Cholesterol and host cell surface proteins contribute to cell-cell fusion induced by the Burkholderia type VI secretion system 5.

Following escape into the cytoplasm of host cells, Burkholderia pseudomallei and the related species Burkholderia thailandensis employ the type VI secretion system 5 (T6SS-5) to induce plasma membrane fusion with an adjacent host cell. This process leads to the formation of multinucleated giant cells and facilitates bacterial access to an uninfected host cell in a direct manner. Despite its importance in virulence, the mechanism of the T6SS-5 and the role of host cell factors in cell-cell fusion remain elusive. To date, the T6SS-5 is the only system of bacterial origin known to induce host-cell fusion. To gain insight into the nature of T6SS-5-stimulated membrane fusion, we investigated the contribution of cholesterol and proteins exposed on the host cell surface, which were shown to be critically involved in virus-mediated giant cell formation. In particular, we analyzed the effect of host cell surface protein and cholesterol depletion on the formation of multinucleated giant cells induced by B. thailandensis. Acute protease treatment of RAW264.7 macrophages during infection with B. thailandensis followed by agarose overlay assays revealed a strong reduction in the number of cell-cell fusions compared with EDTA treated cells. Similarly, proteolytic treatment of specifically infected donor cells or uninfected recipient cells significantly decreased multinucleated giant cell formation. Furthermore, modulating host cell cholesterol content by acute cholesterol depletion from cellular membranes by methyl- ß-cyclodextrin treatment or exogenous addition of cholesterol impaired the ability of B. thailandensis to induce cell-cell fusions. The requirement of physiological cholesterol levels suggests that the membrane organization or mechanical properties of the lipid bilayer influence the fusion process. Altogether, our data suggest that membrane fusion induced by B. pseudomallei and B. thailandensis involves a complex interplay between the T6SS-5 and the host cell.

Entry, Intracellular Survival, and Multinucleated-Giant-Cell-Forming Activity of Burkholderia pseudomallei in Human Primary Phagocytic and Nonphagocytic Cells.

The human pathogen Burkholderia pseudomallei and the related species Burkholderia thailandensis are facultative intracellular bacteria characterized by the ability to escape into the cytosol of the host cell and to stimulate the formation of multinucleated giant cells (MNGCs). MNGC formation is induced via an unknown mechanism by bacterial type VI secretion system 5 (T6SS-5), which is an essential virulence factor in both species. Despite the vital role of the intracellular life cycle in the pathogenesis of the bacteria, the range of host cell types permissive for initiation and completion of the intracellular cycle is poorly defined. In the present study, we used several different types of human primary cells to evaluate bacterial entry, intracellular survival, and MNGC formation. We report the capacity of B. pseudomallei to enter, efficiently replicate in, and mediate MNGC formation of vein endothelial and bronchial epithelial cells, indicating that the T6SS-5 is important in the host-pathogen interaction in these cells. Furthermore, we show that B. pseudomallei invades fibroblasts and keratinocytes and survives inside these cells as well as in monocyte-derived macrophages and neutrophils for at least 17 h postinfection; however, MNGC formation is not induced in these cells. In contrast, infection of mixed neutrophils and RAW264.7 macrophages with B. thailandensis stimulated the formation of heterotypic MNGCs in a T6SS-5-dependent manner. In summary, the ability of the bacteria to enter and survive as well as induce MNGC formation in certain host cells may contribute to the pathogenesis observed in B. pseudomallei infection.

The combination of PCR and serology increases the diagnosis of Pseudomonas aeruginosa colonization/infection in cystic fibrosis.

BACKGROUND: Early diagnosis of Pseudomonas aeruginosa colonization/infection in patients with cystic fibrosis (CF) using microbiological culturing methods may be difficult. Serology and polymerase chain reaction (PCR) may be useful techniques for early detection of P. aeruginosa in children with CF. METHODS: A cross-sectional analysis comparing results obtained by three different methods for P. aeruginosa identification was performed in 87 CF patients with a mean age of 9.7 years. Microbiological culturing and PCR targeting the algD GDP mannose dehydrogenase gene of P. aeruginosa were performed in sputum or oropharyngeal swabs samples, and serum antibodies against three P. aeruginosa antigens (elastase, alkaline protease, and exotoxin A) were assessed once. RESULTS: It was possible to isolate P. aeruginosa by culture in samples from 42 patients (48.2%), while PCR was positive in 53 (60.9%) patients. Serology was positive in 38 patients (43.6%), with a higher positivity for elastase (37.9%), followed by alkaline protease (29.9%) and exotoxin A (19.5%). The difference among the three isolated methods was not statistically significant. The combination of PCR + serology was significantly superior to single methods, to PCR + culture and also to culture + serology. CONCLUSIONS: PCR identified a higher number of patients with P. aeruginosa than serology and conventional culture, but the difference did not reach statistical significance. Any of the combination methods that included PCR resulted in significantly statistical differences in relation to isolated microbiological or serology methods, but not to the PCR method alone, suggesting that PCR may be the main additive method for P. aeruginosa identification.

Diagnostic value of serum antibodies in early Pseudomonas aeruginosa infection in cystic fibrosis patients.

Specific serum antibodies could be helpful in defining the status of Pseudomonas aeruginosa infection as well as the response to early intervention treatment in patients with cystic fibrosis (CF). We used 1,791 serum samples from 375 European CF patients with known respiratory microbiology status to define titers of P. aeruginosa antibodies directed against alkaline protease (AP), elastase (ELA), and exotoxin A (ExoA). Pseudomonas antibody titers were also measured in a separate cohort of 56 patients undergoing antibiotic treatment for eradication of P. aeruginosa. At a specificity of 97.5%, the sensitivity was highest for antibodies against AP (85.4%), followed by ELA (76.2%) and ExoA (72.0%). AP, ELA, or ExoA antibody titers were significantly higher (P < 0.001) in patients chronically infected with P. aeruginosa compared to patients with negative cultures. The sensitivity of the combined three ELISAs was higher than that for any single ELISA alone. Based on the newly defined cut-off levels, positive serum antibody titers against at least one of the three antigens were present in 43% of patients with new onset of P. aeruginosa infection. Longitudinal assessment of antibody titers assessed before and after inhaled antibiotic therapy in patients with first P. aeruginosa isolation showed a significant decrease in antibody titers against AP and ExoA in patients clearing P. aeruginosa infection, whereas titers increased in patients in whom antibiotic therapy failed to eradicate the organism. Antibody testing against AP, ELA, and ExoA offers high sensitivity and specificity for the presence of P. aeruginosa in respiratory cultures of CF patients. Although serum antibody titers are on average low at the time of first P. aeruginosa isolation from respiratory specimens, they may be useful to monitor response to therapy. However, because variability between patients is considerable, treatment decisions should not be based on P. aeruginosa antibody levels alone.