Conformational epitope mapping of OmpC, a major cell surface antigen from Salmonella typhi.

The outer membrane protein OmpC, a trimer made of 16 stranded beta-barrel monomers, is a major cell surface antigen from the human pathogen Salmonella typhi. The relative stability of the epitopes recognising a Salmonella specific MAb (referred as MPN5) and an Enterobacteria specific MAb (referred as P7D8) and the role of the trimeric organisation has been probed using gel electrophoresis and monoclonal antibodies. The assembly of the trimer and the stability of the beta-barrel are found to be important for epitope presentation. The Salmonella specific conformational epitope is found to be more stable than the Enterobacteria specific one. The important residues of the Salmonella specific (Asp 25 of loop 1, Asp 340 of loop 8, Lys 334 of loop 8, and Tyr 210 of loop 5) and the Enterobacteria specific (Asp 25 of loop 1, Tyr 210 of loop 5, and Lys 152 of loop 4) conformational epitope have been identified using monoclonal antibodies, chemical modification, and solid phase binding methods.

Molecular modelling of epitope presentation using membrane protein OmpC.

Three-dimensional models of the chimeric S. typhi OmpC protein carrying an epitope from rotavirus VP4 capsid protein on either of two exposed loops (fourth and sixth) were constructed separately, using computer-aided homology modelling. The theoretical model of S. typhi OmpC was used as a template. The monomers were initially energy minimized. The trimers were generated for both the chimeric S. typhi OmpC proteins and the structures were optimized after several cycles of minimization. The surface accessibility calculations for the resulting models show that epitope recognition should be more effective in the fourth loop than in the sixth loop, in accordance with the experimental results on the immunogenic nature of the rotaviral epitope inserted into the two putative loops of S. typhi OmpC.

Homology model of surface antigen OmpC from Salmonella typhi and its functional implications.

Homology based 3D structural model of the immunodominant major surface antigen OmpC from Salmonella typhi, an obligatory human pathogen, was built to understand the possible unique conformational features of its antigenic loops with respect to other immunologically cross reacting porins. The homology model was built based on the known crystal structures of the E. coli porins OmpF and PhoE. Structure based sequence alignment helped to define the structurally conserved regions (SCRs). The SCR regions of OmpC were modelled using the coordinates of corresponding regions from reference proteins. Surface exposed variable regions were modelled based on the sequence similarity and loop search in PDB. Structural refinement based on symmetry restrained energy minimization resulted in an agreeable model for the trimer of OmpC. The resulting model was compared with other porin structures, having b-barrel fold with 16 transmembrane beta-strands, and found that the variable regions are unique in terms of sequence and structure. A ranking of the loops taking into account the antigenic index, the sequence variability, the surface accessibility in the context of the trimer, and the structural variability suggests that loop 4 (151-172), loop 5 (194-218) and loop 6 (237-264) are the best ranked B-cell epitopes. The model provides possible explanations for the functional and unique immunological properties associated with the surface exposed regions and outlines the implications for structure based experimental design.

Purification of integral outer-membrane protein OmpC, a surface antigen from Salmonella typhi for structure-function studies: a method applicable to enterobacterial major outer-membrane protein.

Extraction of the outer-membrane porin, OmpC, from Salmonella typhi Ty21a was done by using a modified salt-extraction procedure. It was possible to extract only the major outer-membrane protein (OMP) from the crude membrane using this method. Aberrant lipopolysaccharide (LPS) production in the galE mutant Ty21a has resulted in more isoforms of OmpC and subsequently led to anomalous mobility in SDS-PAGE. The purity of the preparation was confirmed by denaturing urea SDS-PAGE and N-terminal sequencing. The major OMP extracts had LPS of both bound and free forms. The free form of LPS could be removed by gel filtration and the bound form, largely, was removed using ion-exchange chromatography and by passing through ultrafiltration devices. This method has been used to extract the native trimer of OmpC, the major OMP, in a large scale, for structure-function studies. S. typhi Ty21a OmpC preparation yielded reproducible diffraction-quality crystals. Extracts of porin from wild-type Escherichia coli HB101, grown under high osmolarity conditions, showed a single species of OMP on SDS-PAGE. This suggests the possible application of the method to other gram-negative bacterial porins.

Crystallization of the immunodominant outer membrane protein OmpC; the first protein crystals from Salmonella typhi, a human pathogen.

OmpC, a surface antigen of Salmonella typhi was crystallized after several attempts, using PEG 3350. Well shaped hexagonal crystals were grown from vapor diffusion method using octyl glucoside and C12E9 as detergents. Crystals are sensitive to X-ray and diffract weakly up to 7 A. Porin isoforms, due to the bound lipopolysaccharides, could be the cause for poor diffraction. Crystal quality depends largely on the purification method, and in case of LPS contamination, the genetic background of the bacteria. Crystallization and initial data collection suggest optimum conditions and the method of choice for OmpC crystallization.