Effect of mutations in Shigella flexneri chromosomal and plasmid‐encoded lipopolysaccharide genes on invasion and serum resistance

Abstract

SummaryThis study shows that both length and distribution of lipopolysaccharide (LPS) are important for Shigella flexneri invasion and virulence. Mutants were generated in the chromosomal LPS synthesis genes rfa, rfb, and rol, and in a plasmid-encoded O-antigen chain-length regulator, cld pHS-2 . LPS analysis showed that mutations in rfb genes and in a candidate rfaL gene either eliminated the entire O-antigen side chains or 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 had different distributions of O-antigen chain lengths. The results of tissue-culture cell invasion and plaque assays, the Seré ny test, and serum-sensitivity assay suggested roles for the different LPS synthesis genes in bacterial survival and virulence; rfaL, rfaX and rfb loci are required for serum resistance and intercellular spread, but not for invasion; cld pHS-2 is required for resistance to serum killing and for full inflammation in the Seré ny test, but not for invasion or intercellular spread, while rol is required for normal invasiveness and plaque 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 and virulence.

Relationship between O-antigen chain length and resistance to colicin E2 in Shigella flexneri

“…Whilst LPS Oag has previously been shown to shield OM proteins from colicin in E. coli (van der Ley et al, 1986), the impact of Oag chain length against colicins has not been investigated. We propose here that Shigella flexneri regulation of S-type Oag chains is required for conferring resistance to colicin, whilst Shigella flexneri regulation of the VL-type Oag chains is required for conferring resistance to complement (Hong & Payne, 1997), as shown by our serum killing assay (Fig. 5).…”

“…In Shigella flexneri, S-type Oag chains have been shown to contribute to IcsA-mediated actin-based motility and affect virulence (Van Den Bosch et al, 1997), whilst loss of the VL-type Oag chains in Shigella flexneri has been shown to enhance bacterial sensitivity to complement (Hong & Payne, 1997). In Pseudomonas aeruginosa, absence of LPS with long (L)-type Oag chains resulted in greater sensitivity to complement killing and reduced virulence in mice (Kintz et al, 2008).…”

Glycan:glycan interactions: High affinity biomolecular interactions that can mediate binding of pathogenic bacteria to host cells

“…S. flexneri LPS O-antigen is a virulence factor whose chain length affects both serum resistance and the function of surface virulence factors; a role for LPS in cell invasion has been reported (11,(44)(45)(46). S. flexneri infection has also been blocked by both the addition of free LPS and treatment with anti-LPS antibodies (11,14,21).…”

Sequence-structure relationships in polysaccharide co-polymerase (PCP) proteins

“…Regulation of surface polysaccharide length is an important adaptation that leads to optimal survival and virulence of pathogens [6][7][8]. During LPS Oag biosynthesis, Wzz proteins control Oag modal length distribution through two proposed mechanisms: as a molecular timer of Oag polymerization [9] or in an organizational manner to determine the crucial ratio of the polymerase Wzy to the Oag ligase WaaL [10].…”