Since important virulence factors produced by pathogenic bacteria (toxins, adhesins, etc.) are generally regulated by environmental signals (temperature, pH, ion/metal concentration etc.), we have begun to identify these regulatory systems in Borrelia burgdorferi. SDS-PAGE and immunoblotting analysis of proteins from B. burgdorferi growth in modified Barbour-Stoenner-Kelly (BSK-II) media treated with metal chelators such as EDDHA, 2-2 dypiryldyl (DIP), or chelex, suggest that B. burgdorferi alters protein expression in response to decreasing metal concentrations When other bacterial pathogens (Neisseria, E. coli, etc.) were studied using similar experimental approaches, regulatory systems that were identified were based upon metal-dependent repressor proteins that sensed the intracellular concentration of Fe. Initially, we believed this to be the case for B. burgdorferi. However, several experimental results strongly suggest that the Fe requirements of B. burgdorferi are very low or non-existent . These findings and the fact that B. burgdorferi lacks a respiratory chain suggest that metabolism is not generating reactive oxygen species (ROS) during normal cell growth. As important, with no intracellular Fe, there is no Fenton reaction with ROS, particularly H2O2. Therefore, oxidative challenges to B. burgdorferi cells must come primarily from the host in the form of ROS and/or reactive nitrogen species , NOS. To understand how B. burgdorferi biochemical eliminates or reduces ROS (or NOS) and regulates the enzymes involved in this process, we have concentrated on three proteins. We have cloned a gene encoding a putative metal-dependent repressor protein (PerR) from B. burgdorferi and identified two target sequences using a mobility shift DNA-binding assay. One sequence is 60 bp upstream of the start codon of a putative 2 gene operon encoding a glutamate transporter (gltP) and a NADH peroxidase (npx). The other is 57 bp upstream of the start codon of napA, the gene encoding an alkylhydroperoxide reductase. These data indicate that PerR may be involved in regulating an oxidative stress response by B. burgdorferi. A PerR homolog identified from Bacillus subtilis mediates cellular responses to oxidative stress and metal starvation in that bacterium. Clearly, characterizing the role this regulatory protein plays in the survival response of B. burgdorferi and identifying other genes it regulates will contribute greatly to the understanding of Lyme disease. To date, we have done the following:: (1) To begin characterization of this system, perR from B. burgdorferi was cloned into expression vector pKK223-3, induced with IPTG, and the protein was purified from E. coli XL1-Blue MRFʹ. PerR was purified using a heparin-agarose affinity column and used to generate polyclonal sera to the recombinant protein. The antisera detected PerR in cell lysates of B. burgdorferi and has been used to probe the high-passage perR- mutant. (2) The genes for Npx and NapA were amplified by PCR from B. burgdorferi genomic DNA and cloned into pCyt3 to generating pSVB5 and pJP23, respectively. We confirmed that no mutations were introduced into either gene during the cloning procedure by sequencing the cloned DNA. Expression of both genes was inducible with IPTG, and the products of these genes accounted for >2% of the total cell protein. The apparent molecular weights of the two overexpressed proteins correlated well with the molecular weights predicted from the deduced amino acid sequences. Both proteins have been purified to homogeneity and each purified protein was used to raise polyclonal serum. Inductively Coupled Plasma-Mass Spectroscopy (ICP-MS) analysis indicated that no metal co-factor co-purified with Npx or NapA. (3) We introduced the plasmid pJP23 (napA) into E. coli strain TA4315 that carried a mutation in the ahpC, encoding one subunit of alkylhydroperoxide reductase. TA4315 harboring pJP23 was able to grow in the presence of 5iM cumene hydroperoxide or t-Butyl hydroperoxide when NapA was overexpressed using IPTG. Similarly, strain TA4315 harboring pSVB5 (npx) was able to grow when challenged with 2iM t-Butyl hydroperoxide. These data suggest that the Npx and NapA function as peroxidases or alkylhydroperoxide reductases. A PerR- mutant of high-passage, avirulent B. burgdorferi has been isolated in collaboration with Dr. J. Skare, Dept. Of Microbiology, Texas A & M University, College Station, TX. Preliminary data suggests that the mutant strain is resistant to 5iM hydrogen peroxide compared to 0.5 iM for the wild-type, high-passage parent strain. Immunoblot and PCR analyses demonstrated that perR had been disrupted and no PerR was produced in the mutant strain. Interestingly, immunoblots probed with anti-NapA serum indicated that NapA (an alkylhydroperoxide reductase) was being over-expressed in the mutant strain partially explaining its peroxide resistant phenotype. (4) In addition, a putative PerR target sequence which flanks the putative promoters of napA were identified. These regions are very homologous to the target sequences of an oxidative repressor binding protein, PerR, from Bacillus subtilus. To demonstrate interactions between these target sequences and PerR, a 251-bp DNA fragment including the sequence upstream of napA was amplified by PCR, end labeled with [32P], and used as a probe in mobility shift DNA-binding assays with PerR. Altered mobility of the target sequence was observed in the presence of purified Per suggesting that PerR recognized the target sequence. These preliminary results are extremely encouraging, as we have demonstrated a potential role for PerR in gene regulation in B. burgdorferi. SUMMARY: Section on oxidate stress. We have proposed a model for the response of B. burgdorferi to oxidative stress. Because this system would promote the in vivo survival of B. burgdorferi cells when challenged by O2.- and H2O2 from host cells, we are particularly interested in the process and how it is regulated. As previously mentioned, we have identified a gene, perR, that may be involved in regulating gene expression in response to oxidate stress and metal limitation in B. burgdorferi. One focus of my research has been to characterize this important regulatory protein identified in B. burgdorferi and assess the role of PerR, Npx, and NapA in survival in vivo. These studies will lead to a better understanding of the pathogenesis of B. burgdorferi.
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