Trapped translocation intermediates establish the route for export of capsular polysaccharides across Escherichia coli outer membranes

Abstract

The outer membrane (OM) of Gram-negative bacteria is designed to exclude potentially harmful molecules. This property presents a challenge for bacteria that must secrete proteins and large glycoconjugates to grow, divide, and persist. Proteins involved in trafficking such molecules have been identified, but their precise roles are often unresolved due to the difficulty in capturing "snapshots" during the export pathway. Wza is the prototype for the large family of OM polysaccharide export proteins. In Escherichia coli, Wza is essential for the assembly of a capsule, a protective surface coat composed of long-chain polysaccharides. Wza creates an octameric α-helical channel spanning the OM, but the bulk of the protein exists as a large periplasmic structure enclosing an extensive lumen. Residues within the lumen of Wza were targeted for site-specific incorporation of the UV photo-cross-linkable unnatural amino acid p-benzoyl-L-phenylalanine. Using this in vivo photo-cross-linking strategy, we were able to trap polysaccharide translocation intermediates within the lumen of Wza, providing the first unequivocal evidence to our knowledge that nascent capsular polysaccharide chains exit the cell through the Wza portal.

Structural and Functional Variation in Outer Membrane Polysaccharide Export (OPX) Proteins from the Two Major Capsule Assembly Pathways Present in Escherichia coli

“…Wza engages the cognate PCP protein (Wzc), but it is still unknown how these proteins interface with Wzx, which exports undecaprenol diphosphate-linked repeat units across the IM, or with the periplasmic domain of the CPS polymerase (Wzy) (1,14). CPS has been detected in transit within the Wza lumen, validating the export conduit to the surface (15). In contrast, KpsD and VexA participate in export complexes containing an ABC transporter and an adaptor protein (KpsE and VexB, respectively) (1,8).…”

“…The wild-type bacterial strains used in this study were E. coli E69 serotype O9a:K30:H12 (group 1 capsule prototype; supplied by F. Ørskov [41]) and S. enterica serovar Typhi Ty2 trp, cys, ⌬aroC1019 (Vi-antigen producer; K. E. Sanderson, Salmonella Genetic Stock Centre, University of Calgary, Canada). Mutants lacking surface CPS expression were used as controls: E69 wza 22 min ::aadA wza K30 ::aacC1 (CWG281 [15]), and S. Typhi ⌬vexC (CWG1235 [9]). lpp deletion mutants in E69 (CWG1380) and S. Typhi (CWG1381) were constructed using the -Red recombineering system (42); the lpp genes were replaced by the chloramphenicol resistance gene, cat.…”

Colanic Acid Intermediates Prevent De Novo Shape Recovery of Escherichia coli Spheroplasts, Calling into Question Biological Roles Previously Attributed to Colanic Acid

“…3B), indicating that genes downstream of wza were expressed normally. Wza is the outer membrane component of the CA export channel, and the absence of Wza blocks extracellular CA secretion (33,34). Thus, the inability of Wza-deficient spheroplasts to revert to a rod shape suggested either that extracellular CA was required for spheroplast recovery or that the accumulation of CA intermediates was deleterious to such cells.…”

Modulation of bacterial multicellularity via spatiotemporal polysaccharide secretion

“…Wzc proteins of the PCP-2A class (typically from Gram-negative species) further contain a cytosolic bacterial tyrosine autokinase (BY-kinase) domain fused to the 2 nd TMS of the PCP, whereas PCP-2B Wzc proteins (largely from Grampositive species) are phosphorylated by a separately-encoded Wze tyrosine kinase [41]. For Gram-negative bacteria, once a polymer has been synthesized in the periplasm, it is then secreted outside the cell through the periplasm-spanning Wza translocon embedded in the OM [42,43] ( Fig 1A). Wzc proteins have also been implicated in polymer secretion likely through contacts formed with their cognate Wza translocon [41].…”