Semester

Summer

Date of Graduation

2006

Document Type

Dissertation

Degree Type

PhD

College

School of Medicine

Department

Microbiology, Immunology, and Cell Biology

Committee Chair

Nyles W. Charon.

Abstract

Borrelia burgdorferi is the spirochete causative agent of Lyme disease. The chemotaxis and motility systems of these bacteria are far less well described than that of Escherichia coli or Salmonella enterica. This dissertation explores the role of the CheA proteins in the chemotactic response and describes the first defined attractants for B. burgdorferi.;In order to test hypotheses, we developed or optimized three protocols. To characterize the motion of cells, two motion tracking systems were optimized. The Hobson BacTracker allowed for tracking cell motions in real time. This hardware/software chimera, while powerful for the specific application, utilizes a cumbersome interface. Therefore, the software package Volocity was adopted. While the tracking itself is somewhat slower, the interface greatly facilitates data collection, organization, and presentation, making it much faster. To assay chemotaxis with the capillary tube assay, one must enumerate cells. This was previously difficult because cell enumeration was slow, laborious, and ineffectual at low concentrations. We overcame these limitations by initially developing a protocol for enumerating cells by flow cytometry. Once this enumeration method was validated with direct comparisons to Petroff-Hausser counting chamber data, we were able to screen for attractants using a modified capillary tube assay.;We found that B. burgdorferi mutants in cheA2 were non-chemotactic to defined attractants. Complementation of cheA2 restored the wild-type phenotype. Mutants in cheA1 failed to show any discernable phenotype. The modified capillary tube chemotaxis assay was used to screen for chemoattractants. To date serine, glycine, N-n-diacetyl-chitobiose, glucose, glutamate, putricine, spermidine, rabbit serum, and glucosamine, have been tested, the latter five had at least some chemoattractant ability. Finally, this is the first work to correlate the ability of the cells to reverse with chemotaxis.;In conclusion, this work developed techniques to track the motion of B. burgdorferi and measure the chemotactic response with a high throughput assay. These tools are being used in a screen of compounds which has already found 5 specific compounds that act as chemoattractants. The techniques developed will be useful not only for B. burgdorferi, but will facilitate measuring the chemotactic response in other slow growing prokaryotic species.

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