| dc.description | The opportunistic human pathogen Legionella pneumophila uses its Hsp60 homologue (HtpB) to attach to and invade host cells, and releases the protein into its replicative endosome during intracellular growth. In this study a yeast model was used to investigate HtpB intracellular function. Saccharomyces cerevisiae cells expressing the htpB gene in the cytoplasm, from the galactose-inducible plasmid pEMBLyex4 displayed pseudohyphal growth (PHG) phenotypes including cell elongation, budding in unipolar fashion, the invasion of solid medium and activation of the PHG reporter construct FG(TyA)::lacZ. An Hsp60 homologue from Escherichia coli (GroEL) did not induce PHG in S. cerevisiae when expressed from plasmid pEMBLyex4. Using genetics-based methods, it was determined that many of the known regulators of PHG in S. cerevisiae, including Ras2p (an activator of kinase signaling), Cdc42p (a GTP-binding protein), Ste20p (a homologue of the mammalian p65 PAK kinases), Ste11p (a MAPKKK), Ste7p (a MAPKK), and the transcription factors Ste12p and Flo8p play a role HtpB-induced PHG. These findings indicate that HtpB has a biological effect in eukaryotic cells. Using the two-hybrid method, two HeLa cell proteins, Map and CksHs1, were identified as putative interacting proteins for HtpB. These proteins are homologous to Bub2p and Cks1, respectively in S. cerevisiae. Bub2p and Cks1 are involved in delaying mitotic exit, which can result in yeast cell elongation, and may serve as the underlying mechanism for HtpB-induced PHG. In mammalian cells Map and CksHs1 influence actin dynamics, which could be relevant for L. pneumophila pathogenesis, since L. pneumophila entry into host cells is an actin-dependent process. A preliminary investigation into the secretion mechanism(s) for Hsp60 homologues in bacteria was also pursued. As determined by immunogold electron microscopy, GroEL was localized to the cytoplasm in enteropathogenic E. coli, while in two Bordetella species, Cpn60 (the Bordetella Hsp60 homologue) was found in association with the bacterial cell envelope. Furthermore, in B. pertussis Cpn60 surface localization was dependent on BvgS, a regulator of virulence gene expression. This study contributes to the awareness that chaperonins, aside from their role in protein folding, have unique functions that may facilitate the survival of organisms in their niches. | en_US |
