Characterisation of the common steps in the glycosylation of Arg-gingipains and synthesis of LPS and APS of P.gingivalis

Lead Research Organisation: Queen Mary, University of London
Department Name: Blizard Institute of Cell and Molecular

Abstract

Periodontal disease, or gum disease, is a very common disease of the mouth which affects between 10-15% of the UK population. In severe cases the disease leads to loss of the teeth and there is growing evidence that people with this disease may be more susceptible to other conditions including heart disease. Over £270 million is currently spent annually by the General Dental Services of the NHS on periodontal disease treatment and its consequences. Hence there are significant clinical and financial pressures to develop improved methods of diagnosis control and of this disease. Porphyromonas gingivalis is one of the most important bacterial causes of periodontal disease. One of the critical characteristics of this bacterium is its ability to live in the mouth of an affected individual for a long period of time despite the actions of the body!
s immune defences. In this work we aim to understand the how three components of the surface of P. gingivalis are constructed. In particular we will examine a the steps in their synthesis which appear to be common to all three and which may prove useful therapeutic targets. In the longer term a better understanding of the biology of this bacterium and the mechanisms it uses to survive in patients can be expected to lead to the development of novel methods for the treatment of this disease and related chronic infectious diseases of human

Technical Summary

Periodontal disease is the most common chronic infectious, inflammatory disease of man. The disease is driven by a chronic bacterial challenge to the periodontal tissues from bacteria in the sub-gingival plaque and of these, Porphyromonas gingivalis represents a key pathogen. A critical requirement for periodontal bacteria is the ability to withstand the lytic and clearance activities of the host defences operating at a chronically inflamed mucosal site for prolongued periods. In this application we aim to build upon recent findings which demonstrate that three of the macromolecules considered important survival determinants of P. gingivalis (lipopolysaccharide [LPS], a novel anionic polysaccharide [APS]and the Arg-gingipain proteases [Rgps]share common enzymatic steps in their biosynthesis/maturation. The data suggest that the Wzy dependent mechanism of LPS O-antigen export and polymerisation in P. gingivalis is also required for synthesis of APS and for glycosylation of the Rgps. The aim of the current proposal is to test this hypothesis by targeted mutagenesis of additional components of the Wzy dependent pathway in P. gingivalis. Specifically we aim to generate mutants in Wzx, Wzy, Wzz and WaaL and determine the influence of these mutations on the assembly of LPS and APS and glycosylation of the Rgps. Elucidation of the details of this system will provide unique insights into the basic mechanism of bacterial glycoprotein formation. The data will also add to our understanding of the maturation of these key survival determinants of P. gingivalis and may provide potential targets for the development of specific antimicrobials against this periodontal pathogen.

Publications

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Bostanci N (2009) Expression and regulation of the NALP3 inflammasome complex in periodontal diseases. in Clinical and experimental immunology

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Bostanci N (2009) Porphyromonas gingivalis stimulates TACE production by T cells. in Oral microbiology and immunology

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Haurat M (2011) Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles in Journal of Biological Chemistry

 
Description NIH R01 project grant
Amount $1,100,000 (USD)
Organisation National Institutes of Health (NIH) 
Sector Public
Country United States
Start 03/2007 
End 03/2011
 
Title Range of isogenic mutants/complemented strains in P. gingivalis 
Description Targeted mutants in P. gingivalis which a well characterised phenotype 
Type Of Material Cell line 
Year Produced 2007 
Provided To Others? Yes  
Impact JoSelective sorting of cargo proteins into bacterial membrane vesicles. Haurat MF, Aduse-Opoku J, Rangarajan M, Dorobantu L, Gray MR, Curtis MA, Feldman MF. J Biol Chem. 2010 Nov 5. [Epub ahead of print] PMID: 21056982 int publication with authors in Canada 
URL http://europepmc.org/abstract/MED/21056982
 
Description LPS and vesicle membrane protein sorting 
Organisation University of Alberta
Department Department of Chemical and Materials Engineering
Country Canada 
Sector Academic/University 
PI Contribution Gene identification and mutagenesis in P. gingivalis and mutant phenotype investigations
Collaborator Contribution The Alberta group have a long track record in the molecular mechanism of LPS biosynthesis in E. coli. Expertise in this area facilitated understanding of the glycosylation systems and polysaccharide formation in P. gingivalis - an oral pathogen - the subject of this award.
Impact Selective sorting of cargo proteins into bacterial membrane vesicles. Haurat MF, Aduse-Opoku J, Rangarajan M, Dorobantu L, Gray MR, Curtis MA, Feldman MF. J Biol Chem. 2010 Nov 5. [Epub ahead of print] PMID: 21056982
Start Year 2007
 
Description Seminars to school children and teachers 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Schools
Results and Impact As part of the Centre of the Cell public engagement facility in the Blizard Building I meet with School groups of approxiamtely 20 6th formers and teachers to explain the importance of bacteria in the development of oral disease. The talks are aimed at increasing understanding of science and also development of careers in science and medicine.

No hard data yet although we are continuously monitoring the visits of children to the Centree of the Cell which attracted over 12,000 visits in 2009/10.
Year(s) Of Engagement Activity 2007,2008,2009,2010