Elevated levels of Legionella pneumophila stress protein HspGO early in infection of human monocytes and L929 cells correlate with virulence

Legionella pneumophila 2064 was selectively radiolabelled in mouse 1,929 cells and human monocytes to identify proteins expressed early in the course of infection. Polypeptide profiles (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography) of adherent or intracellular bacteria indicated that a 60-kDa stress protein (Hsp60) was preferentially synthesized, Hsp60 synthesis was not induced by medium alone. The synthesis of many polypeptides, including OmpS (major outer membrane protein), diminished over the 1-h period postinfection, However, by 17 h postinfection OmpS and Hsp60 were the dominant proteins synthesized by 2064. To establish whether induction of Hsp60 was a correlate of virulence, an isogenic avirulent strain (2064M) of 2064 was isolated following selection on a nonpermissive medium, 2064M did not exhibit a stress response when adherent or intracellular in L929 cells or in human monocytes and failed to abrogate phagosome-lysosome fusion, When grown in vitro, 2064M exhibited no deficiencies in the heat shock response and its polypeptide profile resembled that of 2064, Immunogold electron microscopy was used to localize Hsp60 in L. pneumophila-infected L929 cells. There was an increase in the number of gold particles associated with phagosomes for phagosomes harboring single 2064 bacteria compared with those harboring 2064M. Moreover, by 1 h postinfection, a sixfold increase in the number of gold spheres associated with the membranes of phagosomes was observed for phagosomes harboring 2064 compared with those harboring 2064M. These studies indicate that virulent, but not NaCl-tolerant avirulent, strains of L. pneumophila respond to host-cell-associated environmental signals early in the course of infection, This response includes increased synthesis and possibly extracellular secretion of Hsp60 concomitant with repression of the expression of other genes, including ompS.