Periplasmic depolymerase provides insight into ABC transporter-dependent secretion of bacterial capsular polysaccharides

Abstract

SignificanceCapsules are critical virulence determinants for bacterial pathogens. They are composed of capsular polysaccharides (CPSs) with diverse structures, whose assembly on the cell surface is often powered by a conserved ABC transporter. Current capsule-assembly models include a contiguous trans-envelope channel directing nascent CPSs from the transporter to the cell surface. This conserved apparatus is an attractive target for antivirulence antimicrobial development. This work describes a CPS depolymerizing lyase enzyme found in the Burkholderiales and unique structural features that define its mechanism, CPS specificity, and evolution to function in the periplasm in a noncatabolic role. The activity of this enzyme provides evidence that CPS assembled in an ABC transporter-dependent system is exposed to periplasm during translocation to the cell surface.

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

“…Until recently, the working model for CPS export in an ABC transporter-based system invoked a protected continuous conduit from the cytoplasm to the cell surface to translocate complete CPS molecules. This proposal has been challenged by the discovery that a periplasmic glycanase can degrade CPS export intermediates in S. enterica serovar Typhi (34). The step(s) involved in the transit of periplasmically accessible intermediates between the ABC transporter and the OPX translocon therefore requires further investigation.…”

“…Release of periplasmic contents. Periplasmic contents were released as described elsewhere (34). Briefly, cells were harvested by centrifugation (5,000 ϫ g for 10 min) at late-log phase and 10 optical density at 600 nm (OD 600 ) unit equivalents were resuspended in 0.7 ml 100 mM Tris-HCl (pH 8.2) containing 500 mM sucrose.…”

Genome analysis of a halophilic bacterium Halomonas malpeensis YU-PRIM-29T reveals its exopolysaccharide and pigment producing capabilities

“…KpsE determines the length of the polymer chain while KpsD forms the export channel 16 , 17 . The synthesis and translocation from the periplasm to the cell’s exterior needs the presence of KpsE (PCP-3 family) and KpsD (OPX family) proteins similar to Wza-Wzc proteins 17 , 35 , 36 . Four glycosyltransferases involved in the EPS biosynthesis pathway cluster are identified from the draft genome.…”

Pantoea stewartii WceF is a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like fold

“…Glycan-modifying proteins have been regularly described in the synthesis and export machines that control bacterial and fungal extracellular polysaccharides involved in encapsulation or biofilm formation. In this respect, the presence of enzymes that cleave glycosidic bonds seems counterintuitive, and, indeed, biochemical characterization of these proteins combined with mutagenesis studies points to their multifaceted functional roles in polysaccharide modification ( 7 , 8 , 9 , 10 , 40 ). This work showed that Pantoea stewartii WceF, as part of the biofilm synthesis operon, is a hydrolytic enzyme, active on P. stewartii’s biofilm exopolysaccharide stewartan, and with a bacteriophage tailspike-like fold.…”

“…In turn, changing these macromolecular structures offers an additional control level in biofilms, for example, by matrix-degrading enzymes. For glycan-based biofilm components, regulatory glycosidases have been frequently described, in both bacterial and fungal species that can have impact on biofilm synthesis and export ( 7 , 8 , 9 , 10 , 11 ). These polysaccharide-specific enzymes can alter biofilm viscosity and thus influence the mobility of biofilm-matrix embedded or penetrating particles such as bacteria or bacteriophages, making them promising tools in antimicrobial treatments ( 12 , 13 , 14 , 15 , 16 ).…”