Hemolytic Phospholipase Research Articles

Correlation between antibiotic resistance and virulence of Pseudomonas aeruginosa clinical isolates.

Virulent Pseudomonas aeruginosa. is frequently life-threatening and often challenging to treat, and the emergence of multidrug-resistant isolates presents a critical problem for patients. The aim of the study was concerned with molecular analysis of the virulence factors and antimicrobial resistance profile of multidrug-resistant P. aeruginosa (MDRPA). Out of 44 MDRPA isolates, 12 isolates representing different resistance profiles and sources of samples were selected for further molecular studies. Polymerase chain reaction (PCR) approaches were applied to identify the genes implicated in antimicrobial resistance or virulence factors in the selected MDRPA isolates. Multidrug-resistance (pstS), β-lactamase (IMP7, IMP10, IMP13, and IMP25), and extended spectrum β-lactamase (blaOXA50) genes were detected in all of the selected MDRPA isolates. However, only 4 (33%) MDRPA isolates were positive for the presence of the extended spectrum β-lactamase (blaOXA2) gene. Furthermore, the hemolytic phospholipase C precursor gene (plcH) was detected in all PCR products of the tested MDRPA isolates while the exotoxin A (toxA) gene was absent. Other virulence genes were detected with variable percentage in tested isolates. The statistical analysis revealed a significantly positive correlation (r = 0.779, P = 0.002) between virulence factors and antimicrobial resistance marker profiles of the tested MDRPA isolates.

Global regulator Anr represses PlcH phospholipase activity in Pseudomonas aeruginosa when oxygen is limiting.

Haemolytic phospholipase C (PlcH) is a potent virulence and colonization factor that is expressed at high levels by Pseudomonas aeruginosa within the mammalian host. The phosphorylcholine liberated from phosphatidylcholine and sphingomyelin by PlcH is further catabolized into molecules that both support growth and further induce plcH expression. We have shown previously that the catabolism of PlcH-released choline leads to increased activity of Anr, a global transcriptional regulator that promotes biofilm formation and virulence. Here, we demonstrated the presence of a negative feedback loop in which Anr repressed plcH transcription and we proposed that this regulation allowed for PlcH levels to be maintained in a way that promotes productive host-pathogen interactions. Evidence for Anr-mediated regulation of PlcH came from data showing that growth at low oxygen (1%) repressed PlcH abundance and plcH transcription in the WT, and that plcH transcription was enhanced in an Δanr mutant. The plcH promoter featured an Anr consensus sequence that was conserved across all P. aeruginosa genomes and mutation of conserved nucleotides within the Anr consensus sequence increased plcH expression under hypoxic conditions. The Anr-regulated transcription factor Dnr was not required for this effect. The loss of Anr was not sufficient to completely derepress plcH transcription as GbdR, a positive regulator of plcH, was required for expression. Overexpression of Anr was sufficient to repress plcH transcription even at 21 % oxygen. Anr repressed plcH expression and phospholipase C activity in a cell culture model for P. aeruginosa-epithelial cell interactions.

Enzymatic and hemolytic activity in different Candida species

BackgroundCandida species, in conditions of microbiota imbalance or decreased immune defenses, may be one of the main human fungal pathogens. Virulence factors constitute the mechanisms used by the fungus to avoid host defenses. AimsThis study aimed to investigate the in vitro production of virulence factors, such as hemolytic activity, and deoxyribonuclease (DNase), proteinase, and phospholipase activities in Candida spp. MethodsFifty clinical isolates were analyzed for virulence factors: Candida albicans (15), Candida tropicalis (15), Candida parapsilosis (10), Candida glabrata (5), and Candida krusei (5). Hemolytic activity was determined in Sabouraud dextrose agar plates containing 3% glucose and 7% sheep red cells. Culture media containing, respectively, agar-base DNA, egg yolk, and bovine albumin were used to determine DNase, phospholipase and proteinase activities, respectively. ResultsForty-eight (96%) of 50 isolates showed hemolytic activity, with 10 (20%) positive for DNase, 19 (38%) for proteinase, and 16 (32%) for phospholipase. Statistically significant differences were observed between species for phospholipase (p<0.0001) and proteinase (p<0.05) production. ConclusionsIt is concluded that all species had hemolytic activity. DNase activity was detected in all species except in C. glabrata; proteinase activity was detected in C. albicans, C. tropicalis, and C. parapsilosis; and phospholipase activity was observed in C. albicans and C. tropicalis.

