MudPIT analysis of released proteins in Pseudomonas aeruginosa laboratory and clinical strains in relation to pro-inflammatory effects.
ABSTRACT Pseudomonas aeruginosa (Pa) is the most common virulent pathogen contributing to the pathogenesis of cystic fibrosis (CF). During bacterial lung colonization, the products of its metabolism are released in the extracellular space contributing to the pathogenic events associated with its presence. To gain insights on the mechanisms involved in the Pa pathogenesis we focused our attention on proteins released by Pa using a MudPIT approach combined with cell biology assays. Conditioned medium (CM) collected under aerobic and microaerobic conditions from Pa clinical strains (in early and late colonization), unlike the laboratory strain, induced expression of IL-8 mRNA in CF airway epithelial cells. We have identified proteins released by clinically relevant Pa strains, focusing on the pro-inflammatory effects as metalloproteases (MMPs). In fact, their expression pattern was associated with the highest pro-inflammatory activity measured in the early clinically isolated strain. The relation was further supported by the result of the analysis of a larger and independent set of Pa isolates derived from sporadically and chronically infected CF patients: 76% of sporadic samples expressed protease activity (n = 44), while only 27% scored positive in the chronically infected individuals (n = 38, p < 0.0001, Fisher's exact test). Finally, looking for a possible mechanism of action of bacterial MMPs, we found that CM from early clinical isolates can cleave CXCR1 on the surface of human neutrophils, suggesting a potential role for the bacterially released MMPs in the protection of the pathogen from the host's response.
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ABSTRACT: Selected physiological parameters of 31 classic and rough Pseudomonas aeruginosa strains from respiratory tract cultures of patients with cystic fibrosis were examined. An association of a patient's clinical condition (good or poor) with strain physiology was made. Rough strains from patients in poor clinical condition demonstrated severe alterations in motility when compared with M-2, a highly motile and chemotactic burn strain. Of the 10 rough strains from patients in poor clinical condition, 70% lacked flagella, as determined by electron microscopy. The remaining few flagellated strains from this group exhibited weak motility both in soft agar and by the capillary assay. Their chemotactic response to three amino acids, when compared with that of strain M-2, was reduced approximately 30 to 90%. Classic strains from patients in poor clinical condition were less chemotactic than those from patients in good clinical condition. A majority of classic and rough strains from patients in good clinical condition were comparable to M-2 in both chemotaxis and motility. Changes in other physiological characteristics indicated by reduced growth rates, or auxotrophy, were seldom observed in the cystic fibrosis strains studied. The data suggest that host-selective pressures, associated primarily with patients with cystic fibrosis that are in poor clinical condition, result in the loss of factors related to invasiveness such as motility and chemotaxis. We propose that these results may reflect that there is a more general alteration in the cell envelope of cystic fibrosis strains.Infection and Immunity 12/1985; 50(2):577-82. · 4.07 Impact Factor
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ABSTRACT: The las and rhl quorum sensing (QS) systems regulate the expression of several genes in response to cell density changes in Pseudomonas aeruginosa. Many of these genes encode surface-associated or secreted virulence factors. Proteins from stationary phase culture supernatants were collected from wild-type and P. aeruginosa PAO1 mutants deficient in one or more of the lasRI, rhlRI and vfr genes and analysed using two-dimensional gel electrophoresis. All mutants released significantly lower amounts of protein than the wild-type. Protein spot patterns from each strain were compared using image analysis and visible spot differences were identified using mass spectrometry. Several previously unknown QS-regulated proteins were characterized, including an aminopeptidase (PA2939), an endoproteinase (PrpL) and a unique 'hypothetical' protein (PA0572), which could not be detected in the culture supernatants of Deltalas mutants, although they were unaffected in Deltarhl mutants. Chitin-binding protein (CbpD) and a hypothetical protein (PA4944) with similarity to host factor I (HF-I) could not be detected when any of the lasRI or rhlRI genes were disrupted. Fourteen proteins were present at significantly greater levels in the culture supernatants of QS mutants, suggesting that QS may also negatively control the expression of some genes. Increased levels of two-partner secretion exoproteins (PA0041 and PA4625) were observed and may be linked to increased stability of their cognate transporters in a QS-defective background. Known QS-regulated extracellular proteins, including elastase (lasB), LasA protease (lasA) and alkaline metalloproteinase (aprA) were also detected.Microbiology 06/2003; 149(Pt 5):1311-22. · 2.85 Impact Factor
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ABSTRACT: Certain membrane-anchored proteins, including several cytokines and cytokine receptors, can be released into cell supernatants through the action of endogenous membrane-bound metalloproteinases. The shed molecules are then able to fulfill various biological functions; for example, soluble interleukin-6 receptor (sIL-6R) can bind to bystander cells, rendering these cells sensitive to the action of IL-6. Using IL-6R as a model substrate, we report that the metalloproteinase from Serratia marcescens mimics the action of the endogenous shedding proteinase. Treatment of human monocytes with the bacterial protease led to a rapid release of sIL-6R into the supernatant. This effect was inhibitable with TAPI [N-(D,L-[2-(hydroxyaminocarbonyl)methyl]-4-methylpentanoyl) L-3-(2' naphthyl)-alanyl-L-alanine, 2-aminoethyl amide], a specific inhibitor of the membrane-bound intrinsic metalloproteinase, but not with other conventional proteinase inhibitors. sIL-6R-liberating activity was also detected in culture supernatants of Staphylococcus aureus, Pseudomonas aeruginosa, and Listeria monocytogenes, organisms that are known to produce metalloproteinases. sIL-6R released through the action of S. marcescens metalloproteinase retained biological activity and rendered IL-6-unresponsive human hepatoma cells sensitive to stimulation with IL-6. This was shown by Northern (RNA) blot detection of haptoglobin mRNA and by quantitative measurements of de novo-synthesized haptoglobin in cell supernatants. Analysis of immunoprecipitated, radiolabeled sIL-6R revealed that the bacterial protease cleaved IL-6R at a site distinct from that utilized by the endogenous protease. These studies show that membrane-anchored proteins can be released in active form through cleavage at multiple sites, and they uncover a novel mechanism via which microbial proteases possibly provoke long-range biological effects in the host organism.Infection and Immunity 10/1996; 64(9):3646-51. · 4.07 Impact Factor