No abstract

BackgroundAirway microbiota composition has been clearly correlated with many pulmonary diseases, and notably with cystic fibrosis (CF), an autosomal genetic disorder caused by mutation in the CF transmembrane conductance regulator (CFTR). Recently, a new molecule, ivacaftor, has been shown to re-establish the functionality of the G551D-mutated CFTR, allowing significant improvement in lung function.Objective and MethodsThe purpose of this study was to follow the evolution of the airway microbiota in CF patients treated with ivacaftor, using quantitative PCR and pyrosequencing of 16S rRNA amplicons, in order to identify quantitative and qualitative changes in bacterial communities. Three G551D children were followed up longitudinally over a mean period of more than one year covering several months before and after initiation of ivacaftor treatment.Results129 operational taxonomy units (OTUs), representing 64 genera, were identified. There was no significant difference in total bacterial load before and after treatment. Comparison of global community composition found no significant changes in microbiota. Two OTUs, however, showed contrasting dynamics: after initiation of ivacaftor, the relative abundance of the anaerobe Porphyromonas 1 increased (p<0.01) and that of Streptococcus 1 (S. mitis group) decreased (p<0.05), possibly in relation to the anti-Gram-positive properties of ivacaftor. The anaerobe Prevotella 2 correlated positively with the pulmonary function test FEV-1 (r=0.73, p<0.05). The study confirmed the presumed positive role of anaerobes in lung function.ConclusionSeveral airway microbiota components, notably anaerobes (obligate or facultative anaerobes), could be valuable biomarkers of lung function improvement under ivacaftor, and could shed light on the pathophysiology of lung disease in CF patients.

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation.

The Klebsiella lipoprotein pullulanase (PulA) is exported to the periplasm, triacylated, and anchored via lipids in the inner membrane (IM) prior to its transport to the bacterial surface through a type II secretion system (T2SS). X-Ray crystallography and atomistic molecular dynamics (MD) simulations of PulA in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) model membrane provided an unprecedented molecular view of an N-terminal unstructured tether and the IM lipoprotein retention signal, and revealed novel interactions with the IM via N-terminal immunoglobulin-like domains in PulA. An efficiently secreted nonacylated variant (PulANA) showed similar peripheral membrane association during MD simulations, consistent with the binding of purified PulANA to liposomes. Remarkably, combined X-ray, MD, and functional studies identified a novel subdomain, Ins, inserted in the α-amylase domain, which is required for PulA secretion. Available data support a model in which PulA binding to the IM promotes interactions with the T2SS, possibly via the Ins subdomain.

Helicobacter pylori infection plays a causal role in the development of gastric mucosa-associated lymphoid tissue (MALT) lymphoma (LG-MALT) and duodenal ulcer (DU). Although many virulence factors have been associated with DU, many questions remain unanswered regarding the evolution of the infection toward this exceptional event, LG-MALT. The present study describes and compares the complexome of two H. pylori strains, strain J99 associated with DU and strain B38 associated with LG-MALT, using the two-dimensional blue native/SDS-PAGE method. It was possible to identify 90 different complexes (49 and 41 in the B38 and J99 strains, respectively); 12 of these complexes were common to both strains (seven and five in the membrane and cytoplasm, respectively), reflecting the variability of H. pylori strains. The 44 membrane complexes included numerous outer membrane proteins, such as the major adhesins BabA and SabA retrieved from a complex in the B38 strain, and also proteins from the hor family rarely studied. BabA and BabB adhesins were found to interact independently with HopM/N in the B38 and J99 strains, respectively. The 46 cytosolic complexes essentially comprised proteins involved in H. pylori physiology. Some orphan proteins were retrieved from heterooligomeric complexes, and a function could be proposed for a number of them via the identification of their partners, such as JHP0119, which may be involved in the flagellar function. Overall, this study gave new insights into the membrane and cytoplasm structure, and those which could help in the design of molecules for vaccine and/or antimicrobial agent development are highlighted. Molecular & Cellular Proteomics 9:2796 -2826, 2010.

To date no reliable diagnostic method exists to predict, among the very large and clinically heterogeneous group of Helicobacter pylori-infected patients, the extremely small group at risk for developing low-grade gastric MALT lymphoma (LG-MALT). Search of proteomic biomarkers holds promise for the classification of the H. pylori strains with regard to this severe clinical outcome. In the present study 69 H. pylori strains isolated from patients with two different H. pylori-associated diseases, duodenal ulcer (DU, n=29) and LG-MALT (n=40) were used. Protein expression patterns of the strains were analyzed by using the high-throughput methodology SELDI. Selected proteins were purified by means of chromatographic and electrophoretic methods in view of further sequencing by LC-MS/MS. Univariate analysis (Mann-Whitney test) of the protein expression patterns generated nine significant biomarkers that can discriminate between H. pylori strains from patients with DU and LG-MALT. These biomarkers are of low molecular weight, ranging from 6 to 26.6 kDa. Among them, two are overexpressed in LG-MALT strains and seven - in DU strains. Two biomarker proteins, one overexpressed in LG-MALT strains (13.2 kDa) and another one - overexpressed in DU strains (26.6 kDa), were purified to homogeneity and identified by using LC-MS/MS as a 50S ribosomal protein L7/L12 and a urease subunit, respectively. These biomarkers can be included in novel protein arrays for the differential diagnosis of H. pylori-associated clinical outcomes.