Search PubMed⌕ Search

Biomedical subjects

J E Raulston

Publications and source records attributed to J E Raulston.

15 recordsLinked to original sources
PubMed2002

Protein disulfide isomerase, a component of the estrogen receptor complex, is associated with Chlamydia trachomatis serovar E attached to human endometrial epithelial cells.

Chlamydia trachomatis serovar E, the leading bacterial agent responsible for sexually transmitted diseases, is required to invade genital epithelial cells for its growth and survival, yet little is known about the adhesin-receptor interactions promoting its entry. In contrast, much has been published on the heparan sulfate receptor for binding C. trachomatis L2 elementary bodies (EBs) prior to entry into HeLa cells. Using a different experimental approach in which a biotinylated apical membrane protein receptor(s) attached to EB at 4 degrees C was stripped off the surface of polarized HEC-1B cells and immunoprecipitated with polyclonal anti-EB antibodies, an approximately 55-kDa protein was reproducibly detected by enhanced chemiluminescence and two-dimensional gel electrophoresis. Matrix-assisted laser desorption ionization mass-spectrometry sequence analysis revealed the 55-kDa protein to be protein disulfide isomerase (PDI), a member of the estrogen receptor complex which carries out thiol-disulfide exchange reactions at infected host cell surfaces. Exposure of HEC-1B cells during EB attachment (1.5 to 2 h) to three different inhibitors of PDI reductive reactions--(i) the thiol-alkylating reagent DTNB (5,5'-dithiobis[2-nitrobenzoic acid]), (ii) bacitracin, and (iii) anti-PDI antibodies--resulted in reduced chlamydial infectivity. Since (i) C. trachomatis serovar E attachment to estrogen-dominant primary human endometrial epithelial cells is dramatically enhanced and (ii) productive entry into and infectivity of EB in host cells is dependent on reduction of EB cross-linked outer membrane proteins at the host cell surface, these data provide some preliminary evidence for an intriguing new potential receptor candidate for further analysis of luminal C. trachomatis serovar E entry.

Amino Acid Sequence↗
PubMed2001

Identifying regulators of transcription in an obligate intracellular pathogen: a metal-dependent repressor in Chlamydia trachomatis.

A prominent feature exhibited by Chlamydia trachomatis growing in an iron-limiting environment is a differential pattern of protein expression. In many bacteria, iron-responsive proteins are regulated at the level of transcription by a family of repressors resembling the Escherichia coli ferric uptake regulator (Fur) protein. Although the chlamydial genome sequencing project did not unveil an obvious Fur homologue, a detailed examination indicated five unassigned open reading frames (ORFs) that would encode products with limited sequence homology to Fur. In this report, each chlamydial ORF was engineered in E. coli, and recombinant proteins were examined for functional characteristics resembling Fur. A Fur-specific polyclonal antiserum revealed that the protein encoded by ORF CT296 shares antigenic cross-recognition. Moreover, this protein forms dimers in solution in a fashion analogous to E. coli Fur. Further studies confirmed that the product of ORF CT296 is able to (i) complement Fur activity in a mutant strain of E. coli; and (ii) specifically bind to a 19 bp consensus sequence found in promoters of iron-regulated genes in E. coli. We propose a designation of dcrA (divalent cation-dependent regulator A) for ORF CT296, which encodes a protein distantly related to E. coli Fur. DcrA represents the first repressor described for this obligate intracellular bacterium.

Amino Acid Sequence↗
PubMed1998

Localization of Chlamydia trachomatis heat shock proteins 60 and 70 during infection of a human endometrial epithelial cell line in vitro.

Unlike chlamydial lipopolysaccharide, which is released from the developing inclusion to the surface of infected genital epithelial cells, both Chlamydia trachomatis heat shock protein (hsp) 60 and 70 antigens remained confined within the inclusion during the course of the chlamydial developmental cycle. Exposure of the infected cells to penicillin to induce a persistent infection or to a lipophilic microbicide did not potentiate secretion or exocytosis of the chlamydial hsp.

Bacterial Proteins↗
PubMed1997

Delivery of azithromycin to Chlamydia trachomatis-infected polarized human endometrial epithelial cells by polymorphonuclear leucocytes.

