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WELCOME TO THE 2011 - Harvard Initiative for Global Health ...

WELCOME TO THE 2011 - Harvard Initiative for Global Health ...

8) High-persister

8) High-persister strains in Mycobacterium tuberculosis Heather Torrey, Iris Keren, and Kim Lewis Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, MA USA 02115 Bacterial persister cells are phenotypic variants of the wildtype that are highly tolerant to killing by bactericidal antibiotics. It is thought that persister cells may be responsible for recurrent bacterial infections due to the inability of antibiotics to eradicate them. We suggest that the recalcitrance of tuberculosis to antibiotic therapy may be due to the presence of persister cells. To identify genes responsible for persister formation in Mycobacterium tuberculosis we have generated high-persister mutants in vitro that produce between 10- and 1,000-fold more persisters than the parental strain. Through whole-genome sequencing and transcriptome analysis, we have identified several candidate genes, some of which are involved in glycerol and fatty acid metabolism. The role of these genes in persister cell formation is being tested by overexpression and deletion analysis. We speculate that high-persister mutants may also be selected for in vivo, in clinical isolates. To address this possibility, we are currently testing individual clinical isolates against various classes of antibiotics to which the isolates are known to be susceptible. We have observed up to 100-fold differences in persister level between isolates. Characterization of in vitro and in vivo high-persister strains is in progress. The results of this study will contribute to the identification of persister genes M. tuberculosis and may provide insight to new therapies for persister cell eradication. This work was supported by the National Institutes of Health (NIH-1R01AI085585-01). 9) The role of proteases in Mycobacterium tuberculosis persister formation Lauren Fitch, Iris Keren, Kim Lewis. Antimicrobial Discovery Center, Department of Biology, Northeastern University, Boston, MA 02115. More people are killed each year by Mycobacterium tuberculosis (MTb) than any other bacterial pathogen, due to the long and often ineffective treatment regimen required to eradicate the infection. The recalcitrance of chronic MTb infections may be due to persisters, dormant cells that are tolerant to antibiotics. Previous research has shown that toxin-antitoxin (TA) modules present in many pathogenic bacteria are involved in Escherichia coli persister formation, but MTb’s possession of over 65 TA systems makes a screen of these operons unwieldy and unlikely to produce useful data. Since the successful action of a toxin depends first on the degradation of its cognate antitoxin, we therefore examined the role of the proteases ClpC/ClpP and Lon on persister formation. We used an anhydrotetracycline (ATC)-inducible plasmid to over-express protease genes, including the Clp complex, Lon homologs, and ssrA, and measured the change in antibiotic tolerance. These data will reveal the importance of mycobacterial proteases in rapidly converting the cell from a normally growing state to a dormant state, and identify possible targets for new drugs targeting non-growing persistent MTb. This work was supported by the National Institute of Allergy and Infectious Disease (NIH- AI085585-01) and Northeastern University’s University Excellence Fellowship. 10) CtpD is required for Mycobacterium tuberculosis pathogenesis Daniel Raimunda 1 , Teresita Padilla-Benavides 1 , Jarukit E. Long 2 , Christopher M. Sassetti 2,3 , and José M. Argüello 1 1 Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, Worcester, MA., USA. 01609, 2 Department of Microbiology and Physiological Systems. University of 19

Massachusetts Medical School. Worcester, MA, USA 01655, 3 Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. 20815 Genetic studies in the tuberculosis mouse model have suggested that mycobacterial metal efflux systems, such as the P1B4-ATPase CtpD, are important for pathogenesis. Studies on M. smegmatis showed that CtpD is required for Co 2+ and Ni 2+ transport. A deletion mutant of ctpD showed decrease of growth fitness and intracellular metal accumulation in presence of Co 2+ and Ni 2+ . ATPase activity of an heterologously expressed and purified CtpD was stimulated in the presence of Co 2+ , Ni 2+ , and to a lesser extent Zn 2+ . Interestingly, M. tuberculosis also presents a paralogous gene, ctpJ. In order to assess the functional roles of these PIB4-ATPases, deletion mutants of ctpD (Rv1469) and ctpJ (Rv3473) were generated and their transcriptional induction and metal homeostasis were studied in parallel experiments. In H37Rv WT strain, transcription of both was up-regulated by various redox stressors, but only ctpJ was induced by Co 2+ . Accumulation of Co 2+ was observed in the ctpJ mutant strain while ctpD mutant showed a decrease in total Co 2+ content. Experiments in a mouse competition infection assay (single mutant vs. WT), showed that the ctpD deletion affected the survival rate in lungs. The phenotype was reverted by gene complementation. These observations suggest that in M. tuberculosis CtpD is required for virulence likely by loading Co 2+ , Ni 2+ or Zn 2+ to an extracellular metalloprotein necessary for overcoming stress conditions met in the macrophage phagosome. CtpJ would be required for efficient Co 2+ and Ni 2+ removal from the cytosol, a condition hardly met in the phagosome but probably present in other bacterial niche. This work was supported by NIH awards F32A1093049 (J.E.L.), 1R21AI082484 (J.M.A.), AI064282 (CMS) and the Howard Hughes Medical Institute (CMS). 11) Community based TB prevention: improving population health and decreasing Medicaid costs Cynthia Tschampl 1 , John Bernardo 2 1 The Heller School for Social Policy and Management, Brandeis University, Waltham, MA, USA, 02454 and the Medical Advisory Committee for the Elimination of TB (MACET), Boston, MA, 02130; 2 School of Medicine, Boston University, Boston, MA, 02118 Background: Most TB in our communities results from reactivation of TB infection (LTBI), a treatable condition. Public health programs have expertise and resources to manage LTBI, TB, and other diseases of importance to the public's health, yet they often cannot access those in greatest need. In contrast, Federally-funded Health Centers (FQHCs) often are located within high-incidence communities and may already manage care for many in those communities. Yet, limited FQHC resources, models of care, and provider knowledge and understanding of community priorities undermine important health concerns, including TB prevention. Methods: We collected gray literature and information from TB practitioners and composed a proposal for community-based TB prevention that incorporates key aspects of health care reform. The proposal was reviewed by the Medical Advisory Committee for the Elimination of TB and submitted to the Center for Medicare & Medicaid Innovation. Results: We propose to move public health-TB prevention to FQHCs, led by core provider teams (typically MD-nurse) trained by pubic health experts; to create referral and informationsharing between public health and FQHCs (via a public health liaison-nurse or -FQHC provider); to provide regular performance feedback to FQHCs; and to link these services with targeted, needs-based community education and primary care. Conclusion: Based on evidence from earlier TB programs, the high level of returns for TB prevention work, and new federal incentives, there is reason to believe the proposal would lead 20

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