Understanding events at the cell surface during autotransporter biogenesis

Lead Research Organisation: University of Birmingham
Department Name: Immunity and Infection

Abstract

To survive bacteria must produce proteins which are located on their cell-surface and which are released into the external environment in a process termed secretion. In order to get the proteins from the inside of the cell to the surface the bacteria must transport them across membranes, which in effect actually pose a barrier to secretion. Thus bacteria have evolved specialized machines which allow them to move proteins across the membranes. One such mechanism is the autotransporter system. This is the most widely used protein secretion system within the Gram-negative bacteria. The overall objective of this proposal is an in-depth analysis of certain aspects of autotransporter protein secretion. The study of these proteins and in fact the study of secretion systems in general, is aimed at several important objectives. First, since bacterial pathogens need to export proteins to effect virulence the study of secretion systems provides information about the pathogenic strategies of the particular genus and species. Second, the study of secretion systems provides new opportunities to attenuate bacteria in the pursuit of anti-infective strategies. A third benefit of understanding secretion systems is that these systems can potentially be exploited for the delivery of foreign antigens as part of novel vaccine delivery systems. The importance of understanding the autotransporters is illustrated by the fact that in some cases these are essential virulence factors and in other cases they form part of current human vaccines.

Technical Summary

The increasing incidence of antibiotic resistance has brought a new sense of urgency to the discovery and development of antibacterial drugs. Effectively dealing with infections arising from antibiotic resistance organsims will require expanding the available targets and new approaches. One approach to this problem is to identify surface/secreted proteins involved in the ability of bacteria to cause disease and to design strategies to inhibit the action and synthesis of these proteins thereby effectively inhibiting the infection. The simplest and most widely utilized protein secretion system in Gram-negative bacteria is the autotransporter (AT) system. The overall objective of this proposal is an in-depth analysis of certain aspects of AT protein secretion. Autotransporter proteins possess several domains which are involved in either the function of the protein or the secretion process: the signal sequence mediating inner membrane translocation, the functional passenger domain, the autochaperone domain mediating folding of the passenger domain, the hydrophobic facilitator domain involved in secretion, the alpha-helical linker region and the C-terminal beta-domain which forms a beta-barrel pore in the outer membrane through which the protein is translocated to the cell surface. The current accepted scenario of passenger domain translocation across the outer membrane is that one stretch of the extreme C-terminal end of the passenger domain recognizes the pore formed by the C-terminal beta-barrel pore and inserts into the pore as a hairpin, with both strands of the hairpin existing in an extended conformation. Once the autochaperone domain has been extruded to the cell surface it is presumed to adopt its native conformation and then to facilitate folding of the passenger domain on the cell surface. All autotransporter passenger domains are predicted to adopt a beta-helix conformation with the payload functional domain located at the extreme N-terminus. Indeed, such a scenario has been proven through the crystal structures of two functionally diverse passenger domains, that of the E. coli Hbp and B. pertussis Pertactin proteins. However, the precise mechanism of action of the AC-domain and the contribution of the beta-helix structure to the secretion process remains enigmatic, thus this proposal seeks to characterize the role of these structures in autotransporter biogenesis.

Publications

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Gil-Cruz C (2009) The porin OmpD from nontyphoidal Salmonella is a key target for a protective B1b cell antibody response. in Proceedings of the National Academy of Sciences of the United States of America

 
Description BBSRC Responsive Mode Grant
Amount £658,331 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2010 
End 12/2013
 
Description BBSRC Responsive Mode Grant
Amount £451,999 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2008 
End 12/2011
 
Description BBSRC Responsive Mode Grant
Amount £527,917 (GBP)
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2009 
End 12/2012
 
Description Industrial ITN
Amount £850,000 (GBP)
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 01/2013 
End 01/2017
 
Description MRC Industry Pilot Scheme
Amount £502,391 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 01/2009 
End 12/2012
 
Description MRC Research Grant
Amount £497,652 (GBP)
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 02/2009 
End 12/2011
 
Description Immune response to Salmonella 
Organisation University of Birmingham
Department College of Life and Environmental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Purification of membrnae proteins to analyse immune responses
Collaborator Contribution Intellectually
Impact PMID: 19487686 PMID: 17475847
Start Year 2007
 
Description Why HIV patients succumb to Salmonella infection 
Organisation University of Birmingham
Department College of Medical and Dental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution Purification of membrane components and design of experiments to test hypotheses relating to Why HIV pateints succumb specifically to Salmonella infection
Impact PMID: 20413503
Start Year 2009
 
Title bioprocess 
Description bioprocess 
Type Support Tool - For Medical Intervention
Current Stage Of Development Initial development
Year Development Stage Completed 2010
Development Status Actively seeking support
Impact award of a personal fellowship 
 
Company Name Prokarium 
Description Spin-out company developing vaccines 
Year Established 2011 
Impact company based on live attenuated vaccines
 
Description Presentation for KTF 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Presentation for training individuals in knowledge transfer

Award of a personal fellowship
Year(s) Of Engagement Activity 2009