Disulfide oxidoreductase activity of Shigella flexneri is required for release of Ipa proteins and invasion of epithelial cells.
Secretion of IpaB, IpaC, and IpaD proteins of Shigella flexneri, essential for the invasion of epithelial cells, requires a number of proteins encoded by the spa and mxi loci on the large plasmid. Introduction of dsbA::Tn5 into S.flexneri from Escherichia coli K-12 reduced invasiveness, which resulted from a decrease in the capacity to release IpaB, IpaC, and IpaD proteins into the external medium. Examination of the surface-presented Ipa proteins of the dsbA mutant, however, revealed Ipa proteins at levels similar to those on wild-type cells. Since the defective phenotype was similar to that of the spa32 mutant of S. flexneri and the Spa32 sequence possessed two Cys residues, the effect of dsbA mutation of the folding structure of Spa32 under reducing conditions and on the surface expression of Spa32 was investigated. The results indicated that Spa32 was a disulfide-containing protein whose correctly folded structure was required for its presentation on the outer membrane. Indeed, replacing either one of the two Cys residues in Spa32 with Ser by site-directed mutagenesis reduced its capacity to release Ipa proteins into the external medium and led to the accumulation of Spa32 protein in the periplasm. These results indicated that the DsbA protein performs an essential function during the invasion of mammalian cells, by facilitating transport of the Spa32 protein across the outer membrane.
Watarai
M
M
Department of Bacteriology, University of Tokyo, Japan.
Tobe
T
T
Yoshikawa
M
M
Sasakawa
C
C
D000818:Animals; D000942:Antigens, Bacterial / Q000096:biosynthesis / Q000378:metabolism*; D017209:Apoptosis; D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine; D004220:Disulfides; D004848:Epithelium / Q000382:microbiology; D005798:Genes, Bacterial; D005838:Genotype; D007535:Isomerases / Q000378:metabolism*; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010084:Oxidation-Reduction; D017354:Point Mutation; D019704:Protein Disulfide-Isomerases; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015183:Restriction Mapping; D012763:Shigella flexneri / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*
D000942:Antigens, Bacterial; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D004220:Disulfides; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C087447:ipaB protein, Shigella; C092342:IpaC protein, Shigella; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D003545:Cysteine