Anr and its activation by PlcH activity in Pseudomonas aeruginosa host colonization and virulence.

Pseudomonas aeruginosa hemolytic phospholipase C (PlcH) degrades phosphatidylcholine (PC), an abundant lipid in cell membranes and lung surfactant. A ΔplcHR mutant, known to be defective in virulence in animal models, was less able to colonize epithelial cell monolayers and was defective in biofilm formation on plastic when grown in lung surfactant. Microarray analyses found that strains defective in PlcH production had lower levels of Anr-regulated transcripts than the wild type. PC degradation stimulated the Anr regulon in an Anr-dependent manner under conditions where Anr activity was submaximal because of the presence of oxygen. Two PC catabolites, choline and glycine betaine (GB), were sufficient to stimulate Anr activity, and their catabolism was required for Anr activation. The addition of choline or GB to glucose-containing medium did not alter Anr protein levels, growth rates, or respiratory activity, and Anr activation could not be attributed to the osmoprotectant functions of GB. The Δanr mutant was defective in virulence in a mouse pneumonia model. Several lines of evidence indicate that Anr is important for the colonization of biotic and abiotic surfaces in both P. aeruginosa PAO1 and PA14 and that increases in Anr activity resulted in enhanced biofilm formation. Our data suggest that PlcH activity promotes Anr activity in oxic environments and that Anr activity contributes to virulence, even in the acute infection phase, where low oxygen tensions are not expected. This finding highlights the relationships among in vivo bacterial metabolism, the activity of the oxygen-sensitive regulator Anr, and virulence.

Choline catabolism to glycine betaine contributes to Pseudomonas aeruginosa survival during murine lung infection.

Pseudomonas aeruginosa can acquire and metabolize a variety of molecules including choline, an abundant host-derived molecule. In P. aeruginosa, choline is oxidized to glycine betaine which can be used as an osmoprotectant, a sole source of carbon and nitrogen, and as an inducer of the virulence factor, hemolytic phospholipase C (PlcH) via the transcriptional regulator GbdR. The primary objective was to determine the contribution of choline conversion to glycine betaine to P. aeruginosa survival during mouse lung infection. A secondary objective was to gain insight into the relative contributions of the different roles of glycine betaine to P. aeruginosa survival during infection. Using a model of acute murine pneumonia, we determined that deletion of the choline oxidase system (encoded by betBA) decreased P. aeruginosa survival in the mouse lung. Deletion of the glycine betaine demethylase genes (gbcA-B), required for glycine betaine catabolism, did not impact P. aeruginosa survival in the lung. Thus, the defect of the betBA mutant was not due to a requirement for glycine betaine catabolism or dependence on a downstream metabolite. Deletion of betBA decreased the abundance of plcH transcript during infection, which suggested a role for PlcH in the betBA survival defect. To test the contribution of plcH to the betBA mutant phenotype a betBAplcHR double deletion mutant was generated. The betBA and betBAplcHR double mutant had a small but significant survival defect compared to the plcHR single mutant, suggesting that regulation of plcH expression is not the only role for glycine betaine during infection. The conclusion was that choline acquisition and its oxidation to glycine betaine contribute to P. aeruginosa survival in the mouse lung. While defective plcH induction can explain a portion of the betBA mutant phenotype, the exact mechanisms driving the betBA mutant survival defect remain unknown.

High-level over-expression, purification, and crystallization of a novel phospholipase C/sphingomyelinase from Pseudomonas aeruginosa

The hemolytic phospholipase C/sphingomyelinase PlcH from the opportunistic pathogen Pseudomonas aeruginosa represents the founding member of a growing family of virulence factors identified in a wide range of bacterial and fungal pathogens. In P. aeruginosa PlcH is co-expressed with a 17kDa chaperone (PlcR2) and secreted as a fully folded heterodimer (PlcHR2) of approximately 95kDa, by the twin arginine translocase (TAT) via the cytoplasmic membrane and through the outer membrane, by the Xcp (TypeII) secretory system. PlcHR2 has been shown to be an important virulence factor in model P. aeruginosa infections and is selectively cytotoxic, at picomolar concentrations to mammalian endothelial cells. Here we report how the various challenges starting from protein overexpression in the native organism P. aeruginosa, the use of detergents in the crystallization and data collection using the most advanced μ-focus synchrotron beam lines were overcome. Native diffraction data of this heterodimeric protein complex were collected up to a resolution of 4Å, whereas needle-shaped crystals of l-selenomethionine substituted PlcHR2 with a maximum diameter of 10 micron were used to collect data sets with a maximum resolution of 2.75Å.

