Coiled-coil regions play a role in the function of the Shigella flexneri O-antigen chain length regulator WzzpHS2.

Regulation of the length of the O-antigen (Oag) chain attached to LPS in Shigella flexneri is important for virulence and is dependent on the inner-membrane protein Wzz. A lack of high-resolution structural data for Wzz has hampered efforts so far to correlate mutations affecting function of Wzz with structure and describe a mechanism for chain length regulation. Here we have used secondary structure prediction to show that the periplasmic domain of the Wzz(pHS2) protein has three regions of significant coiled-coil (CC) potential, two of which lie within an extended helical region. We describe here the first site-directed mutagenesis study to investigate the role of individual predicted CC regions (CCRs) in Wzz function and oligomerization. We found that CCRs 2 and 3 are necessary for wild-type Oag chain length regulation by Wzz(pHS2). The in vivo cross-linking profile of mutants affected in the three CCRs was not altered, indicating that individually each CCR is not required for oligomerization. Interestingly, the CCR3 mutation resulted in a temperature-sensitive phenotype and an inhibitory effect on Oag polymerization. Analysis of Wzz(pHS2) and the mutant constructs in a S. flexneri degP mutant showed that DegP did not affect the function of wild-type Wzz(pHS2) but its presence influenced the phenotype of the Wzz(pHS2) CCR3 mutant. Additionally, the phenotype of the Wzz(pHS2) CCR3 mutant was suppressed by a cis mutation near the putative cytoplasmic C-terminus of Wzz(pHS2).

Bacterial polysaccharide co-polymerases share a common framework for control of polymer length.

The chain length distribution of complex polysaccharides present on the bacterial surface is determined by polysaccharide co-polymerases (PCPs) anchored in the inner membrane. We report crystal structures of the periplasmic domains of three PCPs that impart substantially different chain length distributions to surface polysaccharides. Despite very low sequence similarities, they have a common protomer structure with a long central alpha-helix extending 100 A into the periplasm. The protomers self-assemble into bell-shaped oligomers of variable sizes, with a large internal cavity. Electron microscopy shows that one of the full-length PCPs has a similar organization as that observed in the crystal for its periplasmic domain alone. Functional studies suggest that the top of the PCP oligomers is an important region for determining polysaccharide modal length. These structures provide a detailed view of components of the bacterial polysaccharide assembly machinery.

The actin-based motility defect of a Shigella flexneri rmlD rough LPS mutant is not due to loss of IcsA polarity.

Shigella flexneri requires the outer membrane protein IcsA(VirG) and lipopolysaccharide (LPS) for efficient actin-based motility (ABM) within mammalian cells which is essential for virulence. Wild type strains of S. flexneri 2a such as 2457T have smooth LPS whose O antigen (Oag) chains have two modal lengths and IcsA predominantly located at one pole on their cell surface. In contrast, rough LPS mutants lack Oag chains, have IcsA on lateral and polar regions of the cell surface, and are defective for ABM. In this study we directly compared the phenotype of a S. flexneri producing non-IcsP/SopA cleavable IcsA (IcsA*) with that of a rough LPS mutant. IcsA* was located on lateral and polar regions of smooth LPS bacteria, and was fully functional in ABM assays (HeLa cell monolayer plaque and F-actin comet tail formation) which contrasts with the R-LPS phenotype. This indicates that loss of polar IcsA localisation in R-LPS mutants is unrelated to their ABM defect, and suggests that Oag may directly contribute to IcsA-mediated ABM.

Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri.

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.

Multicopy icsA is able to suppress the virulence defect caused by the wzz(SF) mutation in Shigella flexneri.

The lipopolysaccharides (LPS) of Shigella flexneri are important for virulence and their O antigen (Oag) polysaccharide chains affect IcsA (VirG)-mediated actin-based motility (ABM) within mammalian cells. S. flexneri 2a 2457T has smooth LPS whose Oag chains have two modal lengths (short (S)-type and very long (VL)-type), and has IcsA predominantly located at one pole on its cell surface. A S. flexneri 2457T wzz(SF) mutant (RMA696) has VL-type Oag but not S-type Oag chains, less IcsA detectable by immunofluorescence on its cell surface, reduced virulence and defective ABM. Introduction of a plasmid encoding IcsA into S. flexneri wzz(SF) showed that multicopy icsA could suppress the virulence defects (Sereny reaction, HeLa cell monolayer plaquing, and F-actin comet tail formation) caused by the wzz(SF) mutation suggesting that the VL-type Oag chains were masking IcsA and limiting the amount available to initiate ABM.

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

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.

The tailspike protein of Shigella phage Sf6. A structural homolog of Salmonella phage P22 tailspike protein without sequence similarity in the beta-helix domain.

Bacteriophage Sf6 tailspike protein is functionally equivalent to the well characterized tailspike of Salmonella phage P22, mediating attachment of the viral particle to host cell-surface polysaccharide. However, there is significant sequence similarity between the two 70-kDa polypeptides only in the N-terminal putative capsid-binding domains. The major, central part of P22 tailspike protein, which forms a parallel beta-helix and is responsible for saccharide binding and hydrolysis, lacks detectable sequence homology to the Sf6 protein. After recombinant expression in Escherichia coli as a soluble protein, the Sf6 protein was purified to homogeneity. As shown by circular dichroism and Fourier transform infrared spectroscopy, the secondary structure contents of Sf6 and P22 tailspike proteins are very similar. Both tailspikes are thermostable homotrimers and resist denaturation by SDS at room temperature. The specific endorhamnosidase activities of Sf6 tailspike protein toward fluorescence-labeled dodeca-, deca-, and octasaccharide fragments of Shigella O-antigen suggest a similar active site topology of both proteins. Upon deletion of the N-terminal putative capsid-binding domain, the protein still forms a thermostable, SDS-resistant trimer that has been crystallized. The observations strongly suggest that the tailspike of phage Sf6 is a trimeric parallel beta-helix protein with high structural similarity to its functional homolog from phage P22.