Immunolocalization of Hsp60 in Legionella pneumophila

Abstract

One of the most abundant proteins synthesized by Legionella pneumophila, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, including L. pneumophila, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in L. pneumophila grown in vitro, or in vivo inside of HeLa cells. For comparative purposes Escherichia coli, expressing recombinant L. pneumophila Hsp60, was employed. In contrast to E. coli, where Hsp60 was localized exclusively in the cytoplasm, in L. pneumophila Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked L. pneumophila organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by L. pneumophila was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in L. pneumophila and further suggest both the existence of a novel secretion mechanism (not present in E. coli) and a potential role in pathogenesis.

FIG. 1

Schematic representation of a bacterial cell section indicating the four measurements (x_c, y_c, x_p, and y_p) taken on each section analyzed. The formulas used to calculate the areas and perimeters of cytoplasmic and periplasmic compartments are indicated below the drawing. A_c, area of the cytoplasm; L_c, length of the cytoplasmic membrane; A_p, area of the periplasmic space; L_p, length of the outer membrane.

FIG. 2

Immunospecificity of anti-Hsp60 reagents. (a) Immunoblots developed with MAb of the material immunoprecipitated by PAb from whole-cell lysates of E. coli PSH16 (Ec; lane 1) or L. pneumophila SVir (Lp; lane 2). (b) Immunoblots of whole-cell lysates of E. coli PSH16 or L. pneumophila SVir, developed with PAb (lanes 1 and 2) or MAb (lanes 3 and 4). The positions and molecular weights (in thousands) of broad-range, prestained protein markers (New England BioLabs, Beverly, Mass.) are indicated on the left side of each panel. The open arrowhead on the right side of each panel indicates the position at which purified recombinant Hsp60 migrated.

FIG. 3

Comparative labeling of L. pneumophila and control bacteria with PAb. Representative electron micrographs show the labeling patterns of ultrathin sections cut from L. pneumophila SVir (a), E. coli PSH16 (b), and B. pertussis (c) grown at 37°C. Bars, 0.1 μm.

FIG. 4

Comparative labeling of ultrathin sections of non-heat-shocked L. pneumophila SVir with different rabbit PAbs. Representative electron micrographs show the labeling patterns obtained after an overnight incubation with anti-Hsp60 (a), anti-OmpS (b), or anti-serogroup 1 lipopolysaccharide (c) rabbit sera. Specimens were not stained to facilitate visual recognition of the labeling patterns. Bars, 0.1 μm.

FIG. 5

Surface expression of Hsp60 in L. pneumophila SVir. Representative electron micrograph showing profuse gold labeling on the surface of an intact, whole bacterial cell grown at 37°C. Bar, 0.1 μm.

FIG. 6

Distribution of Hsp60 epitopes in ultrathin sections of L. pneumophila 2064 grown in vivo at 37°C. (a) Low-magnification view to show a complex endosome containing replicating bacteria. A few mature forms still contained in a degraded cell are present (arrow), as well as some free mature forms, likely released from a lysed HeLa cell (top right corner). (b) Heavy labeling of the endosomal space between intracellular bacteria, and of the surfaces of replicating intracellular bacteria, after an overnight incubation with PAb. Bars, 1 μm (a) and 0.1 μm (b).