MudPIT analysis of released proteins in Pseudomonas aeruginosa laboratory and clinical strains in relation to pro-inflammatory effects

Cystic Fibrosis Center - Azienda Ospedaliera Universitaria Integrata di Verona, piazzale Stefani, 1-37126 Verona, Italy.
Integrative Biology (Impact Factor: 3.76). 03/2012; 4(3):270-9. DOI: 10.1039/c2ib00127f
Source: PubMed


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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    • "Further, where P. aeruginosa dominates the infection microbiota, its growth is likely to further affect the composition of airway environment. This impact of colonisation could occur both directly through the metabolomic (Kozlowska et al., 2013) and secretomic (Bergamini et al., 2012) footprint of P. aeruginosa, and, in turn, indirectly by stimulating changes in the host immune response (Bergamini et al., 2012) and the activity of other co-colonising species (Bakkal et al., 2010; Tashiro et al., 2013). Whereas the causality in these interactions is difficult to demonstrate, were such relationships to exist, they would result in both an association between P. aeruginosa infection and measures of airway disease, and an association between P. aeruginosa infection and microbiota composition. "
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    ABSTRACT: Chronic bacterial lung infections associated with non-cystic fibrosis bronchiectasis represent a substantial and growing health-care burden. Where Pseudomonas aeruginosa is the numerically dominant species within these infections, prognosis is significantly worse. However, in many individuals, Haemophilus influenzae predominates, a scenario associated with less severe disease. The mechanisms that determine which pathogen is most abundant are not known. We hypothesised that the distribution of H. influenzae and P. aeruginosa would be consistent with strong interspecific competition effects. Further, we hypothesised that where P. aeruginosa is predominant, it is associated with a distinct 'accessory microbiota' that reflects a significant interaction between this pathogen and the wider bacterial community. To test these hypotheses, we analysed 16S rRNA gene pyrosequencing data generated previously from 60 adult bronchiectasis patients, whose airway microbiota was dominated by either P. aeruginosa or H. influenzae. The relative abundances of the two dominant species in their respective groups were not significantly different, and when present in the opposite pathogen group the two species were found to be in very low abundance, if at all. These findings are consistent with strong competition effects, moving towards competitive exclusion. Ordination analysis indicated that the distribution of the core microbiota associated with each pathogen, readjusted after removal of the dominant species, was significantly divergent (analysis of similarity (ANOSIM), R=0.07, P=0.019). Taken together, these findings suggest that both interspecific competition and also direct and/or indirect interactions between the predominant species and the wider bacterial community may contribute to the predominance of P. aeruginosa in a subset of bronchiectasis lung infections.The ISME Journal advance online publication, 18 July 2014; doi:10.1038/ismej.2014.124.
    The ISME Journal 07/2014; 9(1). DOI:10.1038/ismej.2014.124 · 9.30 Impact Factor
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    • "It allows to automatically obtain thousands of features comprising spectra, peptide sequences and related proteins [24,25]. In addition, label-free quantification approaches based on spectral count (SpC) or SEQUEST-based SCORE evaluation permit an high-throughput discovering of multiple biomarkers [26-28], which could contain a higher level of discriminatory information. "
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    Biochemical Journal 12/2012; 450(2). DOI:10.1042/BJ20121087 · 4.40 Impact Factor
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