Translocation of macromolecules between cells is a major area of biomedical interest. In bacteria, the intercellular transfer of macromolecules is commonly orchestrated by surface organelles termed type IV secretion systems. Many bacterial species use type IV systems to deliver DNA substrates within or across species, genus, or even kingdom boundaries through a process termed conjugation. Conjugative DNA transfer contributes to genome plasticity over evolutionary time, and the spread of antibiotic resistance genes and other virulence traits on a more immediate time scale. Many medically-important bacterial pathogens also use type IV systems to deliver effector proteins to eukaryotic target cells during infection processes. The VirB/VirD4 system of Agrobacterium tumefaciens serves as important model for detailed mechanistic studies of type IV machine function. This system, assembled from subunits VirB1 - VirB11 and VirD4, translocates DNA and proteins to phylogenetically-diverse bacterial and eukaryotic target cells by a mechanism dependent on direct cell-to-cell contact. The overall goal of work in this laboratory is to describe in complete molecular terms the VirB/VirD4 machine biogenesis pathway, the basis for substrate recognition and transfer across the cell envelope, and the nature of the VirB/VirD4 machine - target cell contact. Recent studies identified three novel features of VirB10 that focus our attention on this channel subunit: i) it spans the entire Gram-negative cell envelope, a novel property among all described bacterial proteins, ii) it forms an ?-helical pore at the outer membrane, only the second described ?-helical outer membrane pore protein, and iii) it undergoes an ATP-mediated structural transition required for substrate translocation across the outer membrane. We hypothesize that VirB10 forms a structural scaffold across the entire cell envelope for assembly of two structures, the VirB/VirD4 translocation channel and the extracellular T pilus, the latter being important for establishment of target cell contact. We propose to: (A) define the architecture, assembly dynamics, and function of the ?-helical outer membrane pore with respect to substrate transfer and pilus biogenesis, (B) define how VirB10 contributes to channel function through biochemical and structural characterization of VirB10-containing machine subassemblies, and (C) elucidate how the VirB10 transmembrane domain and the VirB4 ATPase located at the inner membrane contribute to pilus biogenesis. Studies of type IV secretion are essential for at least two reasons. First, these are widely used machines among most if not all prokaryotic cells and yet their mechanisms of action remain poorly understood. Second, type IV secretion is a major contributor to the proliferation of antibiotic resistance as well as successful infection by many bacterial pathogens;these systems are therefore excellent targets for therapies aimed at suppressing disease progression.

Public Health Relevance

This project investigates the structure and function of a bacterial type IV secretion system. These systems are widely used by many medically important pathogens for transfer of antibiotic resistance or other virulence traits, as well as effector proteins to human target cells. These systems represent novel targets for therapeutics directed to blocking antibiotic resistance spread and bacterial disease progression.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048746-21
Application #
8598476
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Flicker, Paula F
Project Start
1993-01-01
Project End
2014-12-31
Budget Start
2014-01-01
Budget End
2014-12-31
Support Year
21
Fiscal Year
2014
Total Cost
$374,430
Indirect Cost
$123,363
Name
University of Texas Health Science Center Houston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771594
City
Houston
State
TX
Country
United States
Zip Code
77225
Gordon, Jay E; Costa, Tiago R D; Patel, Roosheel S et al. (2017) Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact-dependent translocation. Mol Microbiol 105:273-293
Grohmann, Elisabeth; Christie, Peter J; Waksman, Gabriel et al. (2017) Type IV secretion in Gram-negative and Gram-positive bacteria. Mol Microbiol :
Bhatty, Minny; Camacho, Martha I; Gonzalez-Rivera, Christian et al. (2017) PrgU: a suppressor of sex pheromone toxicity in Enterococcus faecalis. Mol Microbiol 103:398-412
Whitaker, Neal; Berry, Trista M; Rosenthal, Nathan et al. (2016) Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine. J Bacteriol 198:2701-18
Christie, Peter J (2016) The Mosaic Type IV Secretion Systems. EcoSal Plus 7:
Gonzalez-Rivera, Christian; Bhatty, Minny; Christie, Peter J (2016) Mechanism and Function of Type IV Secretion During Infection of the Human Host. Microbiol Spectr 4:
Beabout, Kathryn; Hammerstrom, Troy G; Wang, Tim T et al. (2015) Rampant Parasexuality Evolves in a Hospital Pathogen during Antibiotic Selection. Mol Biol Evol 32:2585-97
Bhatty, Minny; Cruz, Melissa R; Frank, Kristi L et al. (2015) Enterococcus faecalis?pCF10-encoded surface proteins PrgA, PrgB (aggregation substance) and PrgC contribute to plasmid transfer, biofilm formation and virulence. Mol Microbiol 95:660-77
Whitaker, Neal; Chen, Yuqing; Jakubowski, Simon J et al. (2015) The All-Alpha Domains of Coupling Proteins from the Agrobacterium tumefaciens VirB/VirD4 and Enterococcus faecalis pCF10-Encoded Type IV Secretion Systems Confer Specificity to Binding of Cognate DNA Substrates. J Bacteriol 197:2335-49
Trokter, Martina; Felisberto-Rodrigues, Catarina; Christie, Peter J et al. (2014) Recent advances in the structural and molecular biology of type IV secretion systems. Curr Opin Struct Biol 27:16-23

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