Purification of Chlamydia trachomatis Lymphogranuloma Venereum Elementary Bodies and their Interaction with HeLa Cells

Abstract

A procedure has been developed to yield infectious elementary bodies of the lymphogranuloma venereum strains LGV 434 and 404 of Chlamydia trachomatis, labelled during intracellular growth in HeLa 229 cells. The final preparation, obtained after velocity sedimentation of a polycarbonate membrane-filtered sample through a sucrose gradient, is free of host proteins and, more importantly, of chlamydia1 reticulate bodies. Using such purified preparations, it was found that the association of LGV 434 elementary bodies with HeLa 229 cultures was unaffected by the pretreatment of the host cells with a variety of lectins or with neuraminidases from Clostridium perfringens and Vibrio cholerae. The association was inhibited by dextran sulphate and by mild trypsin treatment of HeLa cultures. Treatment of purified elementary bodies with trypsin, chymotrypsin, neuraminidases and a variety of carbohydrates and lectins did not produce any change in the rate of association with HeLa cultures. Heat-inactivated elementary bodies were significantly less able to associate with the host cells.

Adherence of Multiple Serovars of Chlamydia trachomatis to a Common Receptor on HeLa and McCoy Cells Is Mediated by Thermolabile Protein(s)

“…In three separate experiments adherence of the [35S]methionine/[35S]cysteine-labelled L1 C. trachomatis exposed to 1 pg trypsin per 10 pg EB protein for 60 rnin at 37 "C was inhibited by more than 95% when the assays were done at 4°C (data not shown). No inhibition was detected when the adherence assays were done at 37 "C, confirming results with the L2 serovar (Bose & Paul, 1982;Hackstadt & Caldwell, 1985;Su et al, 1988). A double-reciprocal plot of the data obtained at 4 "C with multiple inputs of untreated and trypsin-treated Ll revealed that, in addition to the decrease in V, the apparent K , of adherence by treated EB was 100-fold higher (data not shown).…”

“…In our previous studies on the adherence characteristics of C. trachomatis to monolayer cultures of HeLa, McCoy and CHO cells and the glycosylation-defective mutant cell lines of CHO (Bose & Paul, 1982;Bose et al, 1983;Bose & Goswami, 1986a, 6) we utilized metabolically radioactively labelled, highly purified preparations of chlamydial EB. Whenever possible, the data obtained with radioactive EB were confirmed by infectivity determinations.…”

“…Such EB are also incapable of competitively inhibiting the adherence of untreated EB ( Table 2). Trypsinization of the L2 serovar is innocuous both with respect to association with HeLa cells at 37 "C and infectivity (Bose & Paul, 1982