Structural and functional characterization of NanU, a novel high-affinity sialic acid-inducible binding protein of oral and gut-dwelling Bacteroidetes species

Many human-dwelling bacteria acquire sialic acid for growth or surface display. We identified previously a sialic acid utilization operon in Tannerella forsythia that includes a novel outer membrane sialic acid-transport system (NanOU), where NanO (neuraminate outer membrane permease) is a putative TonB-dependent receptor and NanU (extracellular neuraminate uptake protein) is a predicted SusD family protein. Using heterologous complementation of nanOU genes into an Escherichia coli strain devoid of outer membrane sialic acid permeases, we show that the nanOU system from the gut bacterium Bacteroides fragilis is functional and demonstrate its dependence on TonB for function. We also show that nanU is required for maximal function of the transport system and that it is expressed in a sialic acid-responsive manner. We also show its cellular localization to the outer membrane using fractionation and immunofluorescence experiments. Ligand-binding studies revealed high-affinity binding of sialic acid to NanU (Kd ~400 nM) from two Bacteroidetes species as well as binding of a range of sialic acid analogues. Determination of the crystal structure of NanU revealed a monomeric SusD-like structure containing a novel motif characterized by an extended kinked helix that might determine sugar-binding specificity. The results of the present study characterize the first bacterial extracellular sialic acid-binding protein and define a sialic acid-specific PUL (polysaccharide utilization locus)

The Virulence Factor PEB4 (Cj0596) and the Periplasmic Protein Cj1289 Are Two Structurally Related SurA-like Chaperones in the Human Pathogen Campylobacter jejuni*

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl-prolyl cis/trans isomerase (PPIase) domains encasing a large central cavity. Unlike SurA, the chaperone domain is formed by interlocking helices from each monomer, creating a domain-swapped architecture. PEB4 stimulated the rate of proline isomerization limited refolding of denatured RNase T1 in a juglone-sensitive manner, consistent with parvulin-like PPIase domains. Refolding and aggregation of denatured rhodanese was significantly retarded in the presence of PEB4 or of an engineered variant specifically lacking the PPIase domain, suggesting the chaperone domain possesses a holdase activity. Using bioinformatics approaches, we identified two other SurA-like proteins (Cj1289 and Cj0694) in C. jejuni. The 2.3-Å structure of Cj1289 does not have the domain-swapped architecture of PEB4 and thus more resembles SurA. Purified Cj1289 also enhanced RNase T1 refolding, although poorly compared with PEB4, but did not retard the refolding of denatured rhodanese. Structurally, Cj1289 is the most similar protein to SurA in C. jejuni, whereas PEB4 has most structural similarity to the Par27 protein of Bordetella pertussis. Our analysis predicts that Cj0694 is equivalent to the membrane-anchored chaperone PpiD. These results provide the first structural insights into the periplasmic assembly of outer membrane proteins in C. jejuni