An in-vitro model was designed to evaluate whether polymorphonuclear leucocytes (PMN) loaded with azithromycin could migrate and deliver the antibiotic in a bioactive form to chlamydia inclusions in polarized human endometrial epithelial (HEC-1B) cells infected with Chlamydia trachomatis. PMN chemotaxis through the extracellular matrix and between infected epithelial cells was readily observed if the HEC-1B cells had been infected with chlamydiae for 36 or 48 h. Inclusions in infected epithelial cells exposed to PMN loaded with azithromycin were initially distinguished by deformed reticulate bodies and an excessive amount of chlamydial outer membrane vesicles. As the amount of PMN-delivered antibiotic increased, chlamydial inclusions were filled with large cell envelope 'ghosts' which were the remnants of lysed reticulate bodies. The lethal effect of azithromycin was confirmed by a reduction in the viability of infectious progeny. Our results demonstrate that the damage to chlamydiae was due to transport and delivery of azithromycin by PMN to infected genital epithelial cells. When infected HEC-1B cells were exposed to PMN not loaded with the antibiotic, chlamydial morphology was not obviously affected yet few viable progeny could be recovered. In this case, PMN-induced damage to host epithelial cells probably interrupted chlamydial nutrient acquisition and subsequent maturation and formation of infectious progeny.

Anti-Bacterial Agents↗
PubMed1997

The microbicidal agent C31G inhibits Chlamydia trachomatis infectivity in vitro.

Safe and effective vaginal microbicidal compounds are being sought to offer women an independent method for protection against transmission of sexually acquired pathogens. The purpose of this study was to examine the efficacy of two formulations of one such compound, C31G, against Chlamydia trachomatis serovar E alone, its host epithelial cell (HEC-1B) alone, and against chlamydiae-infected HEC-1B cells. Preexposure of isolated, purified infectious chlamydial elementary bodies (EB) to C31G, at pHs 7.2 and 5.7, for 1 h at 4 degrees C resulted in reduced infectivity of EB for HEC-1B cells. Examination of the C31G-exposed 35S-EB on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs and by Western blotting revealed a C31G concentration-dependent and pH-dependent destabilization of the chlamydial envelope, resulting in the release of chlamydial lipopolysaccharide and proteins. Interestingly, when the host human genital columnar epithelial cells were infected with chlamydiae and then exposed to dilute concentrations of C31G which did not alter epithelial cell viability, chlamydial infectivity was also markedly reduced. C31G gained access to the developing chlamydial inclusion causing damage to or destruction of metabolically active reticulate bodies as well as apparent alteration of the inclusion membrane, which resulted in premature escape of chlamydial antigen to the infected epithelial surface. These studies show that the broad-spectrum antiviral and antibacterial microbicide C31G also has antichlamydial activity.

Anti-Bacterial Agents↗
PubMed1997

Response of Chlamydia trachomatis serovar E to iron restriction in vitro and evidence for iron-regulated chlamydial proteins.

Iron is a well-established mediator of virulence in several bacterial pathogens, yet little is known about the role of iron in infectious disease processes caused by obligate intracellular bacterial pathogens. In this study, the effect of iron limitation was examined for the sexually transmitted infectious agent Chlamydia trachomatis in an in vitro model of human genital infection using the intracellular iron-chelating reagent deferoxamine mesylate (Desferal). Iron restriction caused a significant reduction in infectivity of C. trachomatis elementary bodies (EB) harvested from Desferal-exposed polarized epithelial cells when compared to that of EB harvested from iron-sufficient control cell cultures. Replacement of the Desferal exposure medium with medium containing iron-saturated transferrin restored chlamydial infectivity, whereas replacement with growth medium alone had no effect. The following three prominent morphological features were observed by electron microscopic examination of chlamydia-infected cells exposed to Desferal: (i) inclusions containing chlamydiae greatly delayed in maturation, (ii) substantial blebbing within chlamydial inclusions, and (iii) electron-dense material surrounding inclusions. Protein analyses of highly purified EB by two-dimensional polyacrylamide gel electrophoresis revealed that there were at least 19 candidate iron-repressible proteins in C. trachomatis and at least one protein which was iron inducible. One putative iron-repressible protein was confirmed by Western blot (immunoblot) analysis to be the chlamydial heat shock protein 60 (hsp60). The enhanced production of this antigen by chlamydiae as a result of iron limitation is of particular importance since there is a well-documented association between chlamydial hsp60 and destructive immunopathological sequelae in infected patients.

Bacterial Outer Membrane Proteins↗
PubMed1996

Accelerated development of genital Chlamydia trachomatis serovar E in McCoy cells grown on microcarrier beads.

Chlamydia trachomatis serovar E is a major cause of bacterially-acquired sexually transmitted infections. Stock cultures of these obligate intracellular bacteria are often propogated in McCoy cells. We recently reported that greater infectious titers of chlamydiae could be obtained if the McCoy cells were cultured on collagen-coated microcarrier beads versus plastic flasks, although the reason for the difference in efficiency was not clear. This study analyzed the development of C. trachomatis grown in McCoy cells by the two methods. Transmission electron microscopy analysis revealed an accelerated chlamydial development, with maturation of reticulate bodies into elementary bodies sooner in McCoy cells grown on the porous substratum. Comparison of particle counts versus infectivity titers indicated the production of fewer numbers of elementary bodies but which were highly infectious sooner from the infected McCoy cell-microcarrier bead cultures than from duplicate infected McCoy cell cultures grown in plastic tissue culture flasks.

