Please refer to the one page Research Plan for the Merit extension, immediately following the progress report. This section contains the information that would normally be in the project summary. Research Plan for the MERIT extension (1 page) During the MERIT extension, we will elucidate - using the murine model of Lyme disease - the importance of specific B. burgdorferi genes that are preferentially expressed in different tissues during Lyme borreliosis and delineate some of the host immune responses that influence spirochete gene expression. The general strategies used in specific aims 1 and 2 of the initial grant were similar, differing mostly in that aim 1 focused on the skin, while aim 2 was directed at the joints. I have organized the extension as a single general specific aim related to preferential gene expression by B. burgdorferi, with several subaims focused on different tissues of clinical importance, and contributing host factors.
Specific aim 1. Characterize B. burgdorferi genes that are preferentially expressed in the skin, joints and hearts (a new target for investigation) during infection with the Lyme disease agent These are 3 of the major locations associated with clinical infection and illness. a) Delineate the expression patterns of selected B. burgdorferi genes, in the skin, joints, and heart, throughput murine infection. encompassing early infection, peak illness, and the regression of disease. Genes that are differentially expressed in the skin, joints or hearts, are likely to be involved in the pathogenesis of cutaneous Lyme disease, Lyme arthritis or Lyme carditis (a new area for investigation), and targets for therapy. Microarray assays, outlined in the progress report, have identified B. burgdorferi genes that are preferentially expressed in the skin (section 2 final paragraph), joints (Table 1) or heart (Table 2). These date show that there are at least 4, 14 or 6 B. burgdorferi genes that are differentially expressed in the skin, joints or heart, respectively. We will perform detailed quantitative RT-PCR analyses to examine the expression levels of these genes in diverse tissues over 90 days in mice, including when infection develops (days 1-14), peaks (14-30) and resolves (30-90). We will select 3 genes that exhibit the highest degree of expression in each tissue for further study. We will extend these data to Lyme disease patients, using available samples as described in our JEM article in the appendix. b) Generate B. burgdorferi lacking each of the 9 genes selected in (a) and determine whether these mutant spirochetes are able to infect mice and cause disease. Based on our success with B. burgdorferi deficient in bmpA/B and murine Lyme arthritis, we will carefully generate B. burgdorferi mutants lacking genes that are induced in the skin, joints and heart. An assessment of 9 mutant B. burgdorferi is realistic over 5 years, assuming that 1-2 are created each year. When a phenotype is associated with specific mutant spirochetes, we will complement the Borrelia to reconstitute disease and confirm our findings. c) Modulate murine Lyme disease with antisera directed at B. burgdorferi gene products preferentially expressed in selected locations. Immunotherapy with antibodies to preferentially expressed antigens may alter Lyme disease, either by blocking the activity of the antigens, or by forming antibody-antigen complexes that activate complement and destroy the spirochetes. It is likely that this activity will be most apparent in the tissues where the antigens are expressed. When specific antibodies are shown to modulate infection, we will explore the mechanism of actions such as complement mediated destruction or interference with the ability of the spirochetes to colonize specific tissues, including the skin, joints and heart. d) Examine whether host immune responses influence B. burgdorferi gene expression in mice. Our JID article in the appendix demonstrates that TLR1/2-medtated responses influence B. burgdorferi gene expression. It is likely that effector molecules, such as cytokines and chemokines including TNF-alpha and IL-6 among others, that are released as an outcome of TLR-mediated cell activation, are causing the alterations is spirochete gene expression. We will examine this in vitro, by stimulating B. burgdorferi with selected cytokines/chemokines and examining spirochete gene expression. In addition, in vivo studies, will examine B. burgdorferi gene expression in mice lacking selected immune mediators, or in mice administered antibodies that block specific immune mediators. Once the specific mediators have been identified, we will elucidate the mechanisms by which B. burgdorferi gene expression is controlled. These studies should lead to a new understanding of the pathogenesis of specific clinical manifestations of Lyme disease, and novel targets for the prevention of Lyme disease. This paradigm should also be applicable to other infectious diseases of medical importance.

Public Health Relevance

This project will lead to a greater understanding of the pathogenesis of, and immunity against, Lyme disease. This information will be useful for the development of new vaccines and therapeutics for this important medical illness.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37AI049200-11
Application #
7983025
Study Section
Special Emphasis Panel (NSS)
Program Officer
Breen, Joseph J
Project Start
2001-05-01
Project End
2016-03-31
Budget Start
2011-04-01
Budget End
2012-03-31
Support Year
11
Fiscal Year
2011
Total Cost
$413,750
Indirect Cost
Name
Yale University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Narasimhan, Sukanya; Rajeevan, Nallakkandi; Liu, Lei et al. (2014) Gut microbiota of the tick vector Ixodes scapularis modulate colonization of the Lyme disease spirochete. Cell Host Microbe 15:58-71
Krause, Peter J; Narasimhan, Sukanya; Wormser, Gary P et al. (2014) Borrelia miyamotoi sensu lato seroreactivity and seroprevalence in the northeastern United States. Emerg Infect Dis 20:1183-90
Narasimhan, Sukanya; Coumou, Jeroen; Schuijt, Tim J et al. (2014) A tick gut protein with fibronectin III domains aids Borrelia burgdorferi congregation to the gut during transmission. PLoS Pathog 10:e1004278
Schuijt, Tim J; Bakhtiari, Kamran; Daffre, Sirlei et al. (2013) Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein. Circulation 128:254-66
Krause, Peter J; Narasimhan, Sukanya; Wormser, Gary P et al. (2013) Human Borrelia miyamotoi infection in the United States. N Engl J Med 368:291-3
Silver, Adam C; Dunne, Dana W; Zeiss, Caroline J et al. (2013) MyD88 deficiency markedly worsens tissue inflammation and bacterial clearance in mice infected with Treponema pallidum, the agent of syphilis. PLoS One 8:e71388
Yang, Xiuli; Hegde, Shylaja; Shroder, Deborah Y et al. (2013) The lipoprotein La7 contributes to Borrelia burgdorferi persistence in ticks and their transmission to naïve hosts. Microbes Infect 15:729-37
Ullmann, A J; Dolan, M C; Sackal, C A et al. (2013) Immunization with adenoviral-vectored tick salivary gland proteins (SALPs) in a murine model of Lyme borreliosis. Ticks Tick Borne Dis 4:160-3
Magnarelli, Louis A; Williams, Scott C; Norris, Steven J et al. (2013) Serum antibodies to Borrelia burgdorferi, Anaplasma phagocytophilum, and Babesia microti in recaptured white-footed mice. J Wildl Dis 49:294-302
Narasimhan, Sukanya; Perez, Oriana; Mootien, Sara et al. (2013) Characterization of Ixophilin, a thrombin inhibitor from the gut of Ixodes scapularis. PLoS One 8:e68012

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