Functional and Structural Characterization of Polysaccharide Co-polymerase Proteins Required for Polymer Export in ATP-binding Cassette Transporter-dependent Capsule Biosynthesis Pathways

Abstract

Neisseria meningitidis serogroup B and Escherichia coli K1 bacteria produce a capsular polysaccharide (CPS) that is composed of ␣2,8-linked polysialic acid (PSA). Biosynthesis of PSA in these bacteria occurs via an ABC (ATP-binding cassette) transporter-dependent pathway. In N. meningitidis, export of PSA to the surface of the bacterium requires two proteins that form an ABC transporter (CtrC and CtrD) and two additional proteins, CtrA and CtrB, that are proposed to form a cell envelope-spanning export complex. CtrA is a member of the outer membrane polysaccharide export (OPX) family of proteins, which are proposed to form a pore to mediate export of CPSs across the outer membrane. CtrB is an inner membrane protein belonging to the polysaccharide co-polymerase (PCP) family. PCP proteins involved in other bacterial polysaccharide assembly systems form structures that extend into the periplasm from the inner membrane. There is currently no structural information available for PCP or OPX proteins involved in an ABC transporter-dependent CPS biosynthesis pathway to support their proposed roles in polysaccharide export. Here, we report cryo-EM images of purified CtrB reconstituted into lipid bilayers. These images contained molecular top and side views of CtrB and showed that it formed a conical oligomer that extended ϳ125 Å from the membrane. This structure is consistent with CtrB functioning as a component of an envelope-spanning complex. Cross-complementation of CtrA and CtrB in E. coli mutants with defects in genes encoding the corresponding PCP and OPX proteins show that PCP-OPX pairs require interactions with their cognate partners to export polysaccharide. These experiments add further support for the model of an ABC transporter-PCP-OPX multiprotein complex that functions to export CPS across the cell envelope. Capsular polysaccharides (CPSs)2 are produced by many bacteria and form a layer (the capsule) that coats the surface of the cell. The capsule helps prevent bacteria from desiccation and is important in infection because it provides protection from the host immune response by interfering with both complement-mediated killing (1-3) and the adhesion of monocytes and neutrophils to the bacteria (4, 5). Consequently, capsules are an important virulence factor for numerous pathogenic bacteria including those that cause disease in livestock, like Pasteurella multocida, and bacteria such as Escherichia coli and Neisseria meningitidis, which cause bacterial meningitis and sepsis in humans (6).CPSs vary significantly in carbohydrate composition and glycosidic linkages. Despite the diversity of structures, the majority of CPSs produced by Gram-negative bacteria are assembled via either an ATP-binding cassette (ABC) transporter-dependent biosynthesis pathway or a Wzy-dependent biosynthesis pathway (for review, see Refs. 7,8). These pathways are fundamentally different in the topology of the polysaccharide polymerization reaction at the inner membrane. In Wzy-dependent pathways, polymer repeat units are assembled at...

Conserved glycolipid termini in capsular polysaccharides synthesized by ATP-binding cassette transporter-dependent pathways in Gram-negative pathogens

“…The higher apparent molecular masses are consistent with previous reports for the corresponding mutants (lipA and lipB) in N. meningitidis (31). Similar effects have also been seen in E. coli strains with mutations affecting later stages of CPS export (kpsE) (32). The reason for this is unknown, but it could reflect unregulated chain extension when synthesis and export are uncoupled.…”

“…To determine whether they were required for synthesizing the glycolipid terminus, ΔkpsC and ΔkpsS mutants were constructed in E. coli K1. As observed previously (32), the mutants are resistant to K1-specific bacteriophage due to a CPS export defect, and each mutant could be complemented in trans, to restore surface CPS (and bacteriophage sensitivity), indicating that the deletions are nonpolar (Fig. S9).…”

KpsC and KpsS are retaining 3-deoxy- d - manno -oct-2-ulosonic acid (Kdo) transferases involved in synthesis of bacterial capsules

“…The PSA from ΔlipAB possessed a significantly higher average apparent molecular mass compared with the parent, suggesting a significantly longer average chain length. A similar effect is also seen in an E. coli ΔkpsE (PCP protein) mutant that cannot export CPS from the periplasm to the surface, so the altered chain lengths may simply reflect disruption of the biosynthesis complex (35). However, with the ΔkpsC, ΔkpsS, and ΔlipAB mutants, the absence of acylation could also influence PAGE migration.…”

“…How the truncations affect the size distribution of CPS is not clear, but variations in the PAGE profiles also occur when the level of KpsC expression is altered (Fig. S2), and in kpsE mutants (35), so the phenotype may just reflect altered stoichiometries in biosynthesis components. In ABC transporterdependent LPS O antigen biosynthesis, glycan chain length is controlled by one of two strategies.…”

“…Bacteriophage K1F-sensitivity assays and immunoblotting were performed as previously described (2,35). The PSA-specific monoclonal antibody mAb 2-2B used in immunoblotting was a gift from M Apicella (University of Iowa, Iowa City, IA).…”