Molecular Cloning and Characterization of the Genes Coding for the Highly Immunogenic Cluster of 90-Kilodalton Envelope Proteins from theChlamydia psittaciSubtype That Causes Abortion in Sheep

Abstract

Proteins present in the outer membrane of chlamydiae that are involved in mucosal epithelial cell infection must clearly be identified and characterized if we are to understand and modify the pathogenic mechanisms utilized by these organisms. We have identified and isolated a family of four genes encoding putative outer membrane proteins (POMPs), a group of proteins of approximately 90 kDa present in the outer membrane of the subtype of Chlamydia psittacithat causes ovine enzootic abortion (strain S26/3). These proteins, although minor components, are major immunogens, as shown by the immunoblotting of chlamydial outer membrane complexes with postabortion sheep sera, and are therefore potential diagnostic and/or protective antigen candidates. Immunoblotting of the expressed amino- and carboxy-terminal halves of one of the POMPs with postabortion sheep sera showed that the major humoral immune response appeared to be directed solely against the amino-terminal half. This result, in combination with the positive immunofluorescence staining of S26/3-infected cells using POMP-specific (specific to the amino-terminal half of the proteins) monoclonal antibodies, suggests the probable surface localization of the POMPs and, more specifically, the surface exposure of the amino-terminal half of these proteins. The four pomp genes are highly homologous, sharing 82 to 100% similarity with each other (two of the genes are identical). Genes with strong and weak homologies were also detected in C. psittaci avian and feline pneumonitis strains, respectively. Nopomp homologs were found in strains of C. trachomatis and C. pneumoniae, but this does not preclude their existence. The absence of homology with various subtypes of C. pecorum, which complicate the diagnosis of the ovine abortion subtype, indicates the possible suitability of the these 90-kDa proteins as serodiagnostic antigens.

Immunoelectron microscopic localisation of the OMP90 family on the outer membrane surface of Chlamydia psittaci

“…The POMP (181 and 0040) and MOMP (4/11) monoclonal antibodies (mAbs) used in this study have been described previously [9,14,17]. A¤nity-pu-ri¢ed antibodies to the recombinant amino-and carboxy-terminal halves of POMP90A [9] were prepared as described previously [8], with the following mod-i¢cations. Puri¢ed recombinant protein (30-Wg aliquots) [9] was subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.…”

“…A¤nity-pu-ri¢ed antibodies to the recombinant amino-and carboxy-terminal halves of POMP90A [9] were prepared as described previously [8], with the following mod-i¢cations. Puri¢ed recombinant protein (30-Wg aliquots) [9] was subjected to preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Protein was transferred to nitrocellulose, and strips containing the recombinant amino and carboxyl fragments were excised and incubated with pooled post-abortion sheep sera [8,9] and sera from ewes vaccinated with the puri¢ed recombinant carboxy-terminal protein [9], respectively.…”

“…However, many chlamydial antigens which elicit an immune response still remain uncharacterised and the major outer membrane protein (MOMP) remains the only characterised antigen unequivocally demonstrated to be exposed on the EB outer membrane surface [6,7]. Recently, genes coding for a group of highly immunogenic envelope proteins of approximately 90 kDa, the putative outer membrane proteins (POMPs), from the ovine enzootic abortion (OEA) subtype of C. psittaci have been identi¢ed, isolated and characterised [8,9]. OEA is a major cause of abortion in sheep in several European countries, and represents a proven zoonotic risk to pregnant women in whom infection can be life-threatening [10].…”

“…OEA is a major cause of abortion in sheep in several European countries, and represents a proven zoonotic risk to pregnant women in whom infection can be life-threatening [10]. The POMPs have been extensively analysed by 2-D electrophoretic analysis [11] and have been suggested to be important serodiagnostic antigen candidates [9,12]. They may also play a role in protective immunity since they are present in chlamydial outer membrane complexes [9,13,14] which have been shown to be protective [15].…”

“…The POMPs have been extensively analysed by 2-D electrophoretic analysis [11] and have been suggested to be important serodiagnostic antigen candidates [9,12]. They may also play a role in protective immunity since they are present in chlamydial outer membrane complexes [9,13,14] which have been shown to be protective [15]. However, such a role is not supported by the recent results of Buend| èa et al [6] who failed to demonstrate localisation to the EB surface.…”