Role of cyclic-di-GMP in Acinetobacter baumannii biofilm formation

Acinetobacter baumannii is a problematic opportunistic pathogen that is the cause of increasing incidences of hospital-acquired infections. Most clinical A. baumannii strains are multi-drug resistant, making it difficult to treat infections by this bacterium. Biofilm formation likely contributes to the prevalence of this organism in the hospital environment, and also may enhance virulence. The small signaling molecule cyclic-di-GMP (cdGMP) has been demonstrated to drive biofilm formation in a number of bacteria, but its role in A. baumannii biofilms was unknown. Expression of a cdGMP synthase in A. baumannii dramatically enhances biofilm formation, demonstrating the importance of cdGMP in A. baumannii biofilms. An online database, SEED-viewer, identified twelve A. baumannii genes predicted to be involved in the synthesis and/or degradation of cdGMP. The objective of this study is to develop genetic techniques to inactivate all twelve genes in A. baumannii without increasing the antibiotic resistance of the resulting strain. Previously, the laboratory created an erythromycin resistance gene (ermC) flanked by flippase recognition target sequences (FRT). The FRT-ermC -FRT cassette will be used to disrupt each of the cdGMP modulatory genes, and expression of the flippase recombinase in the resulting A. baumannii mutant strains will result in the removal of the ermC gene. In this manner, systematic removal of all cdGMP modulatory genes in A. baumannii will be possible, leading to determining the role of cdGMP in A. baumannii biofilm formation and virulence. The results of this study will enhance our understanding of the key role cdGMP plays in virulence and may result in the development of a vaccine.