Identification and Evaluation of Twin-Arginine Translocase Inhibitors

The twin-arginine translocase (TAT) in some bacterial pathogens, including Pseudomonas aeruginosa, Burkholderia pseudomallei, and Mycobacterium tuberculosis, contributes to pathogenesis by translocating extracellular virulence determinants across the inner membrane into the periplasm, thereby allowing access to the Xcp (type II) secretory system for further export in Gram-negative organisms, or directly to the outside surface of the cell, as in M. tuberculosis. TAT-mediated secretion appreciably contributes to virulence in both animal and plant models of bacterial infection. Consequently, TAT function is an attractive target for small-molecular-weight compounds that alone or in conjunction with extant antimicrobial agents could become novel therapeutics. The TAT-transported hemolytic phospholipase C (PlcH) of P. aeruginosa and its multiple orthologs produced by the above pathogens can be detected by an accurate and reproducible colorimetric assay using a synthetic substrate that detects phospholipase C activity. Such an assay could be an effective indicator of TAT function. Using carefully constructed recombinant strains to precisely control the expression of PlcH, we developed a high-throughput screening (HTS) assay to evaluate, in duplicate, >80,000 small-molecular-weight compounds as possible TAT inhibitors. Based on additional TAT-related functional assays, purified PlcH protein inhibition experiments, and repeat experiments of the initial screening assay, 39 compounds were selected from the 122 initial hits. Finally, to evaluate candidate inhibitors for TAT specificity, we developed a TAT titration assay that determines whether inhibition of TAT-mediated secretion can be overcome by increasing the levels of TAT expression. The compounds N-phenyl maleimide and Bay 11-7082 appear to directly affect TAT function based on this approach.

The Structural Domains of Pseudomonas aeruginosa Phosphorylcholine Phosphatase Cooperate in Substrate Hydrolysis: 3D Structure and Enzymatic Mechanism

Pseudomonas aeruginosa is an opportunistic Gram-negative pathogen. It colonizes different tissues by the utilization of diverse mechanisms. One of these may involve the breakdown of the host cell membrane through the sequential action of hemolytic phospholipase C and phosphorylcholine phosphatase (PchP). The action of hemolytic phospholipase C on phosphatidylcholine produces phosphorylcholine, which is hydrolyzed to choline (Cho) and inorganic phosphate by PchP. The available biochemical data on this enzyme demonstrate the involvement of two Cho-binding sites in the catalytic cycle and in enzyme regulation. The crystal structure of P. aeruginosa PchP has been determined. It folds into three structural domains. The first domain harbors all the residues involved in catalysis and is well conserved among the haloacid dehalogenase superfamily of proteins. The second domain is characteristic of PchP and is involved in the recognition of the Cho moiety of the substrate. The third domain stabilizes the relative position of the other two. Fortuitously, the crystal structure of PchP captures molecules of Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol) at the active site and at an additional site. This represents two catalytically relevant complexes with just one or two inhibitory Bistris molecules and provides the basis of the PchP function and regulation. Site-directed mutagenesis along with biochemical experiments corroborates the structural observations and demonstrates the interplay between different sites for Cho recognition and inhibition. The structural comparison of PchP with other phosphatases of the haloacid dehalogenase family provides a three-dimensional picture of the conserved catalytic cycle and the structural basis for the recognition of the diverse substrate molecules.

Overexpression and characterization of a thermophilic and hemolytic phospholipase of Vibrio vulnificus cloned in Escherichia coli (phospholipase of V. vulnificus)

A phospholipase (PLase) gene of Vibrio vulnificus was cloned in Escherichia coli and the properties of the gene product were investigated. The PLase structural gene was composed of 1,251 bp, encoding 417 amino acids for a protein with a predicted molecular mass of 47,187 Da including a putative signal sequence. The predicted protein sequence was 87 and 82% identical to those of hemolysins from Vibrio spp. and that of lecithinase from V. cholerae, respectively. A lipid binding motif, GDSL, conserved among various PLases and lipases was also observed. Over-expression of PLase caused inclusion body formation in E. coli, but not that of the PLase subclone without the signal sequence (45 kDa). Purified PLase exhibited hemolytic activity on red blood cells and hydrolyzed phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and soya-lecithin mainly to fatty acid and 1,2diacylglycerol, indicating that it was a PLase with unique catalytic activity. PLase from V. vulnificus had temperature and pH optimum at 45°C and 7.0 in 50 mM Tris-HCl buffer, respectively, but was quite active at temperatures up to 55°C and in a broad range of pH 5 to 10. The activity of the enzyme was enhanced by divalent cations such as Ca 2+ , Co 2+ , Mg 2+ , and Mn 2+ , but not by ethylenediaminetetraacetic acid (EDTA).

Cellular choline and glycine betaine pools impact osmoprotection and phospholipase C production in Pseudomonas aeruginosa.

Choline is abundantly produced by eukaryotes and plays an important role as a precursor of the osmoprotectant glycine betaine. In Pseudomonas aeruginosa, glycine betaine has additional roles as a nutrient source and an inducer of the hemolytic phospholipase C, PlcH. The multiple functions for glycine betaine suggested that the cytoplasmic pool of glycine betaine is regulated in P. aeruginosa. We used (13)C nuclear magnetic resonance ((13)C-NMR) to demonstrate that P. aeruginosa maintains both choline and glycine betaine pools under a variety of conditions, in contrast to the transient glycine betaine pool reported for most bacteria. We were able to experimentally manipulate the choline and glycine betaine pools by overexpression of the cognate catabolic genes. Depletion of either the choline or glycine betaine pool reduced phospholipase production, a result unexpected for choline depletion. Depletion of the glycine betaine pool, but not the choline pool, inhibited growth under conditions of high salt with glucose as the primary carbon source. Depletion of the choline pool inhibited growth under high-salt conditions with choline as the sole carbon source, suggesting a role for the choline pool under these conditions. Here we have described the presence of a choline pool in P. aeruginosa and other pseudomonads that, with the glycine betaine pool, regulates osmoprotection and phospholipase production and impacts growth under high-salt conditions. These findings suggest that the levels of both pools are actively maintained and that perturbation of either pool impacts P. aeruginosa physiology.

Campylobacter concisus – A New Player in Intestinal Disease

Over the last decade Campylobacter concisus, a highly fastidious member of the Campylobacter genus has been described as an emergent pathogen of the human intestinal tract. Historically, C. concisus was associated with the human oral cavity and has been linked with periodontal lesions, including gingivitis and periodontitis, although currently its role as an oral pathogen remains contentious. Evidence to support the role of C. concisus in acute intestinal disease has come from studies that have detected or isolated C. concisus as sole pathogen in fecal samples from diarrheic patients. C. concisus has also been associated with chronic intestinal disease, its prevalence being significantly higher in children with newly diagnosed Crohn’s disease (CD) and adults with ulcerative colitis than in controls. Further C. concisus has been isolated from biopsy specimens of patients with CD. While such studies support the role of C. concisus as an intestinal pathogen, its isolation from healthy individuals, and failure of some studies to show a significant difference in C. concisus prevalence in subjects with diarrhea and healthy controls has raised contention as to its role in intestinal disease. Such findings could argue against the role of C. concisus in intestinal disease, however, the fact that C. concisus strains are genetically diverse raises the possibility that differences exist in their pathogenic potential. Evidence to support this view comes from studies showing strain specific differences in the ability of C. concisus to attach to and invade cells and produce virulence factors, including toxins and hemolytic phospholipase A. Further, sequencing of the genome of a C. concisus strain isolated from a child with CD (UNSWCD) and comparison of this with the only other fully sequenced strain (BAA-1457) would suggest that major differences exist in the genetic make-up of this species which could explain different outcomes of C. concisus infection.

Synergistic haemolytic activity and its correlation to phospholipase D productivity by Corynebacteruim pseudotuberculosis Egyptian isolates from sheep and buffaloes.

