Salmonella enterica serotype Typhimurium DT104 ArtA-dependent modification of pertussis toxin-sensitive G proteins in the presence of [32P]NAD.
Salmonella enterica serotype Typhimurium (S. Typhimurium) definitive phage type (DT) 104 has become a widespread cause of human and other animal infections worldwide. The severity of clinical illness in S. Typhimurium DT104 outbreaks suggests that this strain possesses enhanced virulence. ArtA and ArtB - encoded by a prophage in S. Typhimurium DT104 - are homologues of components of pertussis toxin (PTX), including its ADP-ribosyltransferase subunit. Here, we show that exposing DT104 to mitomycin C, a DNA-damaging agent, induced production of prophage-encoded ArtA/ArtB. Pertussis-sensitive G proteins were labelled in the presence of [(32)P]NAD and ArtA, and the label was released by HgCl(2), which is known to cleave cysteine-ADP-ribose bonds. ADP-dependent modification of G proteins was markedly reduced in in vitro-synthesized ArtA(6Arg-Ala) and ArtA(115Glu-Ala), in which alanine was substituted for the conserved arginine at position 6 (necessary for NAD binding) and the predicted catalytic glutamate at position 115, respectively. A cellular ADP-ribosylation assay and two-dimensional electrophoresis showed that ArtA- and PTX-induced ADP-ribosylation in Chinese hamster ovary (CHO) cells occur with the same type of G proteins. Furthermore, exposing CHO cells to the ArtA/ArtB-containing culture supernatant of DT104 resulted in a clustered growth pattern, as is observed in PTX-exposed CHO cells. Hydrogen peroxide, an oxidative stressor, also induced ArtA/ArtB production, suggesting that these agents induce in vivo synthesis of ArtA/ArtB. These results, taken together, suggest that ArtA/ArtB is an active toxin similar to PTX.
Uchida
Ikuo
I
United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi 501-1193, Japan.|Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan.
Ishihara
Ryoko
R
Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan.
Tanaka
Kiyoshi
K
Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan.
Hata
Eiji
E
Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan.
Makino
Sou-Ichi
SI
Obihiro University of Agriculture and Veterinary Medicine, Inada, Obihiro 080-8555, Japan.
Kanno
Toru
T
United Graduate School of Veterinary Sciences, Gifu University, 1-1 Yanagido, Gifu-shi 501-1193, Japan.|Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan.
Hatama
Shinichi
S
Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan.
Kishima
Masato
M
National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan.
Akiba
Masato
M
National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan.
Watanabe
Atsushi
A
Hokkaido Research Station, National Institute of Animal Health, Hitsujigaoka-4, Toyohira, Sapporo 062-0045, Japan.
Kubota
Takayuki
T
National Institute of Animal Health, Tsukuba, Ibaraki 305-0856, Japan.
D036002:ADP Ribose Transferases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006861:Hydrogen Peroxide; D016685:Mitomycin; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism*; D037342:Pertussis Toxin / Q000378:metabolism; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism* / Q000472:pathogenicity; D016415:Sequence Alignment; D014774:Virulence
D001426:Bacterial Proteins; D009243:NAD; D016685:Mitomycin; D006861:Hydrogen Peroxide; D036002:ADP Ribose Transferases; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins