CF is associated with respiratory decline and poor prognosis. The switch to mucoidy in most CF isolates is caused by mutations in the mucA gene encoding an anti-sigma factor. The mutations in mucA result in the activation of the alternative sigma factor AlgU, the P. aeruginosa ortholog of Escherichia coli extreme stress sigma factor sigma(E). Because of the global nature of the regulators of mucoidy, we have hypothesized that other genes, in addition to those specific for alginate production, must be induced upon conversion to mucoidy, and their production may contribute to the pathogenesis in CF. Here we applied microarray analysis to identify on the whole-genome scale those genes that are coinduced with the AlgU sigmulon upon conversion to mucoidy. Gene expression profiles of AlgU-dependent conversion to mucoidy revealed coinduction of a specific subset of known virulence determinants (the major protease elastase gene, alkaline metalloproteinase gene aprA, and the protease secretion factor genes aprE and aprF) or toxic factors (cyanide synthase) that may have implications for disease in CF. Analysis of promoter regions of the most highly induced genes (>40-fold, P < or = 10(-4)) revealed a previously unrecognized, putative AlgU promoter upstream of the osmotically inducible gene osmE. This newly identified AlgU-dependent promoter of osmE was confirmed by mapping the mRNA 5' end by primer extension. The recognition of genes induced in mucoid P. aeruginosa, other than those associated with alginate biosynthesis, reported here revealed the identity of previously unappreciated factors potentially contributing to the morbidity and mortality caused by mucoid P. aeruginosa in CF.     

Natriuretic peptides affect Pseudomonas aeruginosa and specifically modify lipopolysaccharide biosynthesis

Natriuretic peptides of various forms are present in animals and plants, and display structural similarities to cyclic antibacterial peptides. Pretreatment of Pseudomonas aeruginosa PAO1 with brain natriuretic peptide (BNP) or C-type natriuretic peptide (CNP) increases bacterium-induced glial cell necrosis. In eukaryotes, natriuretic peptides act through receptors coupled to cyclases. We observed that stable analogs of cAMP (dibutyryl cAMP) and cGMP (8-bromo-cGMP) mimicked the effect of brain natriuretic peptide and CNP on bacteria. Further evidence for the involvement of bacterial cyclases in the regulation of P. aeruginosa PAO1 cytotoxicity by natriuretic peptides is provided by the observed doubling of intrabacterial cAMP concentration after exposure to CNP. Lipopolysaccharide (LPS) extracted from P. aeruginosa PAO1 treated with both dibutyryl cAMP and 8-bromo-cGMP induces higher levels of necrosis than LPS extracted from untreated bacteria. Capillary electrophoresis and MALDI-TOF MS analysis have shown that differences in LPS toxicity are due to specific differences in the structure of the macromolecule. Using a strain deleted in the vfr gene, we showed that the Vfr protein is essential for the effect of natriuretic peptides on P. aeruginosa PAO1 virulence. These data support the hypothesis that P. aeruginosa has a cyclic nucleotide-dependent natriuretic peptide sensor system that may affect virulence by activating the expression of Vfr and LPS biosynthesis.     

Pseudomonas aeruginosa LasB mutant constructed by insertional mutagenesis reveais elastolytic activity due to alkaline proteinase and the LasA fragment

The extracellularly secreted endopeptidase elastase (LasB) is regarded as an important virulence factor of Pseudomonas aeruginosa. It has also been implicated in the processing of LasA which enhances elastolytic activity of LasB. In order to investigate the role of LasB in virulence and LasA processing, a LasB-negative mutant, PAO1E, was constructed by insertional mutagenesis of the LasB structural gene, lasB, in P. aeruginosa PAO. An internal 636 bp lasB fragment of the plasmid pRB1803 was ligated into a derivative of the mobilization vector pSUP201-1. The resulting plasmid, pBRMOB-LasB, was transformed into Escherichia coli and transferred by filter matings to the LasB-positive P. aeruginosa strain, PAO1. Plasmid integration in the lasB site of the chromosome was confirmed by Southern blot analysis. Radioimmunoassay and immunoblotting of PAO1E supernatant fluids yielded no detectable LasB (less than 1 ng ml-1 LasB). The absence of LasB in PAO1E was further proven by the inability of its culture supernatant fluid to cleave transferrin or rabbit immunoglobulin G (IgG) after a 72 h incubation. The residual proteolytic activity of PAO1E culture supernatant fluid was attributed to alkaline proteinase (Apr), since it was totally inhibited by specific antibodies against Apr. Residual elastolytic activity in culture supernatant fluid of PAO1E was due to the LasA fragment and to the combined action of the LasA fragment with Apr on elastin. The sizes of purified LasA from PAO1 and PAO1E were identical (22 kDa). These results show that, besides LasB and the LasA fragment, Apr may also act on elastin in the presence of the LasA fragment and that the proteolytic processing of LasA in P. aeruginosa is independent of LasB.     

Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis.

The alternative sigma factor AlgU (Pseudomonas aeruginosa sigma E) is required for full resistance of P. aeruginosa to oxidative stress and extreme temperatures. AlgU also controls conversion of P. aeruginosa to the mucoid, alginate-overproducing phenotype associated with lethal infections in cystic fibrosis patients. Mutations that cause conversion to mucoidy in cystic fibrosis isolates occur frequently in mucA, the second gene within the algU mucABCD gene cluster. Here we analyze the biochemical basis of conversion to mucoidy. MucA was shown to act as an anti-sigma factor by binding to AlgU and inhibiting its activity. MucB, another negative regulator of AlgU, was localized in the periplasm. MucB exerts its function from this compartment, since deletion of the leader peptide and the cytoplasmic location of MucB abrogated its ability to inhibit mucoidy. These data support a model in which a multicomponent system, encompassing an anti-delta factor and elements in the periplasmic compartment, modulates activity of AlgU. Since factors controlling AlgU are conserved in other gram-negative bacteria, the processes controlling conversion to mucoidy in P. aeruginosa may be applicable to the regulation of AlgU (sigma E) equivalents in other organisms.     

Effect of superoxide dismutase gene inactivation on virulence of Pseudomonas aeruginosa PAO1 toward the silkworm, Bombyx mori

To investigate the role of superoxide dismutase (SOD) in virulence against the silkworm, Bombyx mori, mutants of Pseudomonas aeruginosa PAO1 lacking manganese-SOD (PAO1sodM), iron-SOD (PAO1sodB), or both (PAO1sodMB) were generated. The mutants were injected into the hemocoel of B. mori. The virulence decreased in the order PAO1=PAO1sodM>PAO1sodB>PAO1sodMB. In particular, PAO1sodMB was avirulent at a dose of 10(5) cells or less. The sod double mutant PAO1sodMB was then complemented with either pSodM or pSodB in trans. In both the complemented strains, the virulence was partially restored. Of the two plasmids, pSodB contributed more to the virulence of P. aeruginosa against B. mori. The results of growth in B. mori hemolymph broth and microscopic analysis suggested that a longer lag phase and superoxide sensitivity correlated with decreased virulence in sod mutants. In conclusion, the SODs are required for full virulence of P. aeruginosa against B. mori and Fe-SOD is more important than Mn-SOD in the infection process.     

Analysis of the Pseudomonas aeruginosa regulon controlled by the sensor kinase KinB and sigma factor RpoN

Alginate overproduction by Pseudomonas aeruginosa, also known as mucoidy, is associated with chronic endobronchial infections in cystic fibrosis. Alginate biosynthesis is initiated by the extracytoplasmic function sigma factor (σ(22); AlgU/AlgT). In the wild-type (wt) nonmucoid strains, such as PAO1, AlgU is sequestered to the cytoplasmic membrane by the anti-sigma factor MucA that inhibits alginate production. One mechanism underlying the conversion to mucoidy is mutation of mucA. However, the mucoid conversion can occur in wt mucA strains via the degradation of MucA by activated intramembrane proteases AlgW and/or MucP. Previously, we reported that the deletion of the sensor kinase KinB in PAO1 induces an AlgW-dependent proteolysis of MucA, resulting in alginate overproduction. This type of mucoid induction requires the alternate sigma factor RpoN (σ(54)). To determine the RpoN-dependent KinB regulon, microarray and proteomic analyses were performed on a mucoid kinB mutant and an isogenic nonmucoid kinB rpoN double mutant. In the kinB mutant of PAO1, RpoN controlled the expression of approximately 20% of the genome. In addition to alginate biosynthetic and regulatory genes, KinB and RpoN also control a large number of genes including those involved in carbohydrate metabolism, quorum sensing, iron regulation, rhamnolipid production, and motility. In an acute pneumonia murine infection model, BALB/c mice exhibited increased survival when challenged with the kinB mutant relative to survival with PAO1 challenge. Together, these data strongly suggest that KinB regulates virulence factors important for the development of acute pneumonia and conversion to mucoidy.     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。