Altering the Length of the Lipopolysaccharide O Antigen Has an Impact on the Interaction ofSalmonella entericaSerovar Typhimurium with Macrophages and Complement

Abstract

A panel of isogenic Salmonella enterica serovar Typhimurium strains that vary only in the length of the O antigen was constructed through complementation of a wzz double mutant (displaying unregulated O-antigen length) with one of two homologous (wzz ST and wzz fepE ) or three heterologous (wzz O139 of Vibrio cholerae and wzz SF and wzz pHS-2 of Shigella flexneri) wzz genes. Each gene was functional in the S. enterica serovar Typhimurium host and specified production of O-antigen polymers with lengths typical of those synthesized by the donor bacteria (ranging from 2 to >100 O-antigen repeat units). By use of this panel of strains, it was found that O-antigen length influences invasion/uptake by macrophage cells; this is the first time this has been shown with Salmonella. O-antigen length was confirmed to be related to complement resistance, with a minimum protective length of >4 and <15 repeat units. O antigen of 16 to 35 repeat units was found to activate complement more efficiently than other lengths, but this was unrelated to complement resistance. No evidence was found to suggest that modifying the length of the O-antigen polymer affected expression of the O1, O4, or O5 antigenic factors.The lipopolysaccharide (LPS) of smooth gram-negative bacteria comprises three components-lipid A, the core oligosaccharide, and O antigen (Oag). Oag is a polymer of a sugar repeat unit (RU); the numbers of RUs attached to lipid A-core are clustered around a modal value determined by Wzz proteins (3, 26). Each wzz gene specifies synthesis of an Oag of a characteristic length. Salmonella enterica serovar Typhimurium possesses two Wzz proteins: Wzz ST , which confers a modal range of 16 to 35 RUs, and Wzz fepE , which confers a very long modal length estimated to be Ͼ100 RUs (20). Mutation of both of the corresponding genes resulted in a smooth, unregulated Oag phenotype characterized by predominantly shortlength Oag chains and significantly compromised virulence and complement resistance (20,21).The identification of wzz genes in many pathogenic bacterial species (18) suggests that the control of Oag length confers distinct advantages. Two functional wzz genes have been identified in Shigella flexneri (11), with the product of each wzz gene fulfilling a distinct role in pathogenesis (11,17,29). The interest in the role of S. enterica serovar Typhimurium Oag length is further increased by recent findings that this property is under regulatory control in vivo (2, 15, 21).Several approaches have been used to investigate the importance of Salmonella Oag length for pathogenic potential, but each has had its limitations. This study describes a new approach through complementation of a Salmonella enterica serovar Typhimurium C5 double wzz mutant with heterologous wzz genes. The resulting panel of strains was used to examine new and revisit previously investigated relationships between Oag length and virulence. Our results indicate that Oag modal length impacts upon S. enterica serovar Typhimurium virulence-related properties.Constru...

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

“…typhimurium LPS is also a major virulence factor with the length of the O-antigen contributing to survival in the host (40,55) and has been shown to play a role in adherence (56). We found that host blood group glycans could inhibit the association of S. typhimurium and Caco-2 cells when used at 10× the K D of the LPS:glycan interaction with 50% decreases in adherence noted.…”

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].…”

“…Additionally, the PCP1 group is divided into PCP1a and PCP1b, distinguishing the WzzB proteins (PCP1a) that operate on Oag from the WzzE proteins (PCP1b) that regulate the length of ECA [1]. The most striking effects on polysaccharide length have been visualized for the PCP1a proteins in Oag biosynthesis because Oag ladders can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) such that the number of individual sugar repeat units can be counted, thus, enabling subtle differences in the degree of polymerization to be distinguished [7]. By contrast, the PCP2 and PCP3 proteins influence large molecular weight capsular polysaccharides and exopolysaccharides that currently cannot be resolved to the same level of clarity.…”

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

“…Any model also needs to take into account the interchangeability of Wzz proteins (Klee et al, 1997;Murray et al, 2006). Specific functional domains have not been identified in Wzz so far, and it is apparent from in vivo genetic studies that individual residues throughout Wzz are important (Daniels & Morona, 1999;Franco et al, 1998;Klee et al, 1997).…”