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

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

and turnover of these

and turnover of these proteins in response to specific cues, both S18 proteins can be phosphorylated in vivo at homologous sites. Our in vitro binding experiments imply that this modification changes S18-1 protein structure in a manner that affects its binding to S6, while the effect of S18-2 phosphorylation is still unclear. It is unknown why Mtb has these various mechanisms to regulate incorporation of S18-1 or S18-2 protein into the ribosome, but it seems that S18-1 is the dominant protein during the exponential growth, while S18-2 provides an advantage under certain stress conditions. We showed that two S18 proteins have distinct binding affinities for another ribosomal protein and that their activities are regulated by zinc ion and phosphorylation in vitro. These biochemical properties are without a precedent in mycobacteria and are likely to have an influence on ribosome function and bacterial fitness. S18-2 may play a role in Mtb viability in necrotizing granulomas, which are thought to be a low zinc environment and are important for TB transmission. This study on ribosomal regulation may suggest approaches for interfering with protein synthesis control and allow identification of antibiotics targeting this regulation. This research is supported by the Harvard University Center for AIDS Research (CFAR) and NIH (RO1-AI59702) Biography: Sladjana Prisic received her B.Sc. in Biochemistry from the Faculty of Chemistry, University of Belgrade. She moved to Iowa State University to work on plant enzymes in Dr. Reuben Peters’ group, where she obtained her Ph.D. degree in Biochemistry. While working on plant terpene cyclases, Dr. Prisic briefly studied similar enzymes from Mycobacterium tuberculosis (Mtb) and instantly ‘fell in love’ with this dangerous pathogen. She became especially curious about its ability to infect and persist in such large number of people despite our efforts to eradicate it. Therefore she switched fields and joined Dr. Husson’s group to learn mycobacteriology and join other scientists in the battle against TB. Currently her interests are Ser/Thr kinases from Mtb and their substrates. Initially Dr. Prisic and collaborators identified over 500 phosphorylation sites on 300 Mtb proteins in the largest phosphoproteomic study published for any prokaryote. She uses this list of Mtb phosphoproteins to study in more details roles of these modifications in pathogenesis and to identify new drug targets. In 2010 Dr. Prisic received a CFAR (Center for AIDS Research) Award to study phosphorylation of ribosomal proteins and their role in Mtb physiology, a new field of study in mycobacteria. 3) A chemical genetic approach to identifying essential functions in non-replicating Mycobacterium tuberculosis Sarah Schmidt Grant 1,2 , Tomohiko Kawate 2 , Noriaki Iwase 2 , Katherine Gordon 2 , Thomas Ioerger 3 , James C. Sacchettini 3 , Deborah T. Hung 2 1 Department of Pulmonary and Critical Care Medicine, Brigham and Women’s Hospital, Boston, MA 02115; 2 Infectious Disease Initiative, The Broad Institute, Cambridge MA 02142; 3 Department of Biochemistry and Biophysics, 103 Biochemistry/Biophysics Building, Texas A&M University, 2128 TAMU, College Station, TX 77843. Abstract: During both active and latent infection, a subpopulation of Mycobacterium tuberculosis bacteria likely exists in a non-replicating, metabolically inactive state. The presence of non-replicating bacteria within the host likely contributes to the need for prolonged drug therapy for active and latent M. tuberculosis infection. A better understanding of the nonreplicating state may lead to the development of new antibiotic therapies targeting essential processes in the non-replicating M. tuberculosis bacterium. Here we describe a chemical genetic approach to identify small molecules that affect the viability of non-replicating M. tuberculosis. A carbon starvation assay was used to model non-replicating, antibiotic tolerant M. tuberculosis. 23,680 compounds were screened in duplicate against carbon-starved M. tuberculosis and 308 compounds were identified as hits. Whole genome sequencing was used 7

to identify bacterial mechanisms of resistance for 6 chemical scaffolds. For one scaffold, named compound 5849, resistance is mediated by single nucleotide polymorphisms (SNPs) in the sensor component (prrB) of an essential two-component system. In the resistant mutants prrB exhibits altered transphosphorylation activity on its cognate response regulator, prrA. Gene expression analysis of the resistant mutants compared to wild-type revealed that prrA/prrB regulates fatty acid metabolism and mycolic acid synthesis. Compound 5849 was found to inhibit the synthesis of fatty acid methyl esters (FAMES) as well as mycolic acid methyl esters (MAMES). In conclusion, a novel chemical inhibitor was identified with both replicating and nonreplicating activity in M. tuberculosis. Resistance to this inhibitor is mediated by SNPs in prrB, and the prrA/B two-component system was identified as a novel regulator of fatty acid and mycolic acid synthesis in M. tuberculosis. Biography: Sarah Grant is a pulmonary and critical care medicine physician at the Brigham and Women’s Hospital, and a post-doctoral fellow in Deborah Hung’s lab at the Broad Institute. 4) Retro is in – Using retrogenic mice to study CD8 + T cell responses during tuberculosis Cláudio Nunes-Alves 1,2,3 , Steve Carpenter 4 , Matt Booty 4 , Christophe Benoist 3 , Margarida Correia-Neves 1,2 , Sam M. Behar 4 1 Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal; 2 ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal; 3 Department of Pathology; 4 Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Abstract: Immunity to Mycobacterium tuberculosis (Mtb) infection relies on the establishment of a successful adaptive immune response, with T cells playing a central role in controlling infection. Among these, CD8 + T cell responses are detected in both infected people and experimental animal models of tuberculosis, and CD8 + T cells are required for optimal immunity against virulent Mtb. To elucidate the role of CD8 + T cells during Mtb infection, we developed a novel mouse model with a high frequency of antigen-specific CD8 + T cells that recognize TB10.4 (EsxH; Rv0288). At the peak of Mtb infection, 30-50% of the CD8 + T cells in the infected lung recognize this antigen in both BALB/c and C57Bl/6J mice, defining it as an immunodominant antigen. We find that transfer of purified lung TB10.44-11-specific CD8 + T cells protects irradiated recipient mice against aerosol challenge with virulent Mtb, establishing TB10.44-11 as a protective epitope. Importantly, immunization of intact mice with this peptide also confers protection against Mtb challenge. By combining single cell sorting of tetramer positive cells and PCR amplification of T cell receptors (TCR), we identified and cloned four distinct TCRs specific for TB10.44-11. These four individual TCRs were expressed in mice, using retrovirus-mediated stem cell gene transfer. The resulting retrogenic (Rg) mice (“retro” from retrovirus, “genic” from transgenic) have a high frequency of T cells that express the recombinant TCR. We show that these T cells recognize and respond to TB10.44-11. Interestingly, some of the Rg T cells confer protection against Mtb challenge in our adoptive cell transfer system, while others, despite recognizing the same epitope fail to mediate protection in this model. This suggests that heterogeneity in the function of T cells, independent of their specificity, determines their ability to mediate protection. Currently, we are investigating the mechanism(s) leading to bacterial control by the protective clones, by focusing on the dichotomy between pro-inflammatory cytokine secretion and cytolytic activity exhibited by CD8 + T cells. To do so, we generated Rg mice whose cells are unable to secrete IFN-� (IFN-� -/- Rgs) or incapable of perforin-mediated killing (Prf -/- Rgs). After adoptive 8

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