Major Areas of Research
- Development of a genetic system for Borrelia burgdorferi, the spiral-shaped bacterium that causes Lyme disease.
- Analysis of the structure and function of the plasmid component of the highly segmented B. burgdorferi genome
- Determination of the roles of specific plasmids, genes, and proteins during the natural infectious cycle of B. burgdorferi
- Cultivation and genetic manipulation of free-living and pathogenic Leptospira
Research in this laboratory focuses primarily on Borrelia burgdorferi, the spirochete that causes Lyme disease, the most common arthropod-borne disorder in the United States. B. burgdorferi is maintained in nature through an infectious cycle between wild mammals and ticks. Occasionally, infected ticks feed upon humans and transmit the spirochete, resulting in Lyme disease.
Human infection has medical significance as a multisystemic, potentially chronic illness. The tick vector and the mammalian host represent very different environments, and there is good evidence for differential gene expression by borreliae in these locations.
The infectious cycle of B. burgdorferi
- B. burgdorferi spirochetes persist in a latent state in midgets of infected ticks for many months.
- After a tick attaches to a mammalian host and ingests a blood meal, spirochetes multiply and efficiently move to the salivary glands.
- B. burgdorferi is then transmitted via tick saliva and remains in the mammalian skin for several days before dissemination via the bloodstream.
- Spirochetes persist in low numbers in infected mammals yet are efficiently acquired by feeding ticks following attachment.
This scenario suggests that B. burgdorferi responds to environmental cues to adapt and move between the tick vector and mammalian host. Recent experiments document modulation of spirochetal outer surface proteins in response to environmental conditions and reinforce this hypothesis.
Our broad objective is to use a molecular genetic approach to elucidate the mechanisms of adaptation and variation in B. burgdorferi and their roles in the infectious cycle. The specific aims of our research are as follows:
- Develop basic genetic tools to manipulate borrelial genes of interest. The availability of the complete genomic sequence of B. burgdorferi represents a wealth of information that can be effectively utilized through a genetic approach. However, studies of the biology of B. burgdorferi and the pathogenesis of Lyme disease have been limited by a lack of genetic tools, because most methods that have been developed for other bacteria cannot be directly applied to borreliae. To address our scientific goals, we have developed and continue to add to a set of basic genetic tools for B. burgdorferi. The ability to perform routine genetic manipulations in B. burgdorferi has greatly facilitated our research objectives as well as those of other investigators in the field.
- Understand the structure and function of plasmids in B. burgdorferi. A distinguishing feature of the B. burgdorferigenome (fig 1. below) is the presence of a linear chromosome and multiple linear and circular plasmids. The genomic sequence of B. burgdorferi identified 21 different plasmids, representing the largest known complement of plasmids of all bacteria and constituting one-third of the spirochete’s DNA. More than 90 percent of the plasmid-encoded genes are unique to B. burgdorferi, without homologs in any other organisms, suggesting they encode functions pertinent to the distinctive lifestyle of the spirochete. We have undertaken to
- Define the minimal plasmid elements required for replication, partitioning, and incompatibility of linear and circular replicons
- Assess the roles of individual plasmids and specific plasmid-encoded genes for survival in, or transmission between, the tick vector and mammalian host
- Determine how B. burgdorferi responds to particular environmental cues in order to persist and be transmitted between the tick vector and mammalian host. We hypothesize that discrete environmental signals induce appropriate bacterial responses that are critical for survival and transmission of the spirochete during the infectious cycle. We endeavor to
- Determine which proteins are made in different sites or at different stages of the infectious cycle in ticks and mammals
- Determine what these proteins do and how the genes encoding them are regulated
- Decipher the signals that mediate the adaptive responses
Knowing which bacterial proteins are synthesized in the mammal versus the tick and gaining insight into their functions will contribute to a better understanding of the pathogenesis of Lyme disease. This knowledge is relevant to the diagnosis and prevention of Lyme disease.
Figure 1: B. burgdorferi Genome
The transmission of B. burgdorferi between ticks and mammals represents an ideal system in which to study the adaptive responses of a bacterial pathogen to its vector and host environments. All steps of this infectious cycle can be reproduced in the laboratory, making it accessible to scientific investigation. Molecular genetics represents a powerful method with which to address this system.
Previous studies have identified spirochetal components that should be important in the adaptation of B. burgdorferi to its environment. Ongoing and future studies are designed to test the roles of these genes and their products in the infectious cycle and to identify additional genes that allow the spirochetes to adapt, persist, and be transmitted between ticks and mammals. This research should elucidate the biological basis of these bacterium-host-vector relationships and the factors that contribute to the pathogenesis of disease in an incidental human host.
A second and relatively recent project in the MGS involves spirochetes of the genus Leptospira, which includes both pathogenic and saprophytic species. Leptospirosis is a potentially fatal, neglected zoonotic disease that afflicts residents of poor urban communities around the world. The goal of this new project is to improve the genetic tools available for Leptospirain order to facilitate investigation of the pathogenic species and accelerate measures to prevent disease. It is our hope that the knowledge and experience we have acquired in genetic manipulation of B. burgdorferi will aid in developing and expanding similar tools for Leptospira. This relatively new project was initiated by Dr. Philip Stewart, a staff scientist in MGS, and primarily represents the focus of his research efforts. Dr. Stewart first acquired proficiency in cultivation and genetic manipulation of the saprophytic species, L. biflex, and is currently investigating potential barriers to transformation of the pathogenic species, L. interrogans.
Dr. Rosa received her doctorate in 1980 from the Institute of Molecular Biology at the University of Oregon. In 1988, following research fellowships at Washington University School of Medicine in St. Louis and at the Research Institute of Scripps Clinic, Dr. Rosa joined Rocky Mountain Laboratories. She became a tenured investigator in 2000. Dr. Rosa is a fellow of the American Academy of Microbiology and an internationally recognized leader in the field of bacterial molecular genetics.
Rego ROM, Bestor A, Stefka J, Rosa PA. Population bottlenecks during the infectious cycle of the Lyme disease spirocheteBorrelia burgdorferi. PLoS One. In press.
Hayes BM, Dulebohn DP, Sarkar A, Tilly K, Bestor A, Ambroggio X, Rosa PA. Regulatory protein BBD18 of the lyme disease spirochete: essential role during tick acquisition? MBio. 2014 Apr 1;5(2):e01017-14.
Dulebohn DP, Hayes BM, Rosa PA. Global repression of host-associated genes of the Lyme disease spirochete through post-transcriptional modulation of the alternative sigma factor RpoS. PLoS One. 2014 Mar 26;9(3):e93141.
Tilly K, Bestor A, Rosa PA. Lipoprotein succession in Borrelia burgdorferi: similar but distinct roles for OspC and VlsE at different stages of mammalian infection. Mol Microbiol. 2013 Jul;89(2):216-27.
Stewart PE, Carroll JA, Dorward DW, Stone HH, Sarkar A, Picardeau M, Rosa PA. Characterization of the Bat proteins in the oxidative stress response of Leptospira biflexa. BMC Microbiol. 2012 Dec 13;12:290.
Sarkar A, Hayes BM, Dulebohn DP, Rosa PA. Regulation of the virulence determinant OspC by bbd18 on linear plasmid lp17 ofBorrelia burgdorferi. J Bacteriol. 2011 Oct;193(19):5365-73.