IDENTIFICATION OF A NOVEL VIRULENCE GENE, VIRA, ON THE LARGE PLASMID OF SHIGELLA, INVOLVED IN INVASION AND INTERCELLULAR SPREADING

A novel virulence gene (virA) was identified upstream of the virG gene on the large plasmid of Shigella flexneri 2a YSH6000. Characterizatio n of virA mutants infecting MK2 epithelial cell monolayers revealed th at their invasive capacity was decreased to less than one fifth of the wild-type level. Nevertheless, the bacteria were capable of expressin g and secreting IpaB, IpaC and IpaD proteins. The virA mutants were al so impaired in their ability to spread intercellularly, since the bact eria gave rise to a small number of foci in a focus-plaque-forming tes t with MK2 cells. Although virG expression was slightly decreased in t he virA mutants, introduction of a cloned virG gene into a virA mutant , N1945, failed to restore spreading ability. Although, introduction o f a cloned virA gene into N1945 restored invasiveness and spreading ab ility, the reduced virG transcription level was not affected, indicati ng that the reduced virG expression in virA mutants does not play a ma jor role in defective intercellular spreading. The nucleotide sequence of the virA region revealed that the virA gene was located 528 bp ups tream of the virG gene, in the opposite orientation. The deduced amino acid sequence of the VirA protein indicated a 44.7 kDa protein with n o homology to known proteins. The VirA protein was secreted into the c ulture supernatant, a process that required the Mxi and Spa loci, The expression of virA was under the control of the virB gene, the positiv e regulator of the ipa, mxi and spa operons. These results indicate th at virA is a new member of the invasion regulon directed by virB and t hat the VirA function is involved in invasion and intercellular spread ing.