Genetic modulation of Shigella flexneri 2a lipopolysaccharide O antigen modal chain length reveals that it has been optimized for virulence

Abstract

The lipopolysaccharide (LPS) molecules of Shigella flexneri 2a have O antigen (Oag) polysaccharides with two modal chain length distributions. The chromosomal wzz SF gene results in short (S) type Oag chains [11-17 Oag repeat units (RUs)], and the pHS-2 plasmid-located wzz pHS2 gene results in very long (VL) type Oag chains (>90 Oag RUs). S. flexneri wzz SF mutants are unable to form plaques on HeLa cell monolayers and F-actin comet tails, indicating that IcsA/ VirG function in actin-based motility (ABM) is defective. An S. flexneri wzz SF wzz pHS2 double mutant had LPS with relatively short, random length Oag chains and, paradoxically, was able to form plaques and F-actin comet tails. The influence of Oag modal chain length distribution on virulence and related properties was investigated using complementation with different wzz genes. Wzz O139 from Vibrio cholerae O139 and Wzz ST from Salmonella enterica serovar Typhimurium were fully functional in Shigella flexneri, resulting in LPS with either very short (VS) type Oag chains (2-7 Oag RUs) or long (L) type Oag chains (19-35 RUs), respectively. In the absence of VL-type Oag chains, the VS-, S-and L-type Oag chains were permissive for plaque and F-actin comet tail formation. However, in the presence of LPS with VL-type Oag chains, the VS-and S-type Oag chains but not the L-type Oag chains were permissive for plaque and F-actin comet tail formation. These data, and the results of a previous investigation, show that IcsA function in ABM requires LPS Oag chains with at least two but less than 18 RUs when VL-type Oag chains are co-expressed on the cell surface. However, in the absence of the VL-type Oag chains, LPS Oag chains with at least two but less than 90 RUs are able to support IcsA function in ABM. Indirect immunofluorescence staining of IcsA on the cell surface of the S. flexneri strains did not correlate with the observed effect of Oag chain length on plaque and F-actin comet tail formation. However, when intracellular bacteria lacking VL-type Oag chains were examined, an inverse correlation between Oag modal chain length and detection of IcsA was observed, i.e. staining decreased with increased modal length. It is hypothesized that Oag chains can mask IcsA and interfere with its function in ABM, and a model is presented to explain how LPS Oag and IcsA may interact. It is suggested that S. flexneri 2a has evolved to synthesize LPS with two Oag modal chain lengths, as S-type Oag chains allow IcsA to function in ABM in the presence of VL-type Oag chains that confer resistance to serum.

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).…”

Substrate binding by a bacterial ABC transporter involved in polysaccharide export

“…It is now clear that the terminal modifications are critical for polymer binding by C-Wzt providing insight into the mechanism coordinating polymer biosynthesis and export. The overall process generates a defined O-PS chain length, which is critical for the function of O-PSs in virulence (12). However, it is still unclear how WbdD activity is itself regulated.…”

Nonreducing Terminal Modifications Determine the Chain Length of Polymannose O Antigens of Escherichia coli and Couple Chain Termination to Polymer Export via an ATP-binding Cassette Transporter

“…The involvement of Wzz (formerly Rol or Cld) in regulating the chain length of O-PS synthesized by Wzy-dependent pathways is well accepted. Mutations in wzz make Salmonella enterica serovar Typhimurium (11), E. coli O75 (36), and Shigella dysenteriae serotype 2a (37) sensitive to complement and avirulent. Yersinia enterocolitica O:8 mutants with unregulated chain length are sensitive to killing by polymorphonuclear leukocytes and are attenuated for virulence (38).…”