title: Genomic island 2 of Brucella melitensis is a major virulence determinant: functional analyses of genomic islands.
abstract: Brucella genomic islands (GIs) share similarities in their genomic organization to pathogenicity islands from other bacteria and are likely acquired by lateral gene transfer. Here, we report the identification of a GI that is important for the pathogenicity of Brucella melitensis. The deletion of GI-1, GI-5, or GI-6 did not affect bacterial growth in macrophages as well as their virulence in interferon regulatory factor 1-deficient (IRF-1(-/-)) mice, suggesting that these islands do not contribute to Brucella virulence. However, the deletion of GI-2 resulted in the attenuation of bacterial growth in macrophages and virulence in IRF-1(-/-) mice. The GI-2 mutant also displayed a rough lipopolysaccharide (LPS) phenotype indicated by acriflavin agglutination, suggesting that in vitro and in vivo attenuation is a result of LPS alteration. Further, systematic analysis of the entire GI-2 revealed two open reading frames (ORFs), BMEI0997 and I0998, that encode hypothetical sugar transferases and contribute to LPS alteration, as the deletion of either of these ORFs resulted in a rough phenotype similar to that of the GI-2 mutant. Complementation analyses indicated that in addition to I0997 and I0998, I0999 is required to restore the smooth LPS in the GI-2 mutant as well as its full in vitro and in vivo virulence. The I0999 sequence analysis suggested that it might function as a transporter to help facilitate the transport or linking of the O antigen to the LPS. Our study also indicated that the rough LPS resulting from the GI-2 deletion may affect pathogen-associated molecular pattern recognition by Toll-like receptors.
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH 44691, USA. rajashekara.2@osu.edu
authors.forename: Gireesh
authors.initials: G
authors.lastname: Rajashekara
authors.forename: Jill
authors.initials: J
authors.lastname: Covert
authors.forename: Erik
authors.initials: E
authors.lastname: Petersen
authors.forename: Linda
authors.initials: L
authors.lastname: Eskra
authors.forename: Gary
authors.initials: G
authors.lastname: Splitter
mesh_terms: D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017347:Brucella melitensis / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity* / Q000502:physiology; D002006:Brucellosis / Q000382:microbiology*; D002460:Cell Line; D005260:Female; D016680:Genome, Bacterial; D044404:Genomic Islands; D006801:Humans; D008070:Lipopolysaccharides / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D015398:Signal Transduction; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008070:Lipopolysaccharides; C419843:TLR protein, bacteria; D037521:Virulence Factors