Extracellular secretion and targeted delivery by the type II secretion (T2S) system is considered a major virulence mechanism in gram negative pathogens, as many of the proteins secreted via the T2S pathway constitute important virulence factors, including toxins and degradative enzymes. The T2S apparatus is comprised of at least 13 different proteins, EpsC-EpsN and PilD, that assemble into a complex that spans the entire cell envelope of Vibrio cholerae. The dynamic and perhaps transient nature of this complex may be a prerequisite for function as its assembly and disassembly may drive extracellular secretion. The energy required for this process is thought to be generated from ATP hydrolysis by EpsE, a cytoplasmic protein that is associated with the cytoplasmic membrane via interaction with the membrane proteins EpsL. EpsM and EpsF. EpsE's interactions with these components modulate its ATPase activity and promote its localization to distinct sites within the V. cholerae cell envelope. The experiments described in this proposal are designed to test the hypothesis that specific protein-protein interactions and acidic phospholipids drive T2S in an ATP-dependent process at discrete sites In the cell envelope of V. cho/erae. Specffically, this proposal will i) determine the mechanism by which the enzymatic activity of EpsE is controlled by components of the cytoplasmic membrane including phospholipids. EpsL and EpsF;ii) investigate the ordered assembly of Eps components and determine the mechanism by which EpsD and EpsC drive focal assembly of the T2S complex;iii) map the cleft that forms when EpsM assembles and identify the cellular factor that binds to the cleft. Resolving the mechanisms of regulated assembly and spatial localization of the T2S system will further our understanding of T2S and may identify ways to manipulate the secretion process for preventative, therapeutic and/or biotechnological use.

Public Health Relevance

Many virulence factors such as toxins and degradative enzymes are secreted by gram-negative pathogens via the widely distributed type II secretion (T2S) pathway. Understanding how the components of the T2S system come together to collectively promote secretion should provide important information about the mechanism of T2S and identify novel ways to manipulate this process for preventative and therapeutic use.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI049294-09
Application #
7849928
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Hall, Robert H
Project Start
2001-04-01
Project End
2011-06-30
Budget Start
2010-06-01
Budget End
2011-06-30
Support Year
9
Fiscal Year
2010
Total Cost
$380,179
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
073133571
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Waack, Ursula; Johnson, Tanya L; Chedid, Khalil et al. (2017) Targeting the Type II Secretion System: Development, Optimization, and Validation of a High-Throughput Screen for the Identification of Small Molecule Inhibitors. Front Cell Infect Microbiol 7:380
Rule, Chelsea S; Patrick, Marcella; Sandkvist, Maria (2016) Measuring In Vitro ATPase Activity for Enzymatic Characterization. J Vis Exp :
Rule, Chelsea S; Patrick, Marcella; Camberg, Jodi L et al. (2016) Zinc coordination is essential for the function and activity of the type II secretion ATPase EpsE. Microbiologyopen 5:870-882
Rompikuntal, Pramod K; Vdovikova, Svitlana; Duperthuy, Marylise et al. (2015) Outer Membrane Vesicle-Mediated Export of Processed PrtV Protease from Vibrio cholerae. PLoS One 10:e0134098
Johnson, Tanya L; Waack, Ursula; Smith, Sara et al. (2015) Acinetobacter baumannii Is Dependent on the Type II Secretion System and Its Substrate LipA for Lipid Utilization and In Vivo Fitness. J Bacteriol 198:711-9
Gadwal, Shilpa; Korotkov, Konstantin V; Delarosa, Jaclyn R et al. (2014) Functional and structural characterization of Vibrio cholerae extracellular serine protease B, VesB. J Biol Chem 289:8288-98
Johnson, Tanya L; Fong, Jiunn C; Rule, Chelsea et al. (2014) The Type II secretion system delivers matrix proteins for biofilm formation by Vibrio cholerae. J Bacteriol 196:4245-52
Lu, Connie; Turley, Stewart; Marionni, Samuel T et al. (2013) Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity. Structure 21:1707-17
Korotkov, Konstantin V; Sandkvist, Maria; Hol, Wim G J (2012) The type II secretion system: biogenesis, molecular architecture and mechanism. Nat Rev Microbiol 10:336-51
Gray, Miranda D; Bagdasarian, Michael; Hol, Wim G J et al. (2011) In vivo cross-linking of EpsG to EpsL suggests a role for EpsL as an ATPase-pseudopilin coupling protein in the Type II secretion system of Vibrio cholerae. Mol Microbiol 79:786-98

Showing the most recent 10 out of 27 publications