Recombinant outer membrane protein C of Aeromonas hydrophila elicits mixed immune response and generates agglutinating antibodies (1)
The major virulence factors associated with A. hydrophila infection are surface polysaccharides, toxins (exotoxins and enterotoxins), S-layer and A-layer, pro- teases, secretion systems and several outer membrane proteins (OMPs) involved in adhesion and multidrug-resistance. The OmpF and OmpC are two of the major porins that are involved in transport of molecules across bacterial cell membranes. Vaccination with the OMPs of various organisms has been shown to provide protection in host upon challenge with many pathogenic bacteria including A. hydrophila.
Molecular weight of the recombinant protein from SDS-PAGE was calculated to be *40 kDa, which was close to predicted molecular weight of the histidine-tagged rOmpC (41.4 kDa; 343 aa from the ompC gene and 34 aa from the vector), and confirmed by western blot with anti-His antibodies. Maximum expression of the rOmpC was detected at 6 h post-induc- tion which remained constant thereafter, thus alleviating the possibility of any toxicity associated with the recombinant protein expression. The rOmpC exclusively expressed as inclusion bodies, and therefore it was purified from the insoluble fraction (inclusion bodies) using Ni2+-NTA affinity chromatography.
The endpoint titer in anti-rOmpC antisera from mice immunized with both the doses were found to be >1:40,000. A prominent immunoreactive band of 40 kDa was detected only in the induced cell lysates of E. coli BL21 (kDE3) transformed with pETAhompC in Western blot analysis using antisera raised against the rOmpC.
The ratio of IgG1:IgG2a/ IgG2b levels was determined to be more than 1, indicating a bias toward TH2 type immune response.
Stimulation of sensitized lymphocytes from the spleen of rOmpC-immunized mice with rOmpC resulted in significant increase in T-cell proliferation. IFN-g and IL-4 levels continued to rise gradually, were significantly higher than that of the control samples. The rOmpC-immunized mice activated both the arms of immune response evident by a significant increase in the levels of both IFN-g (TH1 marker) and IL-4 (TH2 marker) when compared to that of the control splenocytes.
Very high numbers of SFUs secreting IFN-g were detected in the rOmpC-sensitized splenocytes after restimulation with the rOmpC.
The anti-rOmpC antisera were able to agglutinate the A. hydrophila cells efficiently, whereas no agglutination was observed with E. coli DH5a and Staphylococcus aureus. The anti-rOmpC antiserum cross-reacted with all the A. hydrophila isolates tested.
Since the signal sequence present at the N-terminus directs the nascent polypeptide chain to the periplasmic space through the translocon in the inner membrane, the ompC gene region without the region encoding the signal peptide (spanning residues 1–23 amino acids of the protein) was cloned. The rOmpC protein was purified to almost 98 % near homogeneity with a yield of 37.3 mg/g wet cell weight (112 mg/L) at shake flask level. The high yield of rOmpC observed was better than that of many T7 expres- sion system-based purification [recombinant PorB (1.1 mg/L) and PorA (30 mg/L or 30 mg/4.7 g wet cell wt) of Neisseria meningitidis. The rOmpC which induce mixed immune response, TH1/TH2 immune responses, would make an efficient vaccine against A. hydrophila infection.Another member of two-component regulatory system of A. hydrophila, recombinant outer membrane protein F, rOmpF, showed predominantly TH1 type of immune response in murine model.