Identification and characterization of an extracellular phospholipase in clinical isolates of Enterobacter aerogenes

FEDRIGO, G. V; CASTELLI, M. E.; CLEMENTÍN, A. L; GARCÍA VÉSCOVI, E.

Orlando, USA

Congreso; 106th General Meeting of the American Society for Microbiology; 2006

American Society for Microbiology

Enterobacter is an opportunistic pathogen associated to nosocomial infections, with increasing incidence in patients from intensive care units. Currently there is no available E. aerogenes genome database, and apart from antibiotic resistance acquisition mechanisms there is scarce information about the pathogenicity traits that allow this bacteria to prosper in extraintestinal niches. In this work, we selected an E. aerogenes strain (RM 66262) which exhibited extracellular lipolyitic activity from a clinical isolates collection. This activity was absent in strains from normal flora, suggesting that it might be a virulence factor. By phenotypic screening of an RM 66262 sub-genomic library we found a locus coding for a lipase activity (phlABEa). DNA sequence analysis revealed high homology to a phospholipase A-encoding locus, present exclusively in Serratia, Yersinia, Xanthomonas and Photorabdus genomes. This result was biochemically confirmed by TLC analysis of the lipase activity present in the cell culture supernatants obtained either form E. aerogenes RM 66262 or from the ATCC 13048 strain transformed with a plasmid harboring phlABEa. Southern blot analysis form the E. aerogenes strain collection showed that phlABEa is only present in a subset of clinical isolates and it is absent from ATCC13048. In parallel, we performed mini Tn5-random mutagenesis and phenotypically selected lipase null strains. Strikingly, analysis of the DNA sequences adjacent to the insertions revealed homology to genes which encode for proteins involved in the outer membrane glycolipid and LPS biosynthesis. These Tn5-mutants were not globally affected in the expression of other exoproteins such as the cytolysin, suggesting that the products of the mutated genes could be implicated in the expression, folding, processing or secretion mechanisms of the E. aerogenes phospholipase.