![]() The protein concentration of bacterial suspensions was estimated by measuring their optical density at 420 nm. tsutsugamushi were purified by Renografin density gradient centrifugation as described previously. The bacteria examined and their cultivation conditions are listed in Table 1. 2 Materials and methods 2.1 Bacterial strains and cultivation Antisera against different Hsp, namely, 70 kDa DnaK, 60 kDa GroEL, 41 kDa DnaJ, 24 kDa GrpE and 10 kDa GroES were tested against eight species of Rickettsiales and 19 other eubacterial species. The purpose of this study was to detect and identify putative Hsp homologues of Rickettsiales and Bartonella by Western blotting with polyclonal antibodies raised against purified GroES from Rickettsia typhi and recombinant purified Hsp from Escherichia coli. Neither the characterization nor the identification of other Hsp by specific antibodies have been reported in rickettsial species. Animal immune responses to GroEL have been examined also using monoclonal and polyclonal antibodies. sennetsu has been cloned and differential expressions of DnaK and GroEL mRNA were documented after temperature upshift. The complete GroES protein sequence and an N-terminal GroEL sequence of R. Rickettsia typhi Hsp, GroEL and GroES have been purified and partially characterized. Hsp60 genes have been cloned and their nucleotide sequences determined from Orientia tsutsugamushi, Bartonella spp. However, little is known about Hsp or their regulation and function in rickettsiae. While circulating in the wild or invading the vertebrate host, rickettsiae undoubtedly encounter various environmental stresses. ![]() In nature, these unusual bacteria are associated with arthropod vectors (fleas, ticks, mites and lice), and may cause febrile disease in animals and humans. Rickettsiae encompass a wide group of microorganisms with very different life styles. Hsp are also thought to protect intracellular pathogens against the hostile environment of host phagocytic cells. Some bacterial Hsp, particularly those in the Hsp60 and Hsp70 families, have been shown to be immunogenic and responsible for delayed type hypersensitivity responses and autoimmune responses. Chaperones facilitate the normal folding and assembly of many proteins and some catalyze the proteolytic degradation of abnormal proteins. The heat shock protein response is highly conserved among prokaryotic and eukaryotic cells. Many of these proteins are expressed at low levels under normal physiological conditions, but their synthesis is dramatically elevated when cells are exposed to enhanced or lowered temperatures and a variety of other stresses. Molecular chaperones, which include many well-studied heat shock proteins (Hsp), are essential for maintenance of bacterial growth and viability. Heat shock protein, Rickettsia, Bartonella, Ehrlichia, Orientia, Wolbachia, Legionella, Proteus, Eubacteria 1 Introduction coli Hsp are not suitable for detection and identification of homologues of these proteins in a wide range of eubacteria. Consequently, commercially available anti-DnaJ, anti-GrpE and anti-GroES polyclonal antibodies and anti-DnaK monoclonal antibody raised against their respective recombinant E. typhi reacted strongly with purified 10 kDa GroES peptide from Rickettsia and Bartonella, and strongly bound to proteins of varying electrophoretic mobility from Wolbachia, Legionella, Proteus and Shigella flexneri and more weakly to other GroES homologues including that found in E. Polyclonal antiserum prepared against GroES from R. coli DnaJ, GrpE and GroES polyclonal antibodies did not recognize DnaJ, GrpE or GroES homologues in Rickettsia, Bartonella, Orientia, Ehrlichia and Legionella. coli DnaJ, GrpE and GroES are much less conserved since anti- E. 590, 352–369) recognize less conserved epitopes. coli DnaK and GroEL monoclonal antibodies (Dasch et al. tsutsugamushi, Bartonella and other Proteobacteria, anti- E. ![]() coli DnaK and GroEL have epitopes that are highly conserved among the homologous proteins found in Rickettsia, Ehrlichia, O. Heat shock proteins (Hsp) of four Rickettsia species, three Bartonella species, two Ehrlichia species, Orientia tsutsugamushi and seventeen other eubacterial species were characterized by the enhanced chemiluminescence Western blotting (WB) technique with antibodies raised against recombinant Hsp from Escherichia coli and purified GroES from R. ![]()
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