Characterization of Legionella pneumophila genes with impact on bacterial uptake into host cells and on bacterial virulence
Final Report Abstract
Phospholipases are a diverse class of enzymes produced both by eukaryotic hosts as well as their pathogens. Major insights into action pathways of bacterial phospholipases have been provided during the last years which on the one hand act as potent membrane destructors and on the other hand manipulate and initiate host signalling paths. A pathogenic bacterium where phospholipases play a dominant role is the lung pathogen "Legionella pneumophila". Here so far 15 different phospholipase A enzymes are encoded in the genome dividing into three major groups, the GDSL, the patatin-like and the PlaB-like enzymes. The first two lipase families are also found in higher plants and the second family shows similarities to eukaryotic cytosolic phospholipases A, therefore when those enzymes are injected or secreted by the bacterium into the host cell they may mimic eukaryotic phospholipases and thereby be important virulence factors. In the project, we developed an agar plate assay which allowed screening for mutants deficient in infecting "Acanthamoeba castellanii" amoebae. Likewise, an "L. pneumophila" clone bank consisting of 23,000 transposon mutants was investigated here, and 19 different established "Legionella" virulence genes, for example, "dot/icm" genes, were identified. Importantly, also novel virulence-associated genes were found. One of those is "bdhA", coding for a protein with homology to established 3-hydroxybutyrate dehydrogenases involved in poly-3-hydroxybutyrate metabolism. Our study revealed that "bdhA" is co-transcribed with "patD", encoding a patatin-like protein showing phospholipase A and lysophospholipase A activities. In addition to strongly reduced lipolytic activities and increased poly-3-hydroxybutyrate levels, a "L. pneumophila bdhA-patD" mutant showed a severe replication defect in amoebae and U937 macrophages. Our data show that the operon is a novel virulence determinant and is further involved in phospholipolysis and poly-3-hydroxybutyrate utilization.
