An essential virulence protein of Brucella abortus, VirB4, requires an intact nucleoside-triphosphate-binding domain.
Brucella abortus is a facultative intracellular bacterium capable of surviving inside macrophages. The VirB complex, which is highly similar to conjugative DNA transfer apparatuses, is required for intracellular replication. A conserved NTP-binding domain in VirB4 suggests that one or both proteins couple energy by NTP hydrolysis to transport of putative effector molecule(s). Here it is shown that a mutant strain of B. abortus that contains an in-frame deletion in virB4 is unable to replicate in macrophages and survives in mice. Intracellular replication and virulence in mice are fully restored by expressing virB4 in trans, indicating that VirB4 is essential for intracellular replication and virulence in mice. An alteration within the NTP-binding region of VirB4 by site-directed mutagenesis abolished complementation of a virB4 mutant, demonstrating that an intact NTP-binding domain is critical for VirB4 function. Intracellular replication was inhibited in wild-type B. abortus after introducing a plasmid expressing a mutant VirB4 altered in the NTP-binding region. The dominant negative phenotype suggests that VirB4 either functions as a multimer or interacts with some other component(s) necessary for intracellular replication. Wild-type B. abortus-containing phagosomes lack the glycoprotein LAMP-1, which is an indicator of the normal endocytic pathway. Mutant strains were found in phagosomes that co-localized with LAMP-1, indicating that VirB4 containing the intact NTP-binding region is essential for evasion of fusion with lysosomes.
Watarai
Masahisa
M
Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan1.
Makino
Sou-Ichi
SI
Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan1.
Shirahata
Toshikazu
T
Department of Veterinary Microbiology, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan1.
D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D002003:Brucella abortus / Q000235:genetics / Q000254:growth & development / Q000378:metabolism* / Q000472:pathogenicity*; D018929:Cell Culture Techniques; D051907:Lysosomal Membrane Proteins; D008247:Lysosomes / Q000378:metabolism; D008264:Macrophages / Q000166:cytology / Q000382:microbiology*; D008562:Membrane Glycoproteins / Q000032:analysis; D051379:Mice; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D009154:Mutation; D009711:Nucleotides / Q000378:metabolism*; D010587:Phagocytosis; D010588:Phagosomes / Q000378:metabolism; D010641:Phenotype; D013329:Structure-Activity Relationship; D014774:Virulence; D037521:Virulence Factors
D015703:Antigens, CD; D001426:Bacterial Proteins; D051907:Lysosomal Membrane Proteins; D008562:Membrane Glycoproteins; D009711:Nucleotides; D037521:Virulence Factors