Repertoires of antibodies to culture filtrate antigens in different mouse strains infected with Mycobacterium bovis BCG

Abstract

Two susceptible (Bcg') mouse strains, BALB/c and C57BL/6, were compared by Western blot (immunoblot) analysis for their immunoglobulin G response to 14-day-old BCG culture filtrate (CF) following intravenous infection with live Mycobacterium bovis BCG. The two strains demonstrated a completely different antibody repertoire. BALB/c antibodies were directed against a wide range of CF antigens between 20 and about 100 kilodaltons (kDa), with a preferential recognition of the 65-kDa heat shock protein and the 32-kDa fibronectin-binding protein. C57BL/6 sera, on the other hand, showed a much more restricted antibody pattern, almost exclusively directed against three antigens with estimated molecular sizes of 37, 38, and 40 kDa. Whereas the 37-and 38-kDa antigens were also recognized by BALB/c mice, the 40-kDa antigen was very intensely stained by C57BL/6 sera only. F1 mice had the restricted antibody pattern of C57BL/6 after one injection of BCG and had a hybrid BALB/c-C57BL/6 phenotype following a boost injection of BCG 2 months after the initial infection. Analysis of seven recombinant inbred strains derived from the BALB/c x C57BL/6 cross and of congenic mice differing in major histocompatibility complex-coding chromosome 17 fragments suggests that a gene in the K-IA region of the H-2 locus is associated with the preferential recognition of certain CF antigens. Inoculation with the same dose of killed BCG failed to elicit an antibody response to these filtrate antigens.

Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter

“…Here we report that the first isolated M. tubereulosis gene, pstA-1, similar to E. coli pstA [15] is also included in a cluster of three genes (pstS-3, pstC-2 and pstA-1) encoding a second putative phosphate permease. Furthermore, we demonstrate unambiguously that the pstS-3 gene encodes the previously described Ag 88, a 40-kDa antigen of M. bovis BCG culture filtrate [16,17], mistakenly considered to be encoded by pstA-1 in a previous report [15]. We also define a signature motif for integral cytoplasmic membrane proteins of prokaryotic phosphate binding protein-dependent permeases.…”

“…Monoclonal antibody 2C1-5 directed against Ag 88 [16] was purified from 500-ml hybridoma culture supernatants using an Avid-Chrom Protein A Antibody purification Kit (Sigma), then coupled to an Hydrazide AvidChrom Cartridge (Sigma). Culture filtrate antigens were obtained as described previously [17] by ammonium sulfate precipitation of the culture supernatant of M. bovis BCG GL2 grown for 2 weeks as a surface pellicle on Sauton culture medium. About 7 mg culture filtrate antigens were applied to the cartridge.…”

“…1). -3) is probably a phosphate binding component Ag 88, a M. boris BCG culture filtrate antigen of 40 kDa, highly immunodominant in H-2 ~ haplotype mice [16,17], has been suggested before as homologous to the E. coli membrane-bound PstA subunit [15]. Following the identification of its correct start codon, the predicted amino acid sequence of pstA-1 gene corresponds now to a protein of 305 amino acids with an expected molecular mass of 32.907 kDa.…”

Antibodies against Secreted and Non‐Secreted Antigens in Mice after Infection with Live Mycobacterium tuberculosis

“…Mice infected with live M. hovis BCG develop antibodies directed against antigens pre.scnt in culture filtrate. Such antibodies cannot be detected in sera from mice infected with dead bacilli [5]. Culture filtrate antigens, or culture supernatant antigens, are those antigens which are secreted into the medium during growth and antigens from lysed dead bacilli, Differenees in amount and composition of proteins in culture supernatant are almost inevitable given the wide range of culture conditions used: pellicle growth in Proskauer and Beck medium [8]; growth on the surface ofSauton medium [9,10]; cultivation in roller bottles and addition of 0,05'MJ Tween 80 to Sauton liquid medium [11]; cultivation in Eagle's and Sauton medium in tissue culture plates [12] and shaking culture [13], Proteins found in culture fluid can be described as secreted oreytosolic according to their localization index [14].…”

“…An association between HLA haplotype and antibody production in humans has been observed in malaria and Rhesus immunization [1,2], In patients with tuberculosis an association has been found between DR2 and litres of antibodies to the M. tuherculo.sis specific epitope of the 38-kDa protein defined by TB72 [3], In mice, H-2 linked genes are known to influence the antibody repertoire following hyperimmunization with live M. tiibercidosi.s [4], live M. hovis BCG (5], M. tuherculo.sis extract [6] or culture filtrate antigens [7], However, the 3S-kDa protein is preferentially recognized by mice of different strains [4].…”