Fourteen isolates of Corynebacteruim pseudotuberculosis of them 7 were isolated from sheep with Caseous Lymphadenitis "biotype 1" and 7 isolated from buffaloes with Oedematous Skin Disease "biotype 2". All isolates were identified by standard microbiological techniques and by polymerase chain reaction targeting, 16S rRNA and phospholipase D genes. Synergistic haemolytic titers of all isolates were assayed by plate technique. The presences of phospholipase D gene in supernatants of all isolates were performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot technique by using hyperimmune serum raised in rabbit immunized with recombinant phospholipase D gene antigen. The concentration of phospholipase D gene was assayed by scanning the bound phospholipase D gene with specific antibodies that appeared at 31.5 kDa. Results presented that there is no correlation between titer of Synergistic haemolytic activity and the actual phospholipase D genes concentration in culture supernatants. Also results presented that Synergistic haemolytic activity and phospholipase D genes produced by biotype 2 (buffalo isolates) was generally higher than those by biotype 1(sheep isolates).

Cooperation between LepA and PlcH Contributes to theIn VivoVirulence and Growth ofPseudomonas aeruginosain Mice

Pseudomonas aeruginosa-derived large extracellular protease (LepA) and hemolytic phospholipase C (PlcH) are considered to play an important role in the pathogenicity of this organism. Although bacterial growth appears to be closely related to virulence, little is known about whether LepA and PlcH participate in the growth and virulence of P. aeruginosa. In this study, we investigated whether LepA and PlcH contribute to the virulence and growth of P. aeruginosa using a wild-type strain and mutants. The growth rate of the isogenic lepA single mutant was lower than that of the wild-type strain in a minimal medium containing serum albumin or hemoglobin as the sole carbon and nitrogen source. Furthermore, the growth rate of the lepA plcH double mutant decreased greatly compared with that of the wild-type strain in a minimal medium containing erythrocytes as a sole nutrient source for growth. Thus, these results indicate that cooperation between LepA and PlcH would contribute to the utilization of erythrocytes as a sole nutrient source for the growth of P. aeruginosa. In addition, mouse infection experiments demonstrated that the virulence of the lepA and plcH single mutants was attenuated, and the numbers of the mutants were lower than the numbers of the wild-type strain in peritoneal lavage fluid and whole-blood specimens. In particular, the virulence and growth rate of the lepA plcH double mutant were markedly lower than those of the wild-type strain. Collectively, these results suggest that LepA and PlcH contribute to the in vivo virulence and growth of P. aeruginosa.

Hemolytic phospholipaseRv0183of Mycobacterium tuberculosis induces inflammatory response and apoptosis in alveolar macrophage RAW264.7 cells

The metabolic pathway of phospholipids is one of the most important physiologic pathways in Mycobacterium tuberculosis, a typical intracellular bacterium. The hemolytic phospholipase lip gene (Rv0183) is one of 24 phospholipase genes that have been demonstrated to play critical roles in the metabolism of phospholipids in M.tuberculosis. Quantitative RT-PCR and flow cytometry were used to elucidate the immunological and pathogenic implications of the Rv0183 gene on the inflammatory response following persistent expression of Rv0183 in mouse alveolar macrophage RAW264.7 cells. Our results demonstrate that a time-course-dependent ectopic expression of Rv0183 significantly elevated the expression of IL-6, NF-κB, TLR-2, TLR-6, TNFα, and MyD88 in these alveolar macrophage cells. Furthermore, the persistent expression of Rv0183 induced RAW264.7 cell apoptosis in vitro. These findings demonstrate that the expression of Rv0183 induces an inflammatory response and cell apoptosis in the host cells, suggesting that Rv0183 may play an important role in the virulence and pathogenesis of M.tuberculosis infection.

Choline catabolism, σ 54 factor and NtrC are required for the full expression of the Pseudomonas aeruginosa phosphorylcholine phosphatase gene

Choline favors the pathogenesis of Pseudomonas aeruginosa because hemolytic phospholipase C and phosphorylcholine phosphatase (PchP) are synthesized as a consequence of its catabolism. The experiments performed here resulted in the identification of the factors that regulate both the catabolism of choline and the gene coding for PchP. We have also identified and characterized the promoter of the pchP gene, its transcriptional organization and the factors that affect its expression. Deletion analyses reveal that the region between −188 and −68 contains all controlling elements necessary for pchP expression: a hypothetical −12/−24 promoter element, a consensus sequence for the integration host factor (−141/−133), and a palindromic sequence resembling a binding site for a potential enhancer binding protein (−190/−174). Our data also demonstrate that choline catabolism and NtrC (nitrogen regulatory protein) are necessary for the full expression of pchP and is partially dependent on σ 54 factor.