Animals↗
PubMed1995

Characterization, expression and envelope association of a Chlamydia trachomatis 28 kDa protein.

Genital serovariants of Chlamydia trachomatis establish infection by attachment, entry and multiplication within human endometrial epithelial cells. In previous studies, a chlamydial recombinant Escherichia coli was identified which exhibited a specific adherent phenotype to endometrial epithelial cells closely resembling that observed for a genital strain of C. trachomatis. One of the plasmid-encoded products expressed by the recombinant is a 28 kDa protein. In this study, localization of the 28 kDa protein in isolated outer membranes of recombinant E. coli and in chlamydial outer membrane complexes lends support for a potential role for this protein in the attachment process. Surprisingly, nucleic acid sequence analysis reveals that the 28 kDa protein shares a modest degree of homology with a member of the E. coli heat shock protein family.

Amino Acid Sequence↗
PubMed1995

Chlamydial envelope components and pathogen-host cell interactions.

Few bacterial pathogens are as widespread in nature or as capable of eliciting such a diversity of disease syndromes as are the chlamydiae. As obligate intracellular organisms, they pose a special research challenge in defining the molecular components and mechanisms for productive growth within host cells and the overall progress of infection throughout host tissue. Although a comprehensive view of chlamydial envelope composition and respective functions in pathogenesis is far from complete, ongoing investigations continue to expose new and intriguing avenues for exploration.

Animals↗
PubMed1994

Effect of clinically relevant culture conditions on antimicrobial susceptibility of Chlamydia trachomatis.

Infection of polarized human endometrial-gland epithelial cells obtained at hysterectomy with Chlamydia trachomatis serovar E may provide a relevant in vitro model for studies of pharmacokinetics in genital chlamydial infections. The minimal bactericidal concentration of azithromycin against C. trachomatis was lower in this model than in studies with nonpolarized cells (0.125 and 0.5 mg/L, respectively). Polarized cells also internalized more azithromycin over 24 hours. The results indicate that the eradication of chlamydial infections may be difficult to prove by antigen detection methods: the persistence of chlamydial envelope material within the intracellular vacuoles of azithromycin-treated cells may lead to a false-positive diagnosis of persisting chlamydial infection.

Anti-Bacterial Agents↗
PubMed1994

Pharmacokinetics of azithromycin and erythromycin in human endometrial epithelial cells and in cells infected with Chlamydia trachomatis.

The pharmacokinetics of azithromycin and erythromycin were examined in uninfected and Chlamydia trachomatis infected human endometrial epithelial cells in vitro. Cells which were grown in a polarized orientation showed a three-fold higher quantity of azithromycin uptake than did non-polarized cells. Cellular penetration profiles of azithromycin exceeded erythromycin by as much as eight-fold. In addition, approximately 20% of azithromycin remained cell-associated after 24 h in drug-free medium whereas erythromycin was not retained beyond 3 h. Hormone-responsive primary human endometrial gland epithelial cells, cultured directly after hysterectomy, showed enhanced uptake of both antimicrobials compared with laboratory adapted epithelial cell lines. Cells infected with a genital serovariant of C. trachomatis showed no significant difference in antibiotic uptake during the early stages of the chlamydial developmental cycle, and only a slight decrease in azithromycin uptake in the late stage of infection compared with non-infected cells. Morphological evidence of the bactericidal activity of azithromycin was evident in infected cells at most stages of the chlamydial developmental cycle, whereas the same concentration of erythromycin produced less evidence of marked bactericidal activity as observed by transmission electron microscopy.

Azithromycin↗
PubMed1993

Molecular characterization and outer membrane association of a Chlamydia trachomatis protein related to the hsp70 family of proteins.

