Latent Tuberculosis Infection
Bacteriological and Molecular Aspects of Mycobacterium tuberculosis During Latent Infection
Tech Area / Field
- BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
- MED-DIS/Disease Surveillance/Medicine
3 Approved without Funding
Research Center of Molecular Diagnostics and Theraphy, Russia, Moscow
- Ministry of Health / The First Moscow State Medical University named after I.M.Sechenov, Russia, Moscow
- University of Colorado / Health Science Center / Department of Microbiology, USA, CO, Aurora\nNational Jewish Medical and Research Center, USA, CO, Denver
Project summaryA third of the world's population and is infected with Mycobacterium tuberculosis (MTB). The majority of these infections are in a clinically latent state. In the past ten years the incidence of tuberculosis in Russia has been increasing and is approaching epidemic levels. Approximately 75% of Russians are infected with latent MTB and mortality from TB can be as high as 15 deaths per 100,000 as in Western Siberia and the Ural Mountains. Tubercle bacilli can remain inactive in lung lesions only to emerge decades later as new outbreaks of tuberculosis. Current therapy for tuberculosis often involves the administration of multiple antimicrobial agents over several months, likely because some bacilli exist in a drug tolerant, dormant-like state. In Russia the emergence of multi-drug resistant (MDR) MTB and existence of scores of MDR latently infected inpiduals has only compounded the threat from MTB. Understanding the physiology of bacilli during the latent infection phase is central to the goal of controlling and ultimately eradicating this infectious agent in Russia and world-wide. Molecular knowledge of latent MTB will aid in the development of vaccines and drugs targeted against metabolically inactive MTB and allow for the development of diagnostics that could help identify persons at high risk for reactivation of latent TB.
Unfortunately very little is currently known about the state of MTB during decades of latent infection. However, it is generally accepted that latent bacilli are less metabolically active, and their replication rate is drastically diminished compared to bacilli in an active infection. Limited access to oxygen impairs the ability of MTB (an obligate aerobe) to grow and has been linked to the control of MTB during disease. In the middle of the last century, removal of oxygen was recognized to promote long term MTB survival in culture. Currently various in vitro and animal models are being used to mimic the physiological state of MTB during latent infection and a number of proteins have been associated with these models. The most intensely studied of these proteins includes: Icl necessary for persistence in a chronic mouse infection, and the dormancy proteins controlled by DosR necessary for survival during low oxygen dormancy. Little is known about the functions and the corresponding physiological pathways that are mediated by latency-associated proteins, and even less evidence exists to demonstrate whether any of these proteins are involved in survival of MTB during latent infections in humans. Thus, methods to determine whether these proteins are involved in latent infection are sorely needed. Identification of mRNA or protein markers in latently infected human tissue specimens would contribute to the validation of these proteins as important for survival during the critical latent stage of MTB infection.
The research proposed herein is designed to bridge knowledge obtained from modeling of latent MTB in vitro with the actual physiology of the tubercle bacilli during latent infection. The primary goal of this project is to identify and confirm markers of latent MTB infection. To address this goal, three main objectives will be met. First, molecular assays for specific markers of low-oxygen-induced dormant MTB will be identified and tested. Second, latently infected human lung tissue specimens will be collected and characterized. Third, the tissue specimens will be analyzed for the presence of dormancy-specific markers. Meeting these objectives will establish a framework for describing the physiological state of MTB during latency that can be used to validate laboratory models of latent infection.
At the completion of this project the presence of dormancy markers will be determined at both the RNA and protein level during a well-established low oxygen dormancy model of latency. Panels of monoclonal antibodies will be prepared against each protein and used for in vitro and in situ experiments. A well characterized collection of human lung tissue specimens from a range of latently-infected tuberculosis foci from surgical and biopsy procedures will be established. Data on the presence of dormancy “latency” markers in latently infected foci will be obtained by a variety of methods including in situ PCR and RT-PCR, immunohistochemical and electron microscopy methods. Identification of these markers will indicate the presence of key physiological processes used by latent MTB. Knowledge of these processes should aid in the development of vaccines and drugs targeted against metabolically inactive latent MTB. Latency markers may allow for diagnostic tests to distinguish active from latent infection and perhaps the identification of latently infected inpiduals likely to develop active disease in the near future.
All participants from RRC MDT, NJRMC, and UCHSC have significant prior experience in investigating MTB latency. The RRC MDT participants gained this experience during implementation of the previous project (Molecular Basis Investigation of Mycobacterium tuberculosis Persistence, ISTC #1847). The laboratory interests of the U.S. collaborators Dr. Leonid Heifets (NJRMC) and Dr. Martin Voskuil (UCHSC) include investigation of antimicrobials effective against dormant MTB and the study of the low oxygen dormant state of MTB, respectively. A central element of the proposed project includes the establishment of a well characterized archive of human lung tissue specimens from a range of latently-infected tuberculosis foci from surgical and biopsy procedures. Dr. Eugenia Kogan and other renowned tuberculosis pathologists from the Pathology Department of Moscow Medical Academy, with experience accumulated over more than a century, will establish and characterize the tuberculosis tissue archive. In the proposed study specimens from the archive will be analyzed for the presence of latency markers and should serve as the beginning of a collection that can be used in future collaborations between Russian and the international tuberculosis research community. Experience gained with this collection will establish researchers at RRC MDT and MMA as leaders in the molecular characterization of TB-infected tissue samples. This experience should lead to continued research and funding opportunities.
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