This proposal involves a broad array of analytical studies designed to reveal new information about the virulence of newly emerged Vibrio cholerae "variant" strains of Vibrio cholerae (the causative agent of cholerae) and its Type VI secretion system (T6SS). The PI proposes to use state-of-the-art deep sequencing- based methods of transcript abundance (RNA-seq), mutant fitness (Tn-seq), and regulatory protein DNA binding site determination (ChIP-seq) to understand the hypervirulence and hyperinfectivity phenotypes of the H1 strain from the 2010 Haiti epidemic. He further proposes to study the biological activities of cyclic-AMP- GMP in promoting intestinal colonization of V. cholerae and hyperinfectivity by studying chemotaxis suppressor mutations and proteins that specifically bind or hydrolyze c-AMP-GMP. Because H1 is elevated in T6SS expression in vitro and other 7th pandemic strain express T6SS in vivo in infected rabbits, the PI also proposes to continue his highly productive investigations on the function of the dynamic T6SS organelle. His lab will test the hypothesis that T6SS spike tip PAAR proteins are adapters for transport of multiple T6SS effector domains into target bacterial and eukaryotic cells by V. cholerae by driving their association with the VgrG trimer of the T6SS spike. A high resolution mutation analysis technology developed in the PI's laboratory (Mut-Seq), will be used to obtain a deeper understanding of the dynamic V. cholerae T6SS apparatus by defining mutations that are compatible or incompatible with various models for how the organelle functions. Moreover, he will apply Mut-seq to understand the functionality of V. cholerae T6SS proteins by defining dominant negative mutations that block T6SS function. These mutations may provide the tools to capture and stabilize intermediates in the T6SS assembly/firing cycle that will aid in fine structure analysis as well as inform efforts to develop drugs that block the function of the T6SS organelle. Because this grant has previously funded cholera vaccine development efforts in the PI's lab, he also proposes to continue research on new live attenuated and nanoparticle-based vaccines derived from V. cholerae and its T6SS, respectively. These include a promising nonreactogenic, nonflagellated derivative, Peru-NT FlaABCDE as well as multivalent nanoparticle derived from the T6SS sheath. He proposes to test whether Hcp and VgrG proteins are recognized by the innate immune system and thus potentially act as adjuvants in vaccine formulations, as well as test the possibility that the V. cholerae T6SS can delivery CTL epitopes to the host cell cytosol and thus stimulate CD8 T cell responses against heterologous antigens. Finally, the PI proposes to develop new technologies that will allow facile phenotypic analysis in V. cholerae and other organisms. These include 'Sortase conjugation technology'(to study protein localization and trafficking in living cells) as well as combinatorial J25 microcin 'lasso peptides'(to develop drug-like small molecule inhibitors of T6SS function). These technologies represent promising and powerful tools to understand more about the T6SS organelle of V. cholerae and its anti-cellular effectors.

Public Health Relevance

This proposal involves a broad array of analytical studies designed to reveal new information about the virulence Vibrio cholerae (the causative agent of cholerae) and its Type VI secretion system (T6SS). The research will employ state-of-the-art deep sequencing-based methods to understand gene expression in a hyper-virulent Haiti isolate and further focus on understanding more about the T6SS virulence factor by using novel mutational, analytical, and inhibitor approaches. Finally, the proposed research will also seek to develop new live attenuated and nanoparticle-based vaccines and vectors derived from V. cholerae and its T6SS.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
2R01AI018045-34
Application #
8694862
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Hall, Robert H
Project Start
Project End
Budget Start
Budget End
Support Year
34
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Harvard Medical School
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02115
Ho, Brian T; Dong, Tao G; Mekalanos, John J (2014) A view to a kill: the bacterial type VI secretion system. Cell Host Microbe 15:9-21
Dong, Tao G; Ho, Brian T; Yoder-Himes, Deborah R et al. (2013) Identification of T6SS-dependent effector and immunity proteins by Tn-seq in Vibrio cholerae. Proc Natl Acad Sci U S A 110:2623-8
Danilchanka, Olga; Mekalanos, John J (2013) Cyclic dinucleotides and the innate immune response. Cell 154:962-70
Basler, Marek; Ho, Brian T; Mekalanos, John J (2013) Tit-for-tat: type VI secretion system counterattack during bacterial cell-cell interactions. Cell 152:884-94
Shneider, Mikhail M; Buth, Sergey A; Ho, Brian T et al. (2013) PAAR-repeat proteins sharpen and diversify the type VI secretion system spike. Nature 500:350-3
Fu, Yang; Waldor, Matthew K; Mekalanos, John J (2013) Tn-Seq analysis of Vibrio cholerae intestinal colonization reveals a role for T6SS-mediated antibacterial activity in the host. Cell Host Microbe 14:652-63
Davies, Bryan W; Bogard, Ryan W; Young, Travis S et al. (2012) Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence. Cell 149:358-70
Basler, M; Pilhofer, M; Henderson, G P et al. (2012) Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483:182-6
Davies, Bryan W; Bogard, Ryan W; Dupes, Nicole M et al. (2011) DNA damage and reactive nitrogen species are barriers to Vibrio cholerae colonization of the infant mouse intestine. PLoS Pathog 7:e1001295
Chin, Chen-Shan; Sorenson, Jon; Harris, Jason B et al. (2011) The origin of the Haitian cholera outbreak strain. N Engl J Med 364:33-42

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