Factors affecting the adhesion and uptake of an oculo-genital strain of Chlamydia trachomatis to McCoy cells

Abstract We have studied adhesion and uptake of C. trachomatis serovar E in McCoy cells under various infection conditions. Adhesion and uptake of chlamydiae was completed about 3 h after the initiation of stationary infection at 37°C, but ingestion of cell membrane-attached organisms was finished within 0.5 h at 37°C. Reincubated chlamydiae, not attached after 3 h at 37°C, attached readily to fresh McCoy cell monolayers, but to a lesser extent than the original inoculum. Our results indicate that the lack of further attachment after 3 h incubation at 37°C under stationary infection conditions has complex causes, involving both host cell and parasite. Centrifugation did not affect the uptake of chlamydiae already bound to the cell membrane, suggesting that the uptake phase of C. trachomatis serovar E by McCoy cells is unaffected by centrifugation.     

Adherence of multiple serovars of Chlamydia trachomatis to a common receptor on HeLa and McCoy cells is mediated by thermolabile protein(s)

Several aspects of the adherence of purified elementary bodies (EB) of Chlamydia trachomatis to HeLa and to McCoy cells were examined using different techniques, including an ELISA. Serovar-specific, biotinylated monoclonal antibodies were used to detect cell-bound chlamydiae. In addition, purified chlamydiae were biotinylated and their adherence properties were studied. The assays were done at 4 degrees C to exclude the energy-dependent internalization of the cell-bound EB and host-cell membrane recycling that occur at 37 degrees C. Saturation kinetics were routinely observed at 4 degrees C, and the rate of adherence remained linear for approximately 60 min. Lineweaver-Burk analysis of the kinetics data showed that adherence of any one serovar was competitively inhibited by other serovars of C. trachomatis. This competition for the same receptor on the two alternative hosts, HeLa and McCoy, was also seen when the adherence assays were done at 37 degrees C in the presence of sodium azide, an energy poison that inhibits endocytosis of cell-bound chlamydiae. Chlamydiae exposed to 56 degrees C for 5 min, or treated with low doses of trypsin, failed to exhibit competitive inhibition, having suffered considerable loss of the ability to adhere to host-cells. These data suggest that heat- and trypsin-labile chlamydial moieties participate in the adherence reaction, and that oculo-genital serovars of C. trachomatis, including that of lymphogranuloma venereum, attach to the same receptor on the host-cell membrane.     

Recovery ofChlamydia trachomatis by inoculation of McCoy cell suspensions

Growth of two laboratory strains ofChlamydia trachomatis (Gambia 17 and UW 5) was compared in McCoy cell monolayers 1–3 days old and McCoy cells inoculated while in suspension (day 0). Both cell preparations were treated with cytochalasin B. Each chlamydial strain produced more inclusions in the cell suspension preparations than in monolayers. Efforts to inoculate cycloheximide-treated cells in suspension were unsuccessful because of the toxic effect of this chemical. When patient specimens were tested in cell suspensions and in monolayers, all positives were detected comparably in both systems, although bacterial contamination was more pronounced in the cell suspensions. The results indicate that cell suspensions can be used as a convenient and rapid supplement to preformed monolayers for chlamydial culture tests.     

Host modification of chlamydiae: differential infectivity for cell monolayers of chlamydiae grown in eggs and monolayers.

Cell monolayer-grown chlamydiae (CGO) differed from egg-grown organisms (EGO) in their increased spontaneous infectivity relative to centrifuge-assisted infectivity for monolayers. For each population spontaneous: centrifuge-assisted infectivity ratios were constant over a wide dose range. Spontaneous infection increased linearly with time and could not be exhausted from either population by prolonged adsorption; there was no change in infectivity ratios in residual supernatants. Further, one passage of EGO through monolayers gave CGO with stable infectivity properties not increased by further cell passage yet reverting on a single passage in eggs. Spontaneous infection of monolayers with EGO gave progeny with the same infectivity ratios as monolayers infected with EGO by centrifugation. The change in properties following EGO infection of monolayers occurred prior to natural release from cells. We conclude that EGO and CGO are two phenotypically distinct, homogeneous populations. The two infection modes are not properties of subpopulations within EGO and CGO. The relationship of these observations on chlamydiae to other possible host-imposed phenomena is considered.     