Crystallization and preliminary X-ray diffraction analysis of Pseudomonas aeruginosa phosphorylcholine phosphatase.

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine to produce choline and inorganic phosphate. Phosphorylcholine is released by the action of haemolytic phospholipase C (PlcH) on phosphatidylcholine or sphingomyelin. PchP belongs to the HAD superfamily and its activity is dependent on Mg2+, Zn2+ or Cu2+. The possible importance of PchP in the pathogenesis of P. aeruginosa, the lack of information about its structure and its low identity to other members of this family led us to attempt its crystallization in order to solve its three-dimensional structure. Crystals of the protein have been grown and diffraction data have been obtained to 2.7 A resolution. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a=137.16, b=159.15, c=73.31 A, beta=117.89 degrees. Statistical analysis of the unit-cell contents and the self-rotation function suggest a tetrameric state of the molecule with 222 point-group symmetry.

Transition from octahedral to tetrahedral geometry causes the activation or inhibition by Zn2+ of Pseudomonas aeruginosa phosphorylcholine phosphatase

Pseudomonas aeruginosa phosphorylcholine phosphatase (PchP) catalyzes the hydrolysis of phosphorylcholine, which is produced by the action of hemolytic phospholipase C on phosphatidylcholine or sphyngomielin, to generate choline and inorganic phosphate. Among divalent cations, its activity is dependent on Mg(2+) or Zn(2+). Mg(2+) produced identical activation at pH 5.0 and 7.4, but Zn(2+) was an activator at pH 5.0 and became an inhibitor at pH 7.4. At this higher pH, very low concentrations of Zn(2+) inhibited enzymatic activity even in the presence of saturating Mg(2+) concentrations. Considering experimental and theoretical physicochemical calculations performed by different authors, we conclude that at pH 5.0, Mg(2+) and Zn(2+) are hexacoordinated in an octahedral arrangement in the PchP active site. At pH 7.4, Mg(2+) conserves the octahedral coordination maintaining enzymatic activity. The inhibition produced by Zn(2+) at 7.4 is interpreted as a change from octahedral to tetrahedral coordination geometry which is produced by hydrolysis of the [Zn(2+)L(2)(-1)L(2)(0) (H(2)O)(2)] complex.

Prevalence of virulence genes among bulgarian nosocomial and cystic fibrosis isolates of Pseudomonas aeruginosa

The aim of this study was to evaluate the prevalence of some virulence genes among 202 Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients (n=42) and non-CF in-patients (n=160) and to analyze the values according to the patient groups, infection localization and antimicrobial resistance. The following frequencies in all studied strains were established: algD (encoding GDP-mannose 6-dehydrogenase AlgD) – 91.1%, pilB (type IV fimbrial biogenesis protein PilB) – 23.8%, nan1 (neuraminidase) – 21.3%, lasB (elastase LasB) – 100%, plcH (haemolytic phospholipase C precursor) – 91.6%, exoS (exoenzyme S) – 62.4%, and exoU (exoenzyme U) – 30.2%. The prevalence of nan1 was significantly higher (P<0.01) in CF isolates (38.1%) than that in non-CF isolates (16.9%). The nan1–positive CF strains were cultured from 16 patients with recurrent lung exacerbations. This study revealed a statistically significant difference (P<0.01) between the portion of multidrug-resistant (MDR) nosocomial P. aeruginosa strains containing a large number (≥5) of virulence genes (38.1%) and the respective part of non-MDR isolates (17.6%). Moreover, pilB, exoU and nan1 manifested a higher spread (P<0.001) among MDR than in non-MDR strains (respectively, 39.1% vs. 13.2%; 40.2% vs. 17.7% and 26.1% vs. 4.4%). In conclusion, the dissemination of nan1 in CF isolates was moderate and correlated with the lower proportion of patients with lung exacerbations. The molecular-genetic detection of this gene may be used as an indirect measure of CF pulmonary disease evolution. Simultaneous determination of virulence factors and antimicrobial resistance is the contemporary approach for examination of the microbiological aspects of infections caused by P. aeruginosa.

