Characterization of lipoprotein EnvA in Chlamydia psittaci 6BC

Abstract

The primary sequence of the small cysteine-rich protein (EnvA) of Chlamydia psittaci 6BC has been shown to possess a potential lipid modification/signal peptidase 11-processing site, and the mature protein was labeled by a [3H]palmitic acid precursor. We further characterized the mature EnvA, showing that it lacks the N-terminal methionine of the primary peptide, is hydrophobic despite a peptide sequence that is predicted to be hydrophilic, and appears to be lipid modified at an N-terminal cysteine in a manner analogous to that of murein lipoproteins of gram-negative bacteria. We also report the fatty acid content of the small cysteine-rich proteins of C. psitaci and Chlamydia trachomatis L2 as determined by combined gas chromatography-mass spectrometry.Chlamydiae are obligate intracellular bacteria which possess a gram-negative cell envelope that appears to lack peptidoglycan (3,8). The genes encoding three envelope proteins, the major outer membrane protein and the large and small cysteine-rich proteins (CRPs), have been cloned and sequenced from several strains of chlamydiae, including Chlamydia psittaci 6BC (6, 7). In the nondividing, infectious, elementary body form of chlamydiae, the CRPs and the major outer membrane protein form a disulfide cross-linked supramolecular complex, which may be the functional equivalent of peptidoglycan (11,13,14,21). The major outer membrane protein is not cross-linked, and the CRPs are not made in the logarithmically dividing reticulate body form of chlamydiae (11,13,14,21).The small CRPs of Chlamydia trachomatis (Omp3) and C. psittaci (EnvA) appear to be lipoproteins: the primary sequences of Omp3 and EnvA contain potential lipid modification/signal peptidase II recognition sites (1, 7), and the C. psittaci small CRP is labeled from a [3H]palmitic acid precursor (7). The present study was undertaken to further characterize the small CRPs of chlamydiae. We have determined the fatty acid content of EnvA of C. psittaci and the small CRP of C. trachomatis, and we present evidence that EnvA is lipid modified at a cysteine residue in a manner similar to that of the murein lipoprotein of gram-negative bacteria.MATERIALS AND METHODS Purification of EnvA. Sodium dodecyl sulfate (SDS)-insoluble envelope protein complexes containing EnvA were prepared from Renografin-purified elementary bodies of C. psittaci 6BC and C. trachomatis LGV434 serotype L2 as described previously (7), except that noncovalently associated lipids were removed from the insoluble complexes by five extractions with 20-ml aliquots of methanol-chloroform-water (10:5:4 [vol/vol] (7) and protein A-Sepharose CL-48 (Sigma Chemical Co., St. Louis, Mo.). EnvA for combined gas chromatography-mass spectrometry (GC-MS) was separated from other proteins in chloroform-methanol-extracted envelope protein complexes by SDSpolyacrylamide gel electrophoresis (PAGE) (17) by using a step gradient gel consisting of 1 cm of 10% acrylamide and 15 cm of 15% acrylamide. The EnvA, with a mass of between 15 and 6 kDa, was excised and elut...

A Novel Recombinant Multisubunit Vaccine against Chlamydia

“…The synthesis and incorporation of OMP2 and OMP3 in the Chlamydia outer membrane coincide with the transition of reticulate body to elementary body (EB) in the developmental cycle (13,14), and these proteins are thought to contribute to cell wall rigidity and osmotic stability of the EB. Topological studies indicate that OMP2 and OMP3 are localized at the inner surface of the outer membrane (15,16) and are encoded by a bicistronic operon (17). Although OMP2 shows V regions between different species, it is well conserved within a chlamydial species (18 -21) and has been shown to be a highly immunoreactive Ag, inducing Ab responses in both humans and animals (21,22), as well as a major immunogen in chlamydial infections (23).…”

Architecture of the cell envelope of Chlamydia psittaci 6BC

“…Considerably less is known about the intracellular location of the CRPs, in part because there are no reports in the literature of successful separation of inner and outer membranes of chlamydiae by physical means. The small 12-kDa CRP of Chlamydia psittaci 6BC, designated EnvA, possesses a predicted signal peptidase II-processing site and appears to be a lipoprotein with a structure similar to that of the Braun lipoprotein of Escherichia coli (12). The large CRP (EnvB) of C. psittaci 6BC is posttranslationally cleaved to a doublet consisting of approximately equimolar concentrations of 60-kDa proteins (13,19).…”

“…35 S]cysteine in cysteinefree medium (MEM Select Amine; GIBCO) in the presence of cycloheximide (100 g/ml) and chased in medium M199-5% fetal calf serum containing cycloheximide (0.5 g/ml) until EBs were harvested and purified at 48 h postinfection. The purified EBs were Triton X-114 phase partitioned in the absence of reducing agents and then reextracted in the presence of 20 mM DTT and 0.64 M 2-mercaptoethanol as described previously (12). Equal portions of the aqueous phosphate-buffered saline (Dulbecco PBS; GIBCO) and the Triton X-114 phases were subjected to SDS-PAGE, and the separation of labeled proteins was examined by autoradiography.…”

Chlamydia trachomatis Scavenges Host Fatty Acids for Phospholipid Synthesis via an Acyl-Acyl Carrier Protein Synthetase

“…The clear prediction from the gene organization and bioinformatic analysis was that lpaT was the C. trachomatis homolog of 2-acyl-GPE acyltransferase and would function to recycle 2-acyl-GPE arising from lipoprotein biogenesis. An abundant C. trachomatis lipoprotein with an acylated amino terminus is known (24), and the phospholipid N-acyltransferase catalyzing this last step in lipoprotein maturation (CT534, Lnt) is present in the genome, suggesting that LpaT would have the same function in C. trachomatis as in E. coli. A complementation approach was used to test this prediction in vivo.…”

“…The resulting 2-acyl-GPE product is transferred to the inner aspect of the membrane by a specific lysophospholipid transporter, LplT (22), and reacylated by the bifunctional Aas (15). Lipoproteins in Chlamydia psittaci and C. trachomatis have the same lipid modifications as observed in E. coli (23,24), and the genes encoding the lipid modification pathway are present (lgt, CT252; lsp, CT408; lnt, CT534). The distinguishing feature of the C. trachomatis system is that the two functions are encoded by separate genes (Fig.…”