One route by which Chlamydia trachomatis is internalized into host endometrial epithelial cells is receptor-mediated endocytosis. Although this implies an adhesin-receptor interaction exists, specific chlamydial surface molecules have not been identified. We are investigating potential adhesin molecules using an in vitro functional assay to select for chlamydial recombinant Escherichia coli expressing an adherent phenotype. We have previously shown that E. coli JM109(pPBW58) attaches to epithelial cells by a specific process paralleling C. trachomatis and expresses at least three plasmid-encoded proteins (18, 28, and 82 kDa; Schmiel, D. H., Knight, S. T., Raulston, J. E., Choong, J., Davis, C. H., and Wyrick, P. B. (1991) Infect. Immun. 59, 4001-4012). In this report, we demonstrate that (i) the 82-kDa protein is associated with the outer membrane of both E. coli JM109-(pPBW58) and C. trachomatis serovar E elementary bodies; (ii) the plasmid-encoded protein is identical to the native chlamydial protein by mass, charge, antigenicity, and partial proteolytic peptide profiles; (iii) a highly homologous protein is present in C. trachomatis biovariant lymphogranuloma venereum; (iv) the 82-kDa protein is not covalently linked by disulfide bonds to other protein species in either E. coli JM109(pPBW58) or C. trachomatis; (v) sequence analysis of the open reading frame indicates this protein is a relative of the heat shock 70 family of proteins; and (vi) the inferred amino acid sequence contains a contiguous 73-amino acid region having 51% identity with the extracellular sperm receptor binding domain in Strongylocentrosus purpuratus (Foltz, K. R., Partin, J. S., and Lennarz, W. J. (1993) Science 259, 1421-1425). The potential involvement of an hsp70 protein in attachment may provide new insight on adherence mechanisms by obligate intracellular pathogens.

Amino Acid Sequence↗
PubMed1991

Recombinant Escherichia coli clones expressing Chlamydia trachomatis gene products attach to human endometrial epithelial cells.

To identify Chlamydia trachomatis genes involved in attachment to host cells, a chlamydial genomic library was screened on the basis of binding characteristics by two methods. In the whole-cell screen, individual recombinant Escherichia coli clones were assayed for adherence to eukaryotic cells. In the membrane-binding screen, each recombinant colony of E. coli was treated with CHCl3 and assayed for binding to purified, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized, 35S-labeled eukaryotic membrane material. Initial screening with McCoy cells was refined by using HEC-1B cells, a human endometrial epithelial cell line, which discriminate among recombinants adhering to McCoy cells. Some recombinants demonstrate significantly greater adherence to HEC-1B cells than to McCoy cells and appear, by transmission electron microscopy, to associate with electron-dense areas of the epithelial cell plasma membrane, resembling coated pits. Recombinants positive by one or both screening methods were examined by Southern and Western (immunoblot) analyses, which revealed the presence of chlamydial sequences inserted in the plasmids and the expression of novel 18-, 28-, and approximately 82 kDa, and perhaps of 18 Maxicell analysis of selected recombinants confirmed that the proteins of 28 and approximately 82 kDa, and perhaps of 18 kDa, are plasmid encoded. Antiserum generated against the recombinant approximately 82-kDa protein reacted in Western analysis with a similar-sized protein from C. trachomatis serovar E elementary bodies (EB) and reticulate bodies, serovar L2 EB, and C. psittaci EB. E. coli JM109(pPBW58) contains a 6.7-kb plasmid insert which encodes proteins of all three sizes. Under a number of different conditions in the whole-cell attachment assay--i.e., at 4 degrees C, in Ca(2+)- and Mg(2+)-free medium, in the presence of trypsin or dextran sulfate, and with rabbit aortic endothelial cells--the binding specificity of JM109(pPBW58) parallels that of C. trachomatis EB. Finally, the adherence phenotype of E. coli JM109(pPBW58) correlates directly with the presence of the recombinant plasmid; the phenotype is lost concurrently with loss of the recombinant plasmid, and the into E. coli JM109. The role of the 18-, 28-, and approximately 82-kDa proteins in mediating attachment, whether they act in concert as a complex or individually, has yet to be determined.

Antigens, Bacterial↗
PubMed1989

Early cell envelope alterations by tobramycin associated with its lethal action on Pseudomonas aeruginosa.

The immediate activities of the aminoglycoside antibiotic tobramycin were investigated in Pseudomonas aeruginosa PAO1. The lethal action of a low concentration of tobramycin (8 micrograms ml-1) occurred rapidly (1-3 min) and was associated with leakage of certain cellular components into the supernatant. The presence of magnesium at the time of initial exposure protected cells by preventing uptake of tobramycin; however, magnesium addition following a brief exposure did not restore viability. Analyses of supernatant material revealed a rapid 2-fold increase in protein released following tobramycin treatment. A prominent 29 kDa protein, observed by SDS-PAGE in the released material was identified as the periplasmic beta-lactamase. Brief exposure to tobramycin did not result in major morphological damage or cell lysis as observed by transmission electron microscopy, and release of LPS was not a primary event. Although activity at the ribosomal level was observed by 2-3 min, leakage was detected after only 1 min. These data indicate that leakage of cellular components, particularly beta-lactamase, occurs simultaneously, if not prior to inhibition of protein synthesis by tobramycin.

Bacterial Proteins↗