Phospholipase PlaB of Legionella pneumophila Represents a Novel Lipase Family: PROTEIN RESIDUES ESSENTIAL FOR LIPOLYTIC ACTIVITY, SUBSTRATE SPECIFICITY, AND HEMOLYSIS

Legionella pneumophila possesses several phospholipases capable of host cell manipulation and lung damage. Recently, we discovered that the major cell-associated hemolytic phospholipase A (PlaB) shares no homology to described phospholipases and is dispensable for intracellular replication in vitro. Nevertheless, here we show that PlaB is the major lipolytic activity in L. pneumophila cell infections and that PlaB utilizes a typical catalytic triad of Ser-Asp-His for effective hydrolysis of phospholipid substrates. Crucial residues were found to be located within the N-terminal half of the protein, and amino acids embedding these active sites were unique for PlaB and homologs. We further showed that catalytic activity toward phosphatidylcholine but not phosphatidylglycerol is directly linked to hemolytic potential of PlaB. Although the function of the prolonged PlaB C terminus remains to be elucidated, it is essential for lipolysis, since the removal of 15 amino acids already abolishes enzyme activity. Additionally, we determined that PlaB preferentially hydrolyzes long-chain fatty acid substrates containing 12 or more carbon atoms. Since phospholipases play an important role as bacterial virulence factors, we examined cell-associated enzymatic activities among L. pneumophila clinical isolates and non-pneumophila species. All tested clinical isolates showed comparable activities, whereas of the non-pneumophila species, only Legionella gormanii and Legionella spiritensis possessed lipolytic activities similar to those of L. pneumophila and comprised plaB-like genes. Interestingly, phosphatidylcholine-specific phospholipase A activity and hemolytic potential were more pronounced in L. pneumophila. Therefore, hydrolysis of the eukaryotic membrane constituent phosphatidylcholine triggered by PlaB could be an important virulence tool for Legionella pathogenicity.

A Complex Extracellular Sphingomyelinase of Pseudomonas aeruginosa Inhibits Angiogenesis by Selective Cytotoxicity to Endothelial Cells

The hemolytic phospholipase C (PlcHR) expressed by Pseudomonas aeruginosa is the original member of a Phosphoesterase Superfamily, which includes phosphorylcholine-specific phospholipases C (PC-PLC) produced by frank and opportunistic pathogens. PlcHR, but not all its family members, is also a potent sphingomyelinase (SMase). Data presented herein indicate that picomolar (pM) concentrations of PlcHR are selectively lethal to endothelial cells (EC). An RGD motif of PlcHR contributes to this selectivity. Peptides containing an RGD motif (i.e., GRGDS), but not control peptides (i.e., GDGRS), block the effects of PlcHR on calcium signaling and cytotoxicity to EC. Moreover, RGD variants of PlcHR (e.g., RGE, KGD) are significantly reduced in their binding and toxicity, but retain the enzymatic activity of the wild type PlcHR. PlcHR also inhibits several EC-dependent in vitro assays (i.e., EC migration, EC invasion, and EC tubule formation), which represent key processes involved in angiogenesis (i.e., formation of new blood vessels from existing vasculature). Finally, the impact of PlcHR in an in vivo model of angiogenesis in transgenic zebrafish, and ones treated with an antisense morpholino to knock down a key blood cell regulator, were evaluated because in vitro assays cannot fully represent the complex processes of angiogenesis. As little as 2 ng/embryo of PlcHR was lethal to ∼50% of EGFP-labeled EC at 6 h after injection of embryos at 48 hpf (hours post-fertilization). An active site mutant of PlcHR (Thr178Ala) exhibited 120-fold reduced inhibitory activity in the EC invasion assay, and 20 ng/embryo elicited no detectable inhibitory activity in the zebrafish model. Taken together, these observations are pertinent to the distinctive vasculitis and poor wound healing associated with P. aeruginosa sepsis and suggest that the potent antiangiogenic properties of PlcHR are worthy of further investigation for the treatment of diseases where angiogenesis contributes pathological conditions (e.g., vascularization of tumors, diabetic retinopathy).