Interaction of Chlamydia trachomatis with human genital epithelium in culture

Primary cultures of human endometrial and ectocervical epithelial cells were examined as a new model system to study genital infection by Chlamydia trachomatis. Initial studies demonstrated that these cells were indeed susceptible to chlamydial infection. Inocula, adjusted to produce inclusions in 50 to 80% of equivalent numbers of standard McCoy cells, resulted in infection rates of approximately 15 to 30% for the columnar cells of the endometrium and 5 to 10% for the squamous cells of the ectocervix. Exposure of cultures to DEAE-dextran and centrifugation-assisted inoculation, manipulations reported to enhance infection of HeLa and McCoy cells, did not alter the number of inclusion-positive genital cells. Addition of cycloheximide to the post-inoculation culture medium slightly increased numbers of inclusion-bearing cells while growth of genital cells in hormone-supplemented medium resulted in a variable effect on inclusion development and a significant reduction in the association of radiolabelled organisms with these cells. The basis for the different levels of infection in McCoy versus genital cell cultures was revealed by immunofluorescence analysis of chlamydial association with host cells immediately after inoculation. Chlamydiae failed to adhere to many cells in the genital cell cultures while adherence to McCoy cells was uniform. In addition, the association of radiolabelled C. trachomatis was significantly lower with genital cells than with McCoy cells. Finally, culture conditions were defined which markedly inhibited inclusion development without an immediate loss of chlamydial growth potential. This investigation indicates that primary genital cell cultures are susceptible to chlamydial infection and will be valuable for studies on the nature of C. trachomatis interactions with natural human target cells.     

Endocytosis of chlamydiae by McCoy cells: measurement and effects of centrifugation

Abstract Chlamydia trachomatis strain 434 and C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) were compared for entry into McCoy cells and expression of productive infection (inclusion body formation). Entry was measured as the difference between extracellular cell-associated organisms, determined directly after fluorescence staining of live cells, and total cell-associated organisms (intracellular and extracellular); the latter were evaluated from radioactivity measurement and known particle: radioactivity ratios for stock radiolabelled suspensions. Under inoculation conditions of natural (spontaneous) infection, 69–82% of cell-associated organisms of both strains were internalised and entry was not enhanced by centrifugation of inocula with monolayers. For 434, inclusion bodies were seen in 10–20% of cells containing organisms and numbers were little influenced by mode of infection. For GPIC, productive infection initiated by centrifugation was comparable with that of 434 but some 15-fold reduced in spontaneous infection. The results suggest that unproductive infection by GPIC occurs, not because of defective entry, but from inhibition at an intracellular step which is circumvented when infection is initiated by centrifugation.     

Assay of Variola Virus by the Fluorescent Cell-Counting Technique

A quantitative assay for infective variola virus particles was developed which is based on the enumeration of cells containing fluorescent viral antigen after infection of McCoy cell monolayers. The direct fluorescent-antibody technique was employed to stain cells. The efficiency of virus adsorption was markedly enhanced by centrifugation of virus inoculum onto McCoy cell monolayers at 500 x g for 15 min. By this procedure, a proportionality was obtained between the number of fluorescent cells and volume of inoculum. Observations on the sequential development of viral antigen within cells and counts of fluorescent cells showed that the optimal time for enumerating fluorescent cells was after an incubation period of 16 to 20 hr. A linear function existed between virus concentration and cell-infecting units. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The assay was demonstrated to be highly sensitive, precise, and reproducible.     

Infection by Mycoplasma hyorhinis strongly enhances uptake of antisense oligonucleotides: a reassessment of receptor-mediated endocytosis in the HepG2 cell line

This paper shows that the ~66 kDa band, previously isolated from the HepG2 cell line as an oligonucleotide (ON) plasma membrane ‘receptor’, is induced by Mycoplasma infection. Moreover, this band has been identified as the invariant membrane protein of Mycoplasma hyorhinis, p70, based on ribosomal DNA sequencing combined with ON ligand blotting after p70 immunoprecipitation by a monoclonal antibody. Whereas antibiotic treatment of infected HepG2 cells strongly decreased ON capture, as measured by a biochemical assay, conversely, deliberate infection of HeLa cells with M.hyorhinis dramatically promoted ON uptake but did not affect receptor-mediated endocytosis of transferrin. This was confirmed by confocal microscopy of infected HepG2 cells, which also showed an indistinguishable labelling pattern after exposure of living cells to fluorescent ON and after p70 immunolabelling in permeabilised fixed cells. We propose that ON binds to p70 on M.hyorhinis attached at the cell surface, after which the complex is internalised by ‘piggy-back’ endocytosis.     

Chlamydia trachomatis in cell culture. II. Susceptibility of seven established mammalian cell types in vitro. Adaptation of trachoma organisms to McCoy and BHK-21 cells

Trachoma organisms of serotype B were grown serially in irradiated cells (McCoy, BHK-21, Microbiological Associates, and BHK-21, Lister) and tested for infectivity in monolayers of five mammalian cell lines (BHK-21, CHO, HeLa S3, McCoy and OWMK) and two diploid strains (ST/BTL and WI-38). All cell types had low susceptibility to chlamydial infection but the number of inclusions increased when the inoculum was centrifuged onto the monolayers, or when the cells were irradiated. Infection was higher in non-irradiated CHO than in irradiated CHO in three out of a total of six experiments. Inclusion number was increased 300 times in HeLa S3 and up to three times in the other cell types after treatment with diethylaminoethyl-dextran (DEAE-D). Serial passage of Chlamydia trachomatis serotype B (strain Har-36) in CO60 McCoy and CO60 BHK-21 Lister resulted in partial adaptation of the strain to the host cell. The phenomenon of adaptation of serotype B to McCoy compensated for the lower susceptibility of this cell revealed when McCoy cells were inoculated with trachoma elementary bodies grown in BHK-21 Lister or in chick embryo yolk sac. Trachoma organisms of immunotypes A, B and C prepared in yolk sac produced more inclusion-forming units per ml in CO60 BHK-21 Lister than in CO60 McCoy.     

Novel Chlamydiales strains isolated from a water treatment plant

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers ( Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae ; two closely related strains belonged to the Criblamydiaceae ; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.     

Host modification of the adherence properties of Chlamydia trachomatis

The adherence of Chlamydia trachomatis LGV440(L1) to human HeLa 229 and mouse McCoy cells was stimulated by the lectin wheat germ agglutinin (WGA) and inhibited by the sugars N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and chitobiose, but only when the chlamydiae had been passaged several times in HeLa cells. After passage in McCoy cells, the lectin and the sugars elicited little response. The non-LGV serovar UW-31(K), however, differed from LGV440(L1) in that, regardless of passage, the lectin and sugar effects were observed only in HeLa cells. Affinity chromatography on WGA-agarose confirmed that HeLa-grown LGV440(L1) bound to a significantly greater extent relative to McCoy-grown chlamydiae. In addition, participation of heterogeneous chlamydial ligands was suggested by the observation that the adherence of heated (60 degrees C, 5 min) UW-31(K) to HeLa cells at 37 degrees C was not inhibited at all, but at 5 degrees C, the adherence rate was greatly reduced, indicating the participation of heat-stable as well as heat-labile ligands. These data are interpreted to indicate that the passage history of C. trachomatis results in the acquisition of altered surface components that participate in the initial interaction of the bacterium with the host.     

Primary Virus-Cell Interactions in the Immuno-fluorescence Assay of Venezuelan Equine Encephalomyelitis Virus

The conditions under which Venezuelan equine encephalomyelitis (VEE) virus attached to host cells markedly influenced the assay of virus by the fluorescent cell-counting technique. When virus inoculum was centrifuged onto McCoy cell monolayers, approximately 97% of virus was attached to cells within 10 min, in contrast to 34% after stationary incubation at 35 C for 2 hr. Maximal binding of virus occurred only in the presence of 0.1 to 0.15 m NaCl. This salt requirement, added to evidence of (p)H dependence and temperature independence of VEE virus attachment to cells, indicated that the initial union involved electrostatic forces. Virus penetration, measured by the insensitivity of virus-cell complexes to viral antiserum, was complete in 30 min at 35 C. The process was temperature-dependent and un-affected by the ionic content of medium. For assay of VEE virus by the fluorescent cell-counting technique, infected cells may be enumerated as early as 12 hr after infection of cell monolayers. The relationship between virus concentration and cell-infecting units was linear; the distribution of fluorescent cells was random. The virus assay was equivalent in sensitivity but more precise and rapid than that of intracerebral inoculation of mice.     

Trifluoperazine inhibits the infectivity of Chlamydia trachomatis for McCoy cells

Abstract Trifluoperazine (TFP), an inhibitor of the Ca²⁺-binding protein calmodulin, was used to study the infectivity of Chlamydia trachomatis for McCoy cells. TFP inhibited the number of chlamydial inclusions and the chlamydia-dependent amino acid incorporation when added within 9 h after inoculation with chlamydiae. However, TFP did not affect the attachment of chlamydiae to the cells or the protease-removable fraction of cell-bound chlamydiae.
These results suggest that an early step in the intracellular development of chlamydiae, partly coinciding with the elementary body-reticulate body conversion, is sensitive to TFP and that clathrin coats are not crucial in the ingestion of chlamydiae by McCoy cells.     

Growth of Chlamydia trachomatis in McCoy Cells Treated with Cytochalasin B

When the fungal metabolite cytochalasin B was added to McCoy cells, multinucleated giant cells developed. Monolayers of these cells proved as efficient as irradiated cells for the growth of three different serotypes of Chlamydia trachomatis and for the primary isolation of chlamydiae from clinical specimens obtained from patients attending a venereal disease clinic. Cytochalasin treatment of McCoy cells provides a convenient alternative to irradiation and should be of value in the isolation of chlamydiae from the eye and genital tract.     

In silico inference of inclusion membrane protein family in obligate intracellular parasites chlamydiae.

Chlamydiae are obligate intracellular pathogens that proliferate only inside a vacuole, called an inclusion. Chlamydial Inc proteins are known to be a major component of the inclusion membrane, but little is known about the gene number and function. The Inc proteins share very low sequence similarity but a similar hydropathy profile among them. Using the hydropathy profile, we computationally searched the open reading frames (ORFs) having a similar profile and predicted 90 and 36 ORFs (Inc-like ORFs) as candidates for Inc proteins in Chlamydia pneumoniae J138 and Chlamydia trachomatis serovar D, respectively. On the other hand, only a few Inc-like ORFs were found in organisms other than chlamydiae, suggesting that the Inc-like ORFs are specific to chlamydiae. Comparative genome analysis also revealed that the Inc-like ORFs have multiplied and diverged as paralogues and orthologues in the chlamydial genomes, and that some Inc-like ORFs lacked the N-terminal portion or encoded the split form. The data suggest that these gene products constitute a large protein family and may play an important role in chlamydial infection, growth and survival in the host cell.     

Use of ultrasound to increase infection of McCoy cell monolayers by Chlamydia trachomatis strain

An ultrasound cell disrupter with a cooled cup tip was used to increase rapidly Chlamydia trachomatis infection in vitro. After three growth cycles of the NI-1 strain (serovar E), the pulsed ultrasound use enhanced the number of infected McCoy cells by approximately 12-times, as compared with control; and 8.8-times over the shaking with glass beads and centrifugation technique. After three growth cycles of the fast-growing LB-1 strain (serovar L2), the enhancement was by 15 and 10.8 respectively. Consequently, ultrasound treatment with a cooled cup tip can offer a working standard procedure to increase rapidly the number of cells infected with Chlamydia.     

Effect of trypsinization on susceptibility of primary human amniotic cells toChlamydia trachomatis TW-3 strain

The effect of trypsinization of human amnion membranes on the susceptibility of amnion cells toChlamydia trachomatis TW-3 infection was examined by infectivity titrations using standard procedures of chlamydial inoculation, and detection of chlamydial inclusions. Epithelial cells derived from freshly trypsinized membranes as well as primary and secondary cultured cells that were freshly removed from monolayers by trypsin treatment were not susceptible to infection at 30 min and at 2 and 6 h after trypsinization. Monolayers grown 18 h and up to 5 or more days after trypsinization were susceptible to infection. Primary 5-day monolayers derived from each of nine placentas inoculated with chlamydiae showed a range of titers from 10⁻³ to 10^−6.5 (SD=1.2 logarithm). Primary monolayers supported the multiplication of chlamydiae to consistently higher titers than secondary and tertiary monolayers from the same amnion.     

Differential neutralization of spontaneous and centrifuge-assisted chlamydial infectivity

Neutralization by specific antibody of a fast-killing variant strain of Chlamydia trachomatis, which showed high spontaneous infectivity for cell monolayers, was examined. It appeared that in spontaneous infection antibody-treated chlamydiae were neutralized by inhibition of attachment to cells. Centrifugation imposed a different effect: infection was inhibited at some step at or subsequent to attachment.     

Intracellular survival by Chlamydia

Chlamydiae are obligate intracellular bacterial pathogens whose entry into mucosal epithelial cells is required for intracellular survival and subsequent growth. After a seemingly stealthy entry, chlamydiae quickly modify their vacuole (i) for exit from the endosomal pathway to the exocytic pathway and (ii) to permit fusion with intercepted endoplasmic reticulum- and Golgi-derived vesicles carrying glycerophospholipids and sphingolipids for chlamydiae-containing vacuole membrane expansion. Chlamydiae possess novel hollow proteinaceous structures, termed projections, which they use to pierce the inclusion membrane, possibly to acquire from the epithelial cytoplasm nutrients they cannot synthesize; whether or not these truncated flagellar-like structures serve a dual exchange function for secretion of molecules to programme host cell signalling is unknown. Despite the accumulation of some 500–1000 progeny in the enormously enlarged inclusion, host cell function is surprisingly little disrupted, and progeny escape can be unobtrusive. This elegant adaptive pathogen strategy, which leads to silent, chronic human infection, is fascinating from a cellular microbiology perspective.