The objective of this work is to determine how a Mycobacterial secretion apparatus called ESX-1 plays critical roles in important, but apparently disparate, molecular processes: conjugal DNA transfer and virulence. This laboratory has established that ESX-1 secretion and DNA transfer are functionally and genetically intertwined;ESX-1 mutants are defective in DNA transfer. Our studies have allowed us to propose a model in which Mycobacteria customize their ESX-1 secretion profile in response to different physiological and environmental cues. This explains the functional versatility of the conserved ESX-1 secretory apparatus in promoting DNA transfer and survival in a macrophage. The proposed studies will elucidate the impact of these two processes on the biology and pathogenesis of Mycobacteria. Conjugation requires cell-cell contact and involves the unidirectional transfer of DNA from a donor cell to a recipient cell, often resulting in the transfer of large segments of chromosomal DNA. DNA transfer between strains of Mycobacterium smegmatis occurs by a novel mechanism distinct from the prototypical, plasmid-mediated conjugation systems. A long-term goal of the work is to understand the contributions of conjugation to mycobacterial evolution, including the spread of drug resistance and virulence determinants. The ESX-1 secretory apparatus is essential for virulence of Mycobacterium tuberculosis. There is no genetic assay for M. tuberculosis ESX-1 activity, and both ESX-1 substrates and the mechanism of secretion are poorly defined. This proposal will address these gaps by taking advantage of a unique observation that we have made: ESX-1 is essential for DNA transfer in the M. smegmatis recipient strain. The requirement for an intact ESX-1 apparatus for DNA transfer in M. smegmatis provides the first genetic screen for the isolation of ESX-1 mutants. The goals of this proposal are to characterize the process of DNA transfer, to exploit DNA transfer as a genetic tool, to elucidate the mechanism of ESX-1 secretion and to determine the roles of the proteins secreted by ESX-1 in both regulation of DNA transfer and M. tuberculosis virulence. These goals will be achieved using a combination of molecular, genetic and biochemical assays.
The specific aims are to: 1. Determine the genetic requirements for conjugal DNA transfer in donor and recipient strains of M. smegmatis in order to characterize this novel mechanism of genetic exchange. 2. Utilize DNA transfer to perform a genome-wide genetic screen to identify genes required for ESX-1 activity, and determine their role in both DNA transfer and virulence. Together, these aims describe an innovative approach to dissect the mechanism of ESX-1 secretion. Moreover, the identification of the secreted proteins, and their extracellular targets will provide significant new insights into their roles in both lateral DNA transfer and M. tuberculosis virulence.

Public Health Relevance

It is estimated that one-third of the world?s population is infected with M. tuberculosis (WHO) and 1.6 million people die of tuberculosis every year, a crisis that is exacerbated by the synergistic association of HIV and M. tuberculosis, and the appearance of multi-drug (MDR) and extremely-drug (XDR) resistant strains of M. tuberculosis. A comprehensive understanding of the biology of M. tuberculosis is critical for the characterization of pathogenesis, the identification of novel drug targets, the development of vaccines, and for determining how it evades the host immune system. This proposal will employ innovative molecular genetic techniques to characterize the process of genetic exchange between mycobacteria and its impact on virulence and the spread of drug resistance in M. tuberculosis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
High Priority, Short Term Project Award (R56)
Project #
1R56AI080694-01A1
Application #
7865197
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Lacourciere, Karen A
Project Start
2009-07-22
Project End
2012-06-30
Budget Start
2009-07-22
Budget End
2012-06-30
Support Year
1
Fiscal Year
2009
Total Cost
$453,861
Indirect Cost
Name
Wadsworth Center
Department
Type
DUNS #
153695478
City
Menands
State
NY
Country
United States
Zip Code
12204
Derbyshire, Keith M; Gray, Todd A (2014) Distributive Conjugal Transfer: New Insights into Horizontal Gene Transfer and Genetic Exchange in Mycobacteria. Microbiol Spectr 2:
Gray, Todd A; Palumbo, Michael J; Derbyshire, Keith M (2013) Draft Genome Sequence of MKD8, a Conjugal Recipient Mycobacterium smegmatis Strain. Genome Announc 1:e0014813
Gray, Todd A; Krywy, Janet A; Harold, Jessica et al. (2013) Distributive conjugal transfer in mycobacteria generates progeny with meiotic-like genome-wide mosaicism, allowing mapping of a mating identity locus. PLoS Biol 11:e1001602
Wirth, Samantha E; Krywy, Janet A; Aldridge, Bree B et al. (2012) Polar assembly and scaffolding proteins of the virulence-associated ESX-1 secretory apparatus in mycobacteria. Mol Microbiol 83:654-64
Javid, Babak; Derbyshire, Keith M (2011) Nanotubes: shaking hands, talking, or sharing? Front Microbiol 2:95
Nguyen, Kiet T; Piastro, Kristina; Gray, Todd A et al. (2010) Mycobacterial biofilms facilitate horizontal DNA transfer between strains of Mycobacterium smegmatis. J Bacteriol 192:5134-42