iTRAQ­based proteomic analysis of the interaction of A549 human lung epithelial cells with Aspergillus fumigatus conidia.

Severe invasive aspergillosis infection occurs when human immune function is impaired. The interaction between Aspergillus fumigatus (A. fumigatus) conidia and type II lung epithelial cells serves an important role in disease progression. The present study compared the proteomes of A549 human lung epithelial cells with and without A. fumigatus infection. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein interaction analyses were performed, and differential protein expression was verified by western blotting and reverse transcription­quantitative PCR (RT­qPCR). In addition, the RNA interference method, an internalization assay and ELISA were performed. Isobaric tags for relative and absolute quantification analysis detected a total of 1,582 proteins, from which 111 proteins with differential expression were obtained (fold change >1.5 or <0.75). Among them, 18 proteins were upregulated and 93 proteins were downregulated in A549 cells challenged with A. fumigatus. GO and KEGG analyses revealed that the altered proteins were mainly involved in biological functions, such as cell metabolism, synthesis, the cellular stress response, metabolic pathways and pyruvate metabolism. N­myc downstream­regulated gene 1 (NDRG1) expression was upregulated 1.88­fold, while CD44 expression was downregulated 0.47­fold following A. fumigatus infection. The expression levels of specific proteins were verified by western blotting and RT­qPCR. The internalization efficiency was affected by NDRG1 gene silencing. The secretion of IL­6 and IL­8 was affected when CD44 was inhibited. These results indicated that A. fumigatus affects lung epithelial cell metabolism and biological synthetic functions. A number of novel molecules, including NDRG1 and CD44, were found to be related to A. fumigatus infection.

Adherence and Cytotoxicity of Candida spp. to HaCaT and A549 cells.

Candida species are opportunistic fungal pathogens that cause superficial or invasive infections. Recently, the incidence of infection by non-Candida albicans species, especially Candida glabrata, has increased. In this study, we analyzed the adhesion and cytotoxicity of various Candida spp. that are part of the normal human microbiota. C. albicans adheres well to cell culture plates and to cultured cells. C. glabrata selectively adheres to epithelial cells rather than to cell culture plates. Candida parapsilosis insufficiently adheres to confluent monolayers of human lung epithelial A549 and keratinocyte HaCaT cells. We then analyzed the cytotoxicity of C. albicans and C. glabrata, which adhered well to epithelial cells. C. glabrata has been found to cause more damage to A549 cells than to HaCaT cells, suggesting that resident Candida spp. have distinct cytotoxic effects in different tissues. It is important to clarify the properties of Candida spp. as there is evidence that normal microbiota can cause infections. Our data suggest that it is necessary to use appropriate cell lines for characterizing the adherence and cytotoxicity of Candida spp.

Evaluation of the ability of colistin, amoxicillin (components of Potencil® ), and fluoroquinolones to attenuate bacterial endotoxin- and Shiga exotoxin-mediated cytotoxicity-In vitro studies.

Escherichia coli is one of the major pathogens in humans and animals causing localized and systemic infections, which often lead to acute inflammation, watery diarrhea, and hemorrhagic colitis. Bacterial lipopolysaccharide (LPS) and Shiga exotoxins (Stx) are mostly responsible for such clinical signs. Therefore, highly effective treatment of E. coli infections should include both eradication of bacteria and neutralization of their toxins. Here, for the first time, we compared the in vitro ability of common antibiotics to decrease LPS- and Stx-mediated cytotoxicity: colistin, amoxicillin (used separately or combined), enrofloxacin, and its metabolite ciprofloxacin. Three experimental scenarios were realized as follows: (a) the direct effect of antibiotics on endotoxin, (b) the effect of antibiotic treatment on LPS-mediated cytotoxicity in an experiment mimicking "natural infection," (c) the effect of antibiotics to decrease Stx2e-mediated cytotoxicity. Two cell lines, A549 and Vero cells, were used to perform cytotoxic assays with the methyl tetrazolium (MTT) and lactate dehydrogenase leakage (LDH) methods, respectively. Colistin and amoxicillin, especially used in combination, were able to attenuate LPS toxic effect, which was reflected by increase in A549 cell viability. In comparison with other antibiotics, the combination of colistin and amoxicillin exhibited the highest boster or additive effect in protecting cells against LPS- and Stx2e-induced toxicity. In summary, in comparison with fluoroquinolones, the combination of colistin and amoxicillin at concentrations similar to those achieved in plasma of treated animals exhibited the highest ability to attenuate LPS- and Stx2e-mediated cytotoxicity.

