OSPC REGULATION AND PATHOGENIC STRATEGY OF BORRELIA BURGDORFERI

• Sponsoring Institution: Cooperating Schools of Veterinary Medicine
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0220981
• Project Start Date: Aug 20, 2009
• Project End Date: Jul 31, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
LOUISIANA STATE UNIVERSITY
202 HIMES HALL
BATON ROUGE,LA 70803-0100

Performing Department
Pathobiological Sciences

Non Technical Summary
): Lyme disease caused by the spirochetal bacterium Borrelia burgdorferi, which is transmitted by deer ticks, is the most common vector-borne illness in North America and Europe. Over 20,000 people contract the disease annually in the United States alone. Lyme disease is a multi-system disorder that can result in arthritis, neurological abnormalities, carditis, and cutaneous lesions such as erythema migrans and acrodermatitis chronica atrophicans. If left untreated, the infection and disease may last years. Even worse, up to 10% of Lyme disease patients may develop post-Lyme syndrome, a mysterious illness that cannot be cured. Tight regulation of gene expression is crucial for the pathogenic strategy of B. burgdorferi. The goal of the proposed study is to understand how B. burgdorferi regulates gene expression to meet its pathogenic strategy.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
722	4010	1100	100%

Knowledge Area
722 - Zoonotic Diseases and Parasites Affecting Humans;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

immune evasion

lyme disease

lipoprotein expression

Goals / Objectives
Lyme disease caused by the spirochete Borrelia burgdorferi is the most common vector-borne illness in North America. Coordinated expression of its surface lipoprotein antigens is crucial for its pathogenic strategy. B. burgdorferi abundantly produces outer surface protein A (OspA) in engorged and flat ticks but downregulates OspA and upregulates OspC in response to bloodmeal. OspC expression ultimately elicits a robust humoral response that poses tremendous pressure on the pathogen. To evade the protective immunity, B. burgdorferi down-regulates ospC. We showed that the downregulation of OspA and OspC in the murine host is achieved via the involvement of newly identified ospC and ospA operators and hypothesize that B. burgdorferi down-regulates ospC and ospA via the interaction of the operators with as-yet unidentified regulatory proteins. This project will first focus on these regulators.

Project Methods
1. Construct a putative DNA-binding protein expression library and identify repressors: Because almost all known repressors are either homodimer or homotetramer DNA-binding proteins, the ospA and ospC repressors should be DNA-binding proteins and each be encoded by a single gene. All borrelial genes without a defined function have been analyzed for putative DNA binding scores using a computer program. Up to 300 unknown genes with a high DNA-binding score will be cloned and expressed under control of a well characterized constitutive promoter, the flab promoter, and introduced into B. burgdorferi. If expression of a putative DNA-binding protein selectively causes the downregulation of ospC or ospA, such a protein will be further investigated for the interaction with the newly identified operator sequences using pulldown and/or electrophoretic mobility shift assays. 2. Confirm the critical role of repressors in immune evasion: Because the downregulation of OspA and OspC is critical for the ability of B. burgdorferi to evade the immune system, identified repressor genes will be investigated for their contributions to this ability. The repressor genes will be inactivated in B. burgdorferi; resulting mutants will be complemented. If the mutants fail to downregulate ospA or ospC during infection of SCID mice and complementation restores them with the wild-type phenotype, these genes will be confirmed as ospA or ospC repressor genes. Their roles attributed to the ability of B. burgdorferi to evade the immune system will be further investigated in immunocompetent mice. 3. Investigate mechanisms governing the downregulation of OspC: We will first address the molecular basis for the selection hypothesis by modifying B. burgdorferi to express a green fluroscent protein (GFP) under control of the flab promoter and a red fluroscent protein (RFP) under control of the odpC regulatory sequence. Because of the constitutive nature of the flab promoter, all modified spirochetes should express GFP. If all spirochetes express both GFP and RFP in SCID mice, the selection hyposthesis will be ruled out. We will then examine whether a killing activity is required for antibody-induced ospC down-regulation in the murine model.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The Lyme disease spirochete, Borrelia burgdorferi, cycles between the tick vector and a mammal in nature. To maintain its life cycle, the pathogen dramatically modifies its surface antigen expression. Most notably, B. burgdorferi downregulates outer surface protein A (OspA) but upregulates OspC in response to fresh bloodmeal. Because OspC is not only a strong immunogen but also an effective immune target, B. burgdorferi must down-regulate OspC in response to the development of anti-OspC humoral response during mammalian infection. The goal of this R01, which includes three aims, is to investigate how these two major surface antigens are regulated. In Aim 1, we planned to construct a putative DNA-binding protein expression library and identify repressors. In Aim 2, we will confirm the critical role of repressors in immune evasion. In Aim 3, we planned to investigate mechanisms governing the downregulation of OspC. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our published studies showed the existence of two regulatory sequences that are involved in ospA down-regulation. Based on our hypothesis, ospA regulator(s), which are one of the major targets of our investigation, bind to the regulatory sequences and, as a result, turns off OspA expression. To screen for regulators, we made a biotin-labeled DNA probe, which matches one of the ospA regulatory sequences. By incubating the probe with lysates prepared from spirochetes with OspA down-regulated, the probe was allowed to bind potential regulator(s). Followed by adding streptavidin-beads, potential ospA regulator(s) may be coprecipitated with the biotin-labeled probe. By using the technique, we have purified 5 candidates and sent out for MS-Spec analysis. Hopefully, useful information will come back soon. In the previous funding period, we reported that incubation of B. burgdorferi modified with increased BBD15 expression led to down-regulation of OspC in DMC, suggesting that this protein may be involved in ospC regulation. We started using the system to search for ospC regulator(s). During the funding period, we found out it takes at least 11 weeks of growth in DMC for spirochetes to down-regulate OspC. If BBD15 does not directly bind to the ospC regulatory sequence resulting in OspC down-regulation, it may up-regulate ospC regulator(s), which bind to the ospC operator and down-regulates OspC. As mentioned above, the biggest hurdle is to prepare a large quantity of spirochetes. Each DMC can collect only about 5 ml of bacterial culture with low bacterial density. We had to use DMCs harvested from 10 rats in each experiment. We made a biotin-labeled DNA probe, which matches the ospC operator we identified previously. Bacterial lysates prepared from DMCs collected from 10 rats were incubated with biotin-labeled DNA probe, followed by adding streptavidin-beads to coprecipitate potential ospC regulator(s). By using the technique, we have purified 7 candidates and sent out for MS-Spec analysis. Results will come out next week. During the funding period, we showed how import it is for B. burgdorferi to maintain its surface lipoprotein layer. OspA/B are the most abundantly produced outer surface lipoproteins, composed of more than 70% surface antigen when B. burgdorferi is grown in vitro. Our study showed removal of both surface antigens forced bacteria to dramatically increase production of two other outer surface lipoproteins, OspD and BBI39. Restoration of OspA/B production by placing the ospAB gene back to the mutant led to down-regulation of both OspD and BBI39. In a separate study, our rpoS mutant was not able to down-regulate OspA/B in DMC no matter how long it was grown in this host-adapted environment. However, by genetically engineering the mutant to constitutively produce OspC, the rpoS mutant successfully down-regulated OspA/B in DMC. Our study indicated that the requirement to maintain the density of the outer surface lipoprotein layer is an overwhelming force that can overwrite normal gene regulation programs in B. burgdorferi.

Publications

• No publications reported this period

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: During the funding period, we made significant progress in addressing OspA downregulation. Previous studies by other groups led to a conclusion that RpoS is required for OspA downregulation. Our data demonstrates the downregulation is completely RpoS-independent. Briefly, an rpoS mutant was first generated, then engineered to constitutively express OspC, and finally allowed to adapt in the dialysis membrane chamber (DMC) intraperitoneally implanted into rats. DMC adaptation allowed the rpoS mutant to completely shutdown OspA production. A manuscript reporting the finding is in preparation. During the funding period, we showed that RpoS controls expression of essential virulence factor(s) that remain to be identified. Briefly, the rpoS mutant was engineered to constitutively express OspC, DbpA and DbpB, the three critical virulence factors that have been identified to date. Failure to restore infectivity by engineering the rpoS mutant to constitutively produce these three known virulence factors led us to conclude that RpoS also controls other critical virulence genes, in addition to ospC, dbpA and dbpB. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Our work of collaboration with Dr. Doerrler, associate professor, from LSU Biological Sciences in the previous funding period has been published in Journal of Bacteriology. We continue collaboration to study another cell division protein, FtsZ, in B. burgdorferi.

Publications

• No publications reported this period

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: The Lyme disease spirochete, Borrelia burgdorferi, cycles between the tick vector and a mammal in nature. To maintain its life cycle, the pathogen dramatically modifies its surface antigen expression. Most notably, B. burgdorferi downregulates outer surface protein A (OspA) but upregulates OspC in response to fresh bloodmeal. Because OspC is not only a strong immunogen but also an effective immune target, B. burgdorferi must downregulate OspC in response to the development of anti-OspC humoral response during mammalian infection. The goal of this R01, which includes three aims, is to investigate how these two major surface antigens are regulated. In Aim 1, we planned to construct a putative DNA-binding protein expression library and identify repressors. In Aim 2, we will confirm the critical role of repressors in immune evasion. In Aim 3, we planned to investigate mechanisms governing the downregulation of Osp. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
During the funding period, we completed identification of the regulatory sequences involved in ospA downregulation. We defined a regulatory structure required for enhancing ospA expression in B. burgdorferi grown in vitro, but repressing its expression during murine infection. This newly identified structure consisted of two sequences flanking the ospAB promoter. Briefly, deletion or replacement of either the upstream or downstream sequence of the ospAB promoter caused a significant decrease in ospA expression in vitro, but a dramatic increase during murine infection. Fusion of either sequence with the flaB reporter promoter led to increased expression of an ospA reporter gene in vitro, but a decrease in the murine host. Furthermore, simultaneous fusion of both sequences with the reporter promoter showed a synergistic effect in enhancing expression of the ospA reporter in vitro, but repressing during murine infection. Taken together, the study demonstrated that the regulatory structure functions oppositely in the two different environments and potentially provides B. burgdorferi with a molecular mechanism to quickly adapt to the distinct environments during its enzootic life cycle. A paper reporting these findings has been accepted for publication. We continue to construct the putative DNA-binding protein expression library in B. burgdorferi as specified in Aim 1 of the grant. We found that expression of BBD15 completely abolishes arthritis virulence of the pathogen. As BBD15 is a putative DNA-binding protein, and can potentially interact with regulatory sequences and thus may be involved in regulation of important genes related to arthritis virulence. For this reason, although BBD15 may not be a regulator of ospC or ospA, it may be a regulator for other important genes. We are in the process to inactivate bbd15 and to investigate its regulatory functions.

Publications

• Xu Q, McShan K, and Liang FT. 2010. Two regulatory elements required for enhancing ospA expression in Borrelia burgdorferi grown in vitro but repressing during mammalian infection. Microbiology. 156: 2194-204.
• Li C, Xu H, Zhang K, and Liang FT. 2010. Inactivation of a putative flagellar motor switch protein FliG1 prevents Borrelia burgdorferi from swimming in a highly viscous medium and blocks its infectivity. Mol. Microbiol. 75:1563-76.