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Campylobacter-A Mystery Half Solved
The present article summarises the key talks presented by Dr. Brendan Wren in the seminar on “Campylobacter- A Tale of two glycosylation systems and tales of unexpected” held at the Institute of food research, Norwich. The major objective of his lecture was to determine the genetic basis by which C.jejuni causes gastrointestinal disease and subsequent neurological disorders. This review outlines the genetic and structural basis of the O and N-linked glycosylation system found in the enteropathogenic bacterium Campylobacter jejuni. It also elucidates the usage of this organism as a model to understand glycoprotein biosynthesis and its function in vivo.
Campylobacter belongs to the rRNA superfamily VI of class proteobacteria. It is found in animal faeces and is a non spore forming bacteria . It is one of the most common causes of human gastroenteritis leading to food poisoning and diarrhoea. C.jejuni is estimated to cause 5-14% diarrhoea worldwide. C.jejuni causes the disease called Campylobacteriosis and is also known as campylobacter enteritis or gastroenteritis. It has been reported that some cases of Gullain Barre syndrome are due to infection by Campylobacter jejuni.
C.jejuni is a commensal in birds and is well adapted in growing at body temperature of birds. Morphologically, they are small, thin (0.2-0.5um X 0.5-5.0um), helically curved gram negative cells (figure 1). Being a microaerophilic organism it requires reduced levels of oxygen.
Presence of Glycosylation in Prokaryotes
It has been considered that the process of glycosylation predominantly occurs in eukaryotes due to the attachment of glycan structures to proteins at an Asn-Xaa-Ser/Thr consensus or at Ser/Thr residues . Dr Brendan and his colleagues have disproved the notion that bacteria cannot glycosylate proteins . Although, there is still very little knowledge about the structures of linked glycans, mechanism behind the glycosylation process and its significance in biological organisms shows that most prokaryotes and arachea have glycoprotein. In eukaryotes, the N-linked glycosylation systems is the most frequent protein modification wherein a preassembled oligosaccharide is transferred from dolicyl phosphate to aspargine residues of nascent polypeptide chains. This transfer is mediated by the enzyme oligosaccharyltransferase. Dr. Brendan and his research team had found that C.jejuni was an exception with respect to other prokaryotes as the pgl gene cluster encodes a protein similar to the eukaryotic glycosyltransferases.
Dr. Brendan emphasised that Campylobacter jejuni had two glycosylation pathways i.e. the N and O-linked pathways. This report thus highlights the genetic and structural components of the two glycosylation pathways found in campylobacter species. Logan et al in 1989 had found out that the flagellin was post transcriptionally modified.
The glycan in the N-linked pathway is a heptasaccharide whereas it is a mono or disaccharide in the O-linked system. There is no modification in structure of the former whereas the latter consists of dihydroxypropionyl, acetamidino and deoxypentose modifications .
As evident from the talk, campylobacter flagellar proteins consist of O-linked carbohydrates. The C.jejuni flagellin glycosylation locus NCTC 11168 has ~50 genes most of which encode flagellin structural proteins FlaA and FlaB. These genes undergo intergenic recombination further adding to their virulence .
The enzyme PglB is the first example of N-linked oligosaccharyltransferase and is similar to the STT3 complex of Saccharomyces cerevisiae. Hence, presence of STT3 homologue in pgl locus indicates similarity of the N linked pathway in prokaryotes and higher organisms.
Importance of protein glycosylation in Campylobacter
The O-linked glycosylation causes structural diversity of the flagellin protein thus enabling antigenic variation in the surface exposed and immunodominant protein. This could be a reason for its immune invasion in avians whereas the disruption of the N-linked glycosyalation reduces the capacity of cells to invade, attach and colonize in the intestine of the chicken and mice. As evident from the talk, it is hypothesised that N-linked glycosylation occurs extracellularly and thus may protect against proteolytic cleavage or as cellular sorting signal for glycoproteins.
Dr. Brendan and his team had reported the presence of genetic determinants to produce a capsular polysaccharide by using a cationic dye Alcian blue and demonstrating presence of polysaccharide capsule retained in coccoid form under the electron microscope. They had also injected healthy Gulleria maggots with C.jejuni and found that the maggots turned black in colour and died. On further study it was found that the reason of death of the maggots was due to presence of a phosphoramide group. Most of the structural information came from mass spectrometry and NMR imaging and suggest that >8% proteins found in Campylobacter encode glycostructures and hence it is also called a “hyperglycaemic bug”.
Dr Brendan had evidently described an unexpected finding which was an outcome of sequencing of entire C.jejuni genome. Their team had reconstituted the C.jejuni glycosylation pathway in E.coli. In presence of Pgl gene cluster, acrA was over expressed in E.coli which in turn had produced two immune reactive proteins. Thus, the production of recombinant glycoproteins in E.coli was possible for the first time.
An important aim of the seminar was to identify and develop glycoconjugate human vaccines. A few examples of such vaccines which are currently available for human use include H.influenzae, N.meningitidis (except type B), S.pneumoniae, triple poultry vaccine, traveller's vaccine etc. By using chemical couplings other novel vaccines can be produced. For example, Burkholderia pseudomallei capsule can be coupled to Bps protein antigen and the Francisella tularensis LPS can be coupled to Ft protein antigen. Hence, this development in the field of glyco-engineering of infectious diseases can lead to various protein-glycan combinations and expediting the manufacture of such vaccines . This has been made possible by the Protein Glycan Coupling Technology (PGCT) and has alleviated the need for chemical coupling of the polysaccharide to the protein carrier. Hence, this new technology has shown spin off way for the development of new vaccines through engineering of different glycans and might find a way to combat the present difficulties in treatment for infections caused by C.jejuni.
The two glycosylation systems present in Campylobacter jejuni namely: O-linked and N-linked pathways are independent of each other. The O-linked system is found in almost all prokaryotes and is diverse whereas the other system is highly conserved. The N-linked glycosyaltion in this Gram negative bacterium proved to be an efficient model to study the same pathway in higher organisms. Hence, further knowledge about these two pathways can lead to development of novel strategies for immunotherapy in the future by designing glycan structures on immunodominant Campylobacter flagellin protein.
Dr. Brendan has started his career as a physical chemist at Leicester University where he was engaged in studying effect of ionizing radiation on DNA. He latter on switched on to more bio oriented research work on single cell bacteria which landed him to the Medical Microbiology Department at St. Bartholomew Hospital, London.
After his stint at St. Bartholomew Hospital, Dr. Brendan Wren had joined the London School of Hygiene and Tropical Medicine with his research team in 1999. His basic interest encompasses the areas of genetics, molecular biology and clinical pathology. Dr. Wren has been primarily engaged in the research area to understand the molecular mechanism how bacteria is responsible for several disease pattern and to understand the genetic basis for such diseases.
He is currently doing research in the following areas:
- Glycosylation in bacterial pathogens, glycoprospecting and glycoengineering
- Comparative phylogenomics and the evolution of bacterial virulence
- Systems biology of host pathogen interactions
Dr Brendan Wren has been studying the role of pathogens like Campylobacter jejuni, Helicobacter pylori, Clostridium difficle, Burkholderia pseudomallei and the enteropathogenic Yersinia. His pioneering work on the above mentioned pathogens allowed him to publish his break through findings in peer journals like Nature, Proc. Natl. Acad. Sci, USA, Molecular Microbiology and many more top ranked international journals.