Molecular analysis of proteins involved in phosphate uptake and regulation in Borrelia burgdorferi

Date
2012
Authors
Guda, Dianne Joy
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Abstract

Lyme disease is a multi-phasic, systemic infection caused by the spirochetal bacterium Borrelia burgdorferi. It is the major arthropod-borne disease in the US and is transmitted to mammals, including humans, by Ixodid ticks. This spirochetal pathogen has a limited metaboliccapability, and therefore is intimately dependent on its hosts for survival. Regulatory mechanisms allowing for drastic adaptive changes between the tick and mammalian host are key to understanding the pathogenesis of B. burgdorferi. PhoU regulates genes of the Pho regulon, including a phosphate transporter and two component regulatory systems (TCRS) responsible for the uptake and expression of genes necessary for the maintenance of inorganic phosphate (Pi). In silico analysis of B. burgdorferi does not demonstrate the presence of a Pho TCRS, though there are open reading frames (ORFs) homologous to members of a phosphate transport system present in other bacteria. PstA, PstB, PstC, and PstS are additional proteins involved in Pi-specific transport under the regulation of PhoU. Pi levels play a large role in bacterial virulence as phosphorylation is required for the activation of the two known TCRS present in B. burgdorferi, responsible for adaptive gene response. Recombinant PhoU, PstB, and PstS have been over-expressed, purified to homogeneity and used to generate anti-sera in mice. A deletion construct was designed by 2-step PCR to replace phoU with an antibiotic resistance cassette and the native gene was also placed under the control of an IPTG-inducible promoter on a shuttle-vector for over-expression in B. burgdorferi. Since B. burgdorferi has only a limited set of ORFs corresponding to regulators of gene expression, we hypothesize that proteins involved in maintaining phosphate homeostasis such as PhoU and other associated Pst proteins, are required for the coupling the metabolic states of B. burgdorferi to its host-specific virulence attributes.

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Integrative Biology