M022: An Immunofluorescent Assay for Determining the Invasiveness
of A549 Cells by Burkholderia (Pseudomonas) Cepacia.
J.L. Tipper, E. Ingham, J.H. Cove, N.J. Todd1,
J.M. Littlewood2 & K.G. Kerr (Dept. of Microbiology, University
of Leeds, Depts. of 'Microbiology & 2Paediatrics, St. James's
University Hospital, Leeds, UK).
The acquisition of B. cepacia in some patients with cystic fibrosis results in asymptomatic colonisation, whereas others experience a rapid deterioration in clinical condition. It is thought that the latter is associated with colonisation with strains of B. cepacia that have increased pathogenic potential. It is also likely that the ability of B. cepacia to invade respiratory epithelial cells is an important virulence factor. This study aimed to determine whether clinical strains of B. cepacia were able to invade the respiratory epithelial cell line A549, using cell culture techniques and a differential immunofluorescence assay. Briefly, rabbit polyclonal antibodies (pAb) were raised against B. cepacia C1359. A549 monolayers were infected with B. cepacia, fixed with paraformaldehyde and incubated with diluted pAb for 45 min. Extracellular bacteria were labelled with a fluorescein isothiocyanate (FITC) conjugated secondary antibody for 30 min. A549 cell membranes were permeabilized with PBS containing 0.3% (v/v) Triton X-100. Monolayers were then incubated with diluted pAb for 45 min and intracellular bacteria were labelled with a Texas Red conjugated secondary antibody. Labelled monolayers were viewed using confocal laser scanning microscopy or with a fluorescence microscope fitted with a dual filter. We have shown that B. cepacia C1359 (provided by Dr. J. Govan, University of Edinburgh) is invasive for A549 respiratory epithelial cells, and in addition, experiments using non-viable (heat or uv killed) cells showed that intracellular B. cepacia occur as a result of active invasion and not by phagacytosis of the bacteria by the A549 cells. We now intend to examine a variety of strains from both clinical and environmental sources to test the hypothesis that invasion of respiratory epithelial cells is an important virulence factor.