Flagellin A is essential for the virulence of Vibrio anguillarum.
A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.
Milton
D L
DL
Department of Cell and Molecular Biology, Umeå University, Sweden.
O'Toole
R
R
Horstedt
P
P
Wolf-Watz
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000942:Antigens, Bacterial; D001483:Base Sequence; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004195:Disease Models, Animal; D005407:Flagella / Q000235:genetics* / Q000276:immunology / Q000648:ultrastructure; D005408:Flagellin / Q000235:genetics*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000235:genetics* / Q000276:immunology / Q000472:pathogenicity* / Q000648:ultrastructure; D014735:Vibrio Infections / Q000662:veterinary
D000942:Antigens, Bacterial; D005408:Flagellin; C067138:flaA protein, bacteria