Nontypeable Haemophilus influenzae (NTHi) directly interfere with the regulation of E-cadherin in lung epithelial cells.

Loss of epithelial barriers characterized by reduction of E-cadherin is a hallmark of chronic obstructive pulmonary disease (COPD). We investigated the effects of nontypeable Haemophilus influenzae (NTHi) infections, associated with acute exacerbations of chronic bronchitis, on the regulation of E-cadherin in host cells. NTHi infection decreased E-cadherin mRNA and protein-levels in lung epithelial cells. E-cadherin reduction was mediated by activation of the fibroblast growth factor 2 (FGF2), the mammalian target of rapamycin (mTOR) and Slug. These data indicate that epithelial integrity and barrier function is disturbed by NTHi infection. Mainly, the destruction of cell-cell contacts is a prominent feature in NTHi infection.

A novel high-throughput assay to quantify the vaccine-induced inhibition of Bordetella pertussis adhesion to airway epithelia.

BACKGROUND: Pertussis or whooping cough is an acute respiratory illness caused by the Gram-negative pathogen Bordetella pertussis. Despite high vaccination coverage whooping cough is currently re-emerging in many developed countries. Although the causes of pertussis resurgence are matter of debate, emerging evidences suggest that acellular vaccines efficiently protect against the hallmark symptoms of pertussis disease but fail to prevent colonization. This presumably impacts on increased risk of bacterial transmission and consequent spread throughout the population. These evidences suggest that improved vaccines may be required for efficient bacterial clearance in the upper respiratory tract. Consequently, there is a need for novel bioassays to evaluate at pre-clinical or clinical level the impact of different vaccines on B. pertussis colonization. RESULTS: We developed a high-throughput bacterial adhesion inhibition (BAI) assay based on human respiratory cell lines and on live bacteria chemically conjugated to a fluorescent dye. Employing A549 cells as model, we evaluated the impact of antibodies elicited by acellular (aP) and whole cell (wP) vaccines on B. pertussis adhesion in vitro. Moreover, we settled the method also on polarized Calu-3 cells grown at air-liquid interface (ALI), showing that this assay can be extended to more complex cell models mimicking the airway epithelium. CONCLUSIONS: We proved that this method is a sensitive, rapid and reproducible system to evaluate the anti-adhesive properties of vaccine-induced antibodies and can be employed to assess improved pertussis vaccines.

Virulence characteristics of extraintestinal pathogenic Escherichia coli deletion of gene encoding the outer membrane protein X.

Outer membrane protein X (OmpX) and its homologues have been proposed to contribute to the virulence in various bacterial species. But, their role in virulence of extraintestinal pathogenic Escherichia coli (ExPEC) is yet to be determined. This study evaluates the role of OmpX in ExPEC virulence in vitro and in vivo using a clinical strain PPECC42 of porcine origin. The ompX deletion mutant exhibited increased swimming motility and decreased adhesion to, and invasion of pulmonary epithelial A549 cell, compared to the wild-type strain. A mild increase in LD50 and distinct decrease in bacterial load in such organs as heart, liver, spleen, lung and kidney were observed in mice infected with the ompX mutant. Complementation of the complete ompX gene in trans restored the virulence of mutant strain to the level of wild-type strain. Our results reveal that OmpX contributes to ExPEC virulence, but may be not an indispensable virulence determinant.