Abstract
Abstract: :
Purpose: We previously reported that a mutant of gene PA4856 of P. aeruginosa, which was isolated from a Tn5 transposon library of cytotoxic strain PA103 screened for abnormal surface associated motility, lacked acute cytotoxic activity towards corneal epithelial cells in vitro, and was associated with altered disease pathogenesis in vivo. The aim of this study was to investigate the role of this homologue of the lemA gene of P. syringae, in corneal pathogenesis. Methods: To understand our previous findings, virulence factor expression and in vitro bacterial behavior were studied. This involved Western blot analysis for detection of extracellular pilin, ExoU and PcrV secretion, and LPS profiling. Bacterial interactions with corneal epithelial cells were studied in vitro by adherence and invasion assays, and by time-lapse video microscopy. Results: Early in the disease, infection with the mutant was less severe; later it was found to spare the peripheral cornea. Although the mutant had characteristics of being twitching motility defective when grown on agar, video microscopy revealed it was able to travel along basolateral membranes of corneal epithelial cells. Western blot analysis confirmed the presence of extracellular pili, which are required for twitching motility. The mutant was, however, unable to secrete ExoU or PcrV under inducing conditions. Furthermore, the LPS profile differed from that of wild-type PA103. Growth on MacConkey agar suggested that the LPS defect was partial. Although adherence levels were similar to an isogenic exoU mutant (p=0.53), it invaded more than the exoU mutant (p<0.0001), and interestingly less than an isogenic exsA mutant (p<0.0001). Conclusion: The data suggest that multiple factors may be involved in the role of the PA4856 gene in corneal disease caused by P. aeruginosa. Since the encoded protein is likely to be part of a two-component regulatory system, further studies of this mutant will contribute to our understanding of disease pathogenesis, and could lead to the development of therapies that simultaneously disable multiple virulence mechanisms.
Keywords: Pseudomonas • cornea: basic science • bacterial disease