EFFECT OF MUTATIONS IN SHIGELLA-FLEXNERI CHROMOSOMAL AND PLASMID-ENCODED LIPOPOLYSACCHARIDE GENES ON INVASION AND SERUM RESISTANCE

This study shows that both length and distribution of lipopolysacchari de (LPS) are important for Shigella flexneri invasion and virulence. M utants were generated in the chromosomal LPS synthesis genes rfa, rfb, and rol, and in a plasmid-encoded O-antigen chain-length regulator, c ld(pHS-2). LPS analysis showed that mutations in rfb genes and in a ca ndidate rfaL gene either eliminated the entire O-antigen side chains o r produced chains of greatly reduced length. Mutation in a previously unidentified gene, rfaX, affected the LPS core region and resulted in reduced amounts of O-antigen. Mutants defective in cld(pHS-2) Or rol h ad different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Sereny test, and s erum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci ar e required for serum resistance and intercellular spread, but not for invasion; cld(pHS-2) is required for resistance to serum killing and f or full inflammation in the Sereny test, but not for invasion or inter cellular spread, while rol is required for normal invasiveness and pla que formation, but not for serum resistance. Thus, O-antigen synthesis and chain-length regulation genes encoded on both the chromosome and the small plasmid pHS-2 play important roles in S. flexneri invasion a nd virulence.