The use of high-throughput DNA sequencing in the investigation of antigenic variation: application to Neisseria species.
Antigenic variation occurs in a broad range of species. This process resembles gene conversion in that variant DNA is unidirectionally transferred from partial gene copies (or silent loci) into an expression locus. Previous studies of antigenic variation have involved the amplification and sequencing of individual genes from hundreds of colonies. Using the pilE gene from Neisseria gonorrhoeae we have demonstrated that it is possible to use PCR amplification, followed by high-throughput DNA sequencing and a novel assembly process, to detect individual antigenic variation events. The ability to detect these events was much greater than has previously been possible. In N. gonorrhoeae most silent loci contain multiple partial gene copies. Here we show that there is a bias towards using the copy at the 3' end of the silent loci (copy 1) as the donor sequence. The pilE gene of N. gonorrhoeae and some strains of Neisseria meningitidis encode class I pilin, but strains of N. meningitidis from clonal complexes 8 and 11 encode a class II pilin. We have confirmed that the class II pili of meningococcal strain FAM18 (clonal complex 11) are non-variable, and this is also true for the class II pili of strain NMB from clonal complex 8. In addition when a gene encoding class I pilin was moved into the meningococcal strain NMB background there was no evidence of antigenic variation. Finally we investigated several members of the opa gene family of N. gonorrhoeae, where it has been suggested that limited variation occurs. Variation was detected in the opaK gene that is located close to pilE, but not at the opaJ gene located elsewhere on the genome. The approach described here promises to dramatically improve studies of the extent and nature of antigenic variation systems in a variety of species.
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
John K
JK
Davies
Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia.
Paul F
PF
Harrison
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Ya-Hsun
YH
Lin
School of Pathology and Laboratory Medicine.
Stephanie
S
Bartley
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Chen Ai
CA
Khoo
Victorian Bioinformatics Consortium, Monash University, Clayton, Victoria, Australia.
Torsten
T
Seemann
Department of Microbiology, Monash University, Clayton, Victoria, Australia.
Catherine S
CS
Ryan
School of Pathology and Laboratory Medicine ; The Marshall Centre for Infectious Diseases, Research and Training, University of Western Australia, Nedlands, Western Australia, Australia ; Telethon Institute of Child Health Research, University of Western Australia, Nedlands, Western Australia, Australia.
Charlene M
CM
Kahler
Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois, United States of America.
Stuart A
SA
Hill
D000940:Antigenic Variation; D000942:Antigens, Bacterial / Q000235:genetics* / Q000276:immunology; D019295:Computational Biology; D036781:Fimbriae Proteins / Q000235:genetics / Q000276:immunology; D059014:High-Throughput Nucleotide Sequencing; D009343:Neisseria / Q000145:classification / Q000235:genetics* / Q000276:immunology
D000942:Antigens, Bacterial; D036781:Fimbriae Proteins