Proteins in gram-negative bacteria destined for the extracellular environment are met with the additional challenge of crossing two membranes in order to reach the exterior of the cell. Unique among the several pathways evolved for this purpose is the type II secretion (T2S) pathway, which has the ability to transport folded proteins through the outer membrane. As many of the substrates secreted via the T2S system constitute toxins and degradative enzymes, this secretion system is considered a major virulence determinant. An exciting role for the involvement of the T2S system in biofilm formation is also emerging. T2S substrates are produced with N-terminal signal peptides, which target them to the Sec pathway for inner membrane translocation. Following removal of the signal peptides and folding in the periplasm the proteins engage with the T2S system, a multiprotein complex that spans the entire cell envelope. While many T2S substrates are released to the extracellular space once transported through the outer membrane, a subset of proteins remain surface associated or may reattach to the bacterial cell surface following extracellular release. In Vibrio cholerae, the causative agent of the diarrheal disease cholera, the main virulence factor cholera toxin is an example of a completely secreted substrate, while the trypsin-like protease VesB is primarily localized to the cell surface. VesB is produced with a conserved C-terminal extension that contains two prominent glycines and a hydrophobic helix followed by positively charged residues (GlyGly-CTERM domain), a tripartite pattern that is reminiscent of the sortase targeting motif that is required for surface anchoring of proteins in Gram positive bacteria. As VesB transits the cell envelope, the GlyGly-CTERM domain is cleaved off by the rhomboid-like intramembrane protease, rhombosortase. When the rhombosortase gene is inactivated or when VesB is expressed without its GlyGly-CTERM domain, surface associated VesB activity is no longer detected. In addition to the defect in VesB cleavage, biofilm formation is negatively affected in the rhombosortase mutant. Using two model organisms, V. cholerae and the nosocomial pathogen Acinetobacter baumannii, the experiments described in this proposal are designed to test the hypothesis that the T2S system, in collaboration with rhombosortase, promotes the maturation and surface localization of GlyGly-CTERM proteins that support biofilm development and maintenance. Specifically, this proposal will determine the mechanism of maturation and surface localization of proteins with GlyGly-CTERM extensions, resolve the relationship between rhombosortase and the T2S apparatus, and identify the role(s) of GlyGly-CTERM proteins in biofilm formation, growth and maintenance. The findings will facilitate understanding of the function and specificity of rhombosortase as well as the broader class of medically relevant rhomboid proteases and may identify ways to manipulate the T2S/rhombosortase system for preventative and therapeutic use.

Public Health Relevance

The type II secretion system is responsible for the extracellular secretion of toxins and digestive enzymes that cause disease and support formation of biofilms, surface-associated bacterial communities. A subset of these enzymes relies on the newly discovered rhombosortase for maturation and surface localization. This study aims to understand the function of rhombosortase and its substrates and resolve its relationship to the type II secretion system with the goal of identifying novel ways to manipulate this system for preventative and therapeutic use.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
High Priority, Short Term Project Award (R56)
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Prokaryotic Cell and Molecular Biology Study Section (PCMB)
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Hall, Robert H
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University of Michigan Ann Arbor
Schools of Medicine
Ann Arbor
United States
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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
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
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; Gray, Miranda D; Kreger, Allison et al. (2009) Calcium is essential for the major pseudopilin in the type 2 secretion system. J Biol Chem 284:25466-70
Abendroth, Jan; Mitchell, Daniel D; Korotkov, Konstantin V et al. (2009) The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae. J Struct Biol 166:303-15