Molecular and functional analysis of the Rickettsia typhi groESL operon
Introduction
Rickettsia typhi, the causative agent of murine typhus, is maintained in nature via horizontal transmission between invertebrate hosts (e.g. fleas) and warm-blooded mammalian hosts (e.g. rats and other murids) and is transmitted accidentally to humans (Azad and Beard, 1998). In the fleas, R. typhi enters midgut epithelial cells and in human infection the majority of rickettsiae are found within the endothelial cells lining blood vessels (Azad et al., 1997, Walker et al., 2000). The rickettsial ability to tolerate temperatures ranging from 25 to 37 °C or higher serves as the underlying mechanism by which efficient maintenance and transmission of this bacterium take place. As intracellular bacteria, R. typhi has adapted to thermal as well as other forms of stresses (e.g. nutritional, pH and oxidative environments) (Azad and Traub, 1985). The adaptation to such variant conditions involves the induction of the synthesis of a large number of highly conserved heat shock proteins (HSPs) encoded by the dnaK (Hsp70) and groESL (Hsp10 and Hsp60) genes (Dasch et al., 1990). HSPs are essential for bacterial growth and viability and are known to be among the main targets of the immune response against a variety of pathogenic bacteria (Craig et al., 1993). To date, HSPs of only four Rickettsia species have been characterized by the Western blotting technique with antibodies raised against recombinant HSPs from Escherichia coli and purified GroES of R. typhi (Eremeeva et al., 1998, Sumner et al., 1997). Polyclonal antiserum to GroES of R. typhi, while reacting strongly with purified 10 kDa GroES peptide from Bartonella and proteins of varying mobility from Wolbachia, Legionella, Proteus, and Shigella, was weakly reactive to E. coli (Eremeeva et al., 1998).
The production of stress proteins, including the molecular chaperone GroESL, is a central feature of the bacterial stress responses (Craig et al., 1993, Hecker et al., 1996, LaRossa and Van Dyk, 1991). Unlike for E. coli there is no information on the modulation of stress gene expression in rickettsia. Considering R. typhi's obligate intracellular existence in both the warm-blooded vertebrate and poikilothermic invertebrate hosts, the Hsp10 and Hsp60 may be carrying multiple functional tasks necessary for rickettsial survival. We were interested in the heat shock response of R. typhi and the role that rickettsial HSPs play during host cell infections. We have initially cloned and sequenced the genes encoding the GroES and GroEL homologues from an Ethiopian rat isolate of R. typhi, analyzed the transcription of the groESL operon and characterized its function in E. coli background. We are providing here the first detailed characterization of the R. typhi groESL operon.
Section snippets
Bacterial strains
Rickettsia typhi strain AZ322, Ethiopian isolate (Azad and Traub, 1985) was used in this study. The E. coli temperature sensitive groEL44 mutant strain NRK117 (Kusukawa et al., 1989) that was used was a derivative of E. coli K12 strain MC4100 [groE+, araD139, D(argF-lac)U169, rpsL150, relA1, flbB5301, deoC1, ptsF25, rbsR].
Rickettsia typhi genomic DNA extraction
African green monkey kidney cells (Vero cells, ATCC #CRL-1573) were cultured in DMEM medium (Dulbecco's modified MEM with 4.5 g glucose/l with glutamine; Biofluids Inc.,
Molecular cloning and sequence analysis of the groESL operon of R. typhi
The groESL operon of R. typhi was cloned and sequenced as described in Section 2. The nucleotide sequence of the groESL operon of R. typhi is shown in Fig. 1. The G+C content of groESL was found to be 34.2%. Genome sequences of R. prowazekii and Rickettsia conorii showed overall G+C content of 29% and 32.4%, respectively (Andersson et al., 1998, Ogata et al., 2001). The sequence analysis of the groESL operon (2229 bp) of R. typhi revealed two open reading frames (ORFs) of 288 nucleotides (95
Discussion
Rickettsia typhi is transmitted to humans by infected fleas (Azad et al., 1997). Rickettsial transition from arthropod vector to human host involves adaptation to various environmental stresses (Azad and Traub, 1985). The environmental stress to bacteria results in the production of a variety of stress proteins, including the molecular chaperones GroES and GroEL (Craig et al., 1993). GroES and GroEL are among the most highly conserved proteins in nature (Segal and Ron, 1996b, Zeilstra-Ryalls et
Acknowledgements
The research presented in this paper was supported by the National Institute of Health (R37AI 17828 and RO1 AI 43006) and an Intramural grant from the University of Maryland Baltimore. We thank Dr Yoshimitsu Mizunoe (Department of Bacteriology, Faculty of Medicine, Kyushu University, Fukuoka, Japan) for providing E. coli groESL mutants used in this work.
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