Project: AN INTRACELLULAR NICHE FOR BORRELIA BURGDORFERI
Institution: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
PI: Skare, Jonathan
Description (by applicant):
Lyme disease, caused by the spirochetal bacterium Borrelia burgdorferi, is the leading arthropodborne infection in the United States and causes significant morbidity in endemic areas. If untreated B. burgdorferi can persistently infect individuals even though the host mounts a potent adaptive immune response such that antibodies obtained from infected patients or experimentally infected animals effectively kills in vitro cultivated B. burgdorferi. In addition, a robust cell-mediated proinflammatory response is observed that induces IL-6, IL-12 and IFN- and inhibits IL-10. Furthermore, the spirochete can resist complement killing demonstrating that this important component of the innate immune response is not sufficient to eliminate B. burgdorferi infection.
The observation that B. burgdorferi persists in such a hostile environment indicates that the spirochete is adept at evading the host immune response via mechanisms that have not been completely elucidated. One possibility is that B. burgdorferi invades host cells and survives at low levels. Recently we have determined that B. burgdorferi invade both immortalized and, more importantly, primary cells (both fibroblasts and endothelial cells) and persist as viable cells in o-culture. In addition we have preliminary data suggesting that the ability to invade host cells involves both integrin binding and Src kinase activity.
In this application we propose to further characterize the internalization of B. burgdorferi and track the fate of B. burgdorferi within thes infected cells to determine how they affect the localized host response following infection. To accomplish this we will use both in vitro correlates of invasion and intracellular survival as well as in vivo imaging of experimentally infected mice as readouts for our studies.
Specifically, we propose to:
(1) Characterize the invasion of Borrelia burgdorferi into primary fibroblasts. The working hypothesis here is that B. burgdorferi exploits invasion as an additional mechanism to avoid host clearance. Our preliminary studies demonstrate that B. burgdorferi invasion is not dependent on host fibronectin, but does involve B1 integrins other than a5B1. In this Aim we will identify the subunit that pairs with B1 to promote invasion and will also evaluate how B. burgdorferi traffics within these cells; and
(2) Determine if invasion is required for B. burgdorferi persistence in vivo. Our working hypothesis is that invasion contributes to persistence by providing an immunoprotected niche for B. burgdorferi. Since Src kinases are required for borrelial internalization in vitro, we will determine whether Src kinase inhibitors alter the infectivity potential of B. burgdorferi in vivo. In addition to standard cultivation and molecuar approaches, novel in vivo imaging will be employed to assess how the inhibitor affects colonization.
The overall goal of these studies is to determine the extent in which an intracellular locale contributes to borrelial persistence.
PUBLIC HEALTH RELEVANCE: Borrelia burgdorferi, the etiologic agent of Lyme disease, is the most common arthropod-borne infectious agent in the United States, and, as such, represents an important Public Health issue. The studies described in this application are designed to address how B. burgdorferi is able to persist effectively in infected mammals despite effective innate immune killing mechanisms and a potent adaptive immune response directed against this pathogen. The hypothesis being tested herein is that B. burgdorferi is capable of low-level intracellular survival in non-immune cells as an additional strategy to prevent borrelial host clearance.
Comment: This really begins fulfilling my wishlist, and I look forward to the imaging study videos that I hope will be made and posted online. If there is some sort of confirmation of intracellular Bb in vivo this may explain why some patients need additional antibiotics and why existing treatments may be inadequate as a matter of timing.
This next project is bound to generate discussion, as it involves the potential role of toxins in Borrelia burgdorferi. In this case, the researcher is looking for gene clusters in Borrelia burgdorferi which may create cytolysins similar to the toxins which are found in Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum.
Project: A COMMON DENOMINATOR OF PATHOGENESIS; A RARE OPPORTUNITY FOR NOVEL THERAPEUTIC DE(VELOPMENT)
Institution: UNIVERSITY OF ILLINOIS URBANA-CHAMPAIGN
PI: Mitchell, Douglas
Description (by applicant):
Abstract: The 20th century witnessed several major advances in medicine. Perhaps most important were the discovery of antibiotics for bacterial infections and effective vaccines for several major viruses. Unfortunately, the creation of effective vaccines for bacteria has lagged behind analogous anti-viral strategies. Compounded with the rise in antibiotic resistance and a lack of interest from the pharmaceutical industry in pursuing novel antibiotics, we risk losing the fight against bacterial pathogens.
Described herein is an unconventional strategy to exploit bacterial toxins as both novel targets for antibacterial agents and antigens for vaccine development. To intelligently address the increasing threat posed by bacterial pathogens, more effort is needed to uncover the molecular underpinnings of virulence. Our group specializes in the use of bioinformatics, in vitro reconstitution, and genetic manipulation to identify and characterize gene clusters that are responsible for the biosynthesis of virulence-promoting cytolysins. The best-known toxin in this family is the highly modified peptide, streptolysin S (SLS, produced by Streptococcus pyogenes).
SLS production is required for the infective process, but not essential life processes. Our work has uncovered SLS-like toxins are synthesized by at least three other notorious human pathogens, including Staphylococcus aureus, Listeria monocytogenes, and Clostridium botulinum. We aim to study the potential role of the SLS-like toxin in an additional organism, Borrelia burgdorferi (Bb), which causes Lyme disease.
Although widely known, the Bb molecular mechanism of pathogenesis is inadequately defined. If the SLS-like toxin was indeed employed during Bb infections, this would represent the first demonstration of toxin utilization in this family of organisms and would prompt a major revision of borrelioses.
Because bacteria typically employ disparate pathogenic mechanisms, the conserved, SLS-like pathway provides a rare opportunity to develop more broadly applicable, yet targeted countermeasures. From our perspective, new antimicrobial strategies should directly target the pathogenic mechanism, rather than DNA replication, protein synthesis, or the cell wall. This approach holds enormous potential, as these drugs will theoretically be resistant to resistance.
This project will identify inhibitors of SLS toxin biosynthesis for the specific purpose of developing novel antibacterials. Moreover, SLS is non-immunogenic, rendering it an unfeasible candidate for vaccine development.
We have succeeded in generating attenuated variants with the anticipation that these can be used for raising toxin-neutralizing antibodies. The notion of immunizing against a bacterial toxin represents a potentially general strategy for future vaccine development.
With this proposal, we aim to not only fundamentally shift the accepted view of Bb pathogenesis, but also to challenge the paradigm that antibiotics must kill bacteria and non-immunogenic toxins are intractable vaccine candidates. These seemingly unrelated goals are actually quite intertwined. Our approach rests on the philosophy that a more complete understanding of toxin biosynthetic pathways and chemical structure can be rationally exploited to design novel therapeutics.
Public Health Relevance: Bacterial pathogens employ numerous mechanisms to evade the human immune system. We have discovered a novel strategy within the organism that causes Lyme Disease, who's pathogenesis remains largely enigmatic. A greater understanding of these processes will lay the foundation for developing the next generation of antimicrobial drugs.
Wait... I thought Radolf & co. said Borrelia burgdorferi does not produce a toxin? I know Donta patented some genes in Bb he saw as being analogous to a toxin.
Is there now evidence of newly researched genes which create a toxin in Bb? Or is this an old hypothesis which is being revisited?
Project: ASSESSMENT OF PATIENTS WITH BORRELIA INFECTION
PI: Marques, Adriana
Description (by applicant):
Lyme disease is a multisystem illness caused by infection with the spirochete Borrelia burgdorferi and it is the leading vector-borne disease in the United States. Our current work addresses the following areas in Lyme disease: development of new tests and biomarkers for infection, investigation of persistence of infection with B. burgdorferi in humans, search for the cause of Southern Tick-associated Rash Illness (STARI), and investigation of the role of immune response in Lyme disease and PLDS.
One of the main problems in Lyme diagnosis has been the lack of highly specific and sensitive assays for B. burgdorferi and the lack of a test that could be used to assess response to therapy. Such assays should greatly facilitate the accurate diagnosis of Lyme disease and assessment of response to therapy in individual patients. Currently, no such test is available.
We have developed a new test using the luciferase immunoprecipitation systems (LIPSs) for profiling of the antibody responses to a panel of B. burgdorferi proteins for the diagnosis of Lyme disease. A synthetic protein consisting of a repeated antigenic peptide sequence, named VOVO, had the best diagnostic performance, similar to the C6 test (a diagnostic test using a peptide ELISA that we have helped develop and is highly sensitive and specific). The VOVO LIPS test displays a wide dynamic range of antibody detection spanning over 10,000-fold without the need for serum dilution; and offers an efficient quantitative approach for evaluation of the antibody responses in patients with Lyme disease.
Recent studies have shown that B. burgdorferi may persist in animals after antibiotic therapy and can be detected by using the natural tick vector (Ixodes scapularis) to acquire the organism through feeding. Whether this occurs in humans is unknown.
We have implemented a new clinical protocol to investigate the utility of this approach for identifying persistence of B. burgdorferi in treated human Lyme disease.
STARI is a rash similar to the rash of Lyme disease that occurs in persons residing in southeastern and south-central states and is associated with the bite of the lone star tick, Amblyomma americanum. The cause of the rash is unknown, as it is the natural course of the disease.
We have a clinical protocol to investigate the cause of STARI, and we are applying new genomic tools that identify bacteria based on species-specific sequences in the 16S rRNA ribosomal genes to the skin biopsies from patients with STARI.
Inflammatory innate immune responses are critical in the control of early disseminated infection, while adaptive immune responses are vitally important, particularly the humoral immune response, in controlling spirochete levels in tissues and resolution of Lyme arthritis in animal models. We are examining the antibody response to immunogenically dominant antigens of B. burgdorferi in PLDS patients and controls.
Further investigation of the anti-borrelia immune response may help in elucidating the pathogenic mechanism of PLDS and yield important information for future approaches to diagnosis and treatment. We have a clinical protocol in which we use DNA microarrays to characterize gene expression patterns in skin biopsies from individuals with EM, with the aim of capturing the human host response to pathogen exposure.
We are also investigating the differences in immunological response between predominantly lymphocytic meningitis and predominantly neutrophilic meningitis. Results from these studies will serve as a window into the fundamental biology of the infection.
The existence of the VOVO LIPS test is nothing new - reports on the development of this test have been around since 2010. Also, there is already information about a chronic Lyme disease xenodiagnosis study out there.
It seems like this project has a large scope - or consists of more than one project under the same umbrella. So far, no project end date has been posted for this entry.
What would be of most interest to me would be finding differences in immunological response between patients with acute Lyme disease and those with assumed PLDS - something Alaedini has already been studying.
(Side note: I thought that it was already determined that Borrelia lonestari, a relapsing fever spirochete, was the cause of STARI or Masters disease - did I miss something?)
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