Chlamydia trachomatis proteins were electrophoresed and then transferred to nitrocellulose paper to detect chlamydial proteins which bind to eucaryotic cell membranes. Resolved polypeptides of C. trachomatis serovars J and L2 were reacted with iodinated HeLa cell membranes and autoradiographed. Infectious elementary bodies of both serovars possess 31,000-and 18,000-dalton proteins which bind to HeLa cells. In contrast, noninfectious reticulate bodies do not possess eucaryotic cell-binding proteins. Both proteins are antigenic when reacted with hyperimmune rabbit antisera in immunoblots and antisera raised against the 31,000-and 18,000-dalton proteins are inhibitory to chlamydia-host cell association. In addition, these antisera exhibit neutralizing activity. Our data suggest that these putative chiamydial adhesins play a key role in the early steps of chlamydia-host cell interaction and that antibody directed against them may be protective.Chlamydiae are obligate intracellular parasites which have been linked to an expanding spectrum of human disease (12,23). The infectious forms of the organism, elementary bodies (EB), attach to host cells and, once ingested, initiate a unique developmental cycle (24). The intracellular reticulate bodies (RB) are noninfectious, larger, and more fragile than EB and display differences in their protein profile (10).Although chlamydiae appear to be internalized by an endocytic-like process (3,25,27), neither a chlamydial adhesin nor a host cell receptor has yet been identified. Information that EB are ingested more efficiently than are Escherichia coli or latex spheres implicates a specific mechanism for chlamydial attachment and uptake (3). Although a chlamydial ligand has not been characterized, the finding that EB cell walls attach to and are ingested by host cells nearly as efficiently as are whole EB (18) implicates cell wall components as putative adhesins. Earlier information that attachment is inhibited by heating EB provides evidence that the adhesin is a protein (2, 3). However, heat inhibition was only partial and was not observed with a serovar A Chlamydia trachomatis strain (17). Antiserum to whole EB inhibits attachment (3), but antibody to the major outer membrane (OM) protein does not affect either attachment or uptake of EB (4). To elucidate the initial steps of association between chlamydiae and host cells the objective of the present study was the identification and preliminary characterization of adhesin(s). We utilized a procedure which allows the detection of separated bacterial polypeptides on nitrocellulose that bind to eucaryotic cell membranes to identify putative adhesins on the surface of C. trachomatis. MATERIALS AND METHODSGrowth and purification of chlamydiae. C. trachomatis serovars L2/434/Bu (L2) and J/UW-36 (J) were grown in HeLa 229 cells as described by Kuo et al. (15). Cultures were harvested at 48 h for EB or 18 h for RB, and cells were removed with sterile glass beads. The cell suspension was sonicated and submitted sequentially to differenti...
The trophozoites of Giardia lamblia possess several protein antigens, predominant among them a protein of approximately 32,000 Da. In the present study, we used monospecific antibodies that recognize this protein to demonstrate its presence on a variety of G. lamblia isolates from human and animal sources. Immune electron microscopy was used to localize 32-kDa antigen on the trophozoite membrane and disk. Immunofluorescent assays employing monospecific antibodies confirmed the presence of 32-kDa antigen on the membrane and disk and its absence on flagella or nuclei. The N-terminal 17 amino acids of the 32-kDa antigen are identical to alpha-1-giardin, a protein component of microribbons on the ventral disk. These results suggest that the 32-kDa immunodominant trophozoite antigen is alpha-1-giardin.
Giardia duodenalis is a common intestinal parasite in most parts of the world. In Canada it is associated with both endemic and epidemic infections that are often transmitted by the waterborne route. Although G. duodenalis strains have been isolated from several animals, the role of other mammals in human infection is unclear. We have isolated and cultured G. duodenalis trophozoites from domestic and wild animals in Alberta and compared them with a human isolate by protein gel electrophoresis and immunoblot analysis. All strains examined share a similar polypeptide profile and important protein antigens. Prominent antigens of 62, 52, 38, and 31 kilodaltons are conserved. The 52- and 31-kilodalton proteins are the major surface-exposed trophozoite components. The high degree of antigenic sharing among strains from different hosts suggests that there may be a wide range of potential reservoirs for G. duodenalis infections.
We describe the cloning and sequence analysis of the region surrounding the gene for the ␣ subunit of RNA polymerase from Chlamydia trachomatis. This region contains genes for proteins in the order SecY, S13, S11, ␣, and L17, which are equivalent to Escherichia coli and Bacillus subtilis r proteins. The incorporation of chlamydial ␣ subunit protein into the E. coli RNA polymerase holoenzyme rather than its truncated variant lacking the amino terminus suggests the existence of structural conservation among ␣ subunits from distantly related genera.The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen responsible for a wide range of infections (37). It exhibits a unique developmental cycle alternating between noninfectious intracellular, metabolically active reticulate bodies and infectious extracellular, inert elementary bodies (EB). The process of chlamydial differentiation and development is complex and involves temporally regulated genes. Both early-and late-development-specific genes have been identified; however, the mechanism underlying stagespecific gene expression remains unelucidated (3,11,30,43,47).A better understanding of chlamydial gene regulation will require a more detailed characterization of its RNA polymerase (RNAP). Studies of RNAP subunits have contributed to our understanding of the mechanism of gene regulation in Escherichia coli (12). The bacterial DNA-dependent RNAPs are complex enzymes comprising four different subunits with the subunit structure ␣ 2 Ј that perform various functions leading to regulated transcription (12,15,16). Assembly of the core RNAP takes place in a sequential manner in the order ␣ 3 ␣ 2 3 ␣ 2  3 ␣ 2 Ј (15). The core enzyme is active in RNA polymerization, while the binding of confers the promoter specificity to the holoenzyme. The purification of RNAP from Chlamydia spp. is a difficult process because of the limitations imposed by very little starting material. Recently Mathews et al. (25) studied the in vitro transcription of chlamydial genes by using a crude preparation of native RNAP. Recent work has focused on the cloning and sequencing of various RNAP subunits with the aim of developing an in vitro transcription system to study surrogate chlamydial genetics.Two groups have independently reported the cloning and sequencing of 66 , , and Ј homologs from Chlamydia spp. (6,7,21). While this work was in progress, Tan et al. (42) reported the cloning and sequence analysis of the ␣ subunit from the C. trachomatis mouse pneumonitis biovar (MoPn). However, these studies have been found to be in error, and the sequence described applies to Mycoplasma spp. rather than Chlamydia spp. (see the author's correction in this issue).Cloning and genomic organization of C. trachomatis ␣ operon genes. The purpose of this investigation was to clone and characterize the ␣ subunit of the RNAP holoenzyme with the aim of developing an in vitro transcription system in which purified and reconstituted RNAP are used. We have previously isolated the ...
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