Recipient Organization
UNIV OF MARYLAND
(N/A)
COLLEGE PARK,MD 20742
Performing Department
VETERINARY MEDICINE
Non Technical Summary
Lyme disease is the most prevalent tick-borne disease in the United States and is a public health threat worldwide. The infectious agent is a microbe that cycles in nature between Ixodes ticks and a mammalian host, usually wild rodent. A wide range of veterinary animals and humans became the incidental host, when infected ticks bite and transmit the pathogen, which results in a multi-system disorder including arthritis, carditis and neurological disorders. The project seeks to understand the molecular basis for the induction of Lyme arthritis in an animal model of Lyme disease. Arthritis is the most frequent and significant clinical complications of the disease, which results from the invasion of the joint tissue by the spirochete pathogen. The purpose of the current study is to understand the role of a specific molecular component of the pathogen in microbial persistence and genesis of the joint disease. Overall, this study will add to our understanding of the detail
molecular basis how Lyme arthritis occurs and contribute towards development of preventive measures against this joint disorder induced by B. burgdorferi.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Lyme disease, caused by the infection with the bacterium, Borrelia burgdorferi, is continued to be the most prevalent arthropod-borne infections in United States and also in parts of Europe and Asia. The disease afflicts both human as well as a wide range of pet, livestock or wild animals. One of the goals of current bio-medicine reserach to improve animal health by contributing in cutting edge veterinary medicine research. Arthritis is the one of the most prominent and significant clinical complications of Lyme disease that affect both human and animal health. An interesting feature of Lyme disease is that not all the B. burgdorferi infected hosts are symptomatic and antibiotic therapy is not always effective. Therefore, development of vaccines or effective therapeutic strategies to prevent B. burgdorferi infection remains a major focus of Lyme disease research. The pathogen survives in a complex enzootic cycle in nature and has evolved unusual capabilities to persist
in a wide range of host niches. Selective expression of gene products by B. burgdorferi has been shown to play essential roles in the survival of spirochetes. This proposal will assess functional role of a B. burgdorferi gene, bb0574, that is highly and selectively upregulated in infected joints. The outcome of this proposal could thus establish important roles of bb0574 in the molecular pathogenesis of Lyme arthritis. Major technical concerns related to B. burgdorferi mutagenesis have already been standardized; therefore, we anticipate that this work can be accomplished over a 1-year time period. Overall, this study will add to the understanding of pathogenesis of Lyme arthritis and contribute towards development of preventive measures against joint inflammation induced by B. burgdorferi.
Project Methods
The current research hypothesis builds upon our published works showing that tissue-specific gene expression by B. burgdorferi could potentially contribute to the microbial persistence in host and genesis of disease, such as development of arthritis in joints. Major focus of this hatch proposal is to dissect functional role of a B. burgdorferi gene-product, BB0574 that is preferentially induced in infected joints. Following steps will be employed: (1). Generation of bb0574-deficient and complemented isolates of B. burgdorferi. We will use an infectious B. burgdorferi isolate for the targeted mutagenesis study using our recently published procedures [Pal U et. al. J Clin Invest 113: p220, 2004., Li X, Pal U, et al. Mol Microbiol 63, p694, 2007]. Our strategy for the insertional inactivation of bb0574 will involve the standard procedures of allelic exchange to replace bb0574 gene in B. burgdorferi genome with a Borrelia adapted antibiotic resistance cassette
(streptomycin). If our mutagenesis efforts result in a desired loss-of-function phenotype, we will further perform genetic complementation studies to re-introduce the wild-type copy of bb0574 gene into genome of B. burgdorferi bb0574 mutant using the allelic exchange procedure with a different antibiotic marker (kanamycin) as we have described recently. (2). Characterization of mutant isolates of B. burgdorferi for use in animal studies. Characterization of B. burgdorferi mutants will follow similar procedures as indicated in the publications noted above. Briefly, in vitro grown spirochetes will be assessed for the loss of bb0574 transcripts and protein by standard RT-PCR and western blotting assay. We will further study their infectivity and maintenance of phenotype in vivo. Mice will be infected with spirochetes and murine tissues will be isolated at 1, 2 and 3 weeks. Presence of viable spirochetes and lack of bb0574 expression will be assessed by culture and RT-PCR. (3). Assessment
of bb0574 function in a murine model of Lyme arthritis. BB0574 is preferentially produced by B. burgdorferi in the infected joints where ensuing host inflammatory responses could result in genesis of Lyme arthritis. We will assess if bb0574 mutant can persist in joints and capable of inducing inflammation. Groups of C3H/HeN mice, a reliable animal model of Lyme arthritis, will be infected with spirochetes (either wild-type, BB0574-deficient or complemented isolates). Murine arthritis peaks around second week of infection and resolves around fourth week. Mice will be sacrificed at 7-, 15-, 21- and 30-day after spirochete injection and joint samples will be isolated and used for microscopic assessment of arthritis and quantitative PCR measurement of B. burgdorferi burden. We will assess whether bb0574 mutants failed to persist in murine joint and induce lower degree of arthritis, compare to the wild type microbe. These studies will provide important insight into whether BB0574, a
joint-induce B. burgdorferi gene-product, is involved in the pathogenesis of Lyme arthritis and might contribute to develop preventive measure to interfere with the disease.