C-terminal domain residues important for secretion and attachment of RgpB in Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontal pathogen, expresses a group of surface proteins with a common C-terminal domain (CTD) that are exported by a novel secretion system to the surface, where they are covalently attached. Using RgpB as a model CTD protein, we have produced a series of site-directed mutations in the CTD sequence at conserved residues and at residues that may be modified and, hence, surface attached. The mutant RgpB proteins were expressed in a P. gingivalis host lacking functional RgpB and RgpA Arg-specific proteases. The RgpB mutants produced were Y674F, Y674F Y718F, T675Q S679Q T682Q T684Q, T693Q, F695A, D696A, N698A, G699P, G716P, T724Q, T728Q T730Q, and K732Q and a protein with a deletion of residues 692 to 702 (Δ692-702). The mutants were characterized for cell-associated Arg-specific protease activity and for cellular distribution using anti-Rgp antibodies and Western blotting of culture fractions. All the mutants exhibited cell-associated Arg-specific activity similar to that of the positive control except for the D696A and Δ692-702 mutants. For all mutants, except D696A and Δ692-702, the RgpB proteins were found modified and attached to the cell surface, which was the same profile found in the positive-control strain. Only trace amounts of the precursor form of the Δ692-702 mutant were detected in the outer membrane, with none detected in the periplasm or culture fluid although cell transcript levels were normal. The results suggest that residues 692 to 702 of the CTD, in particular, residue D696, have an important role in the attachment of RgpB at the cell surface and that without attachment secretion does not occur.

Development of a multiplex bead-based assay to monitor dengue virus seroconversion.

Dengue virus (DENV) envelope protein is responsible for viral attachment to host cells and as such is a target of neutralizing antibody responses. However, the presence of envelope-specific antibodies against a given serotype may contribute to enhanced disease during secondary infection with another serotype. There is a need therefore for a standardized, high-throughput low-volume assay which permits the simultaneous screening of reactivity to multiple DENV serotypes. Here, we describe a method of identifying DENV serotype-specific response in exposed individuals using a multiplexed bead-based immunoassay. The ED3 domain of a specific DENV serotype is cloned into pQEAM containing 6xHIS, TEV protease, and AviTag biotinylation sites. Biotinylated ED3 proteins are expressed in E. coli CVB101 and purified by sequential column fractionation followed by coupling onto fluorescent avidin-coated microspheres. Methods for determining the optimum amount of biotinylated ED3 protein coupled onto the microsphere are described. The assay demonstrates both a high degree of sensitivity and specificity using well-characterized patient plasma samples. The nature of the assay permits further development to include a variety of DENV serotypes and regionally important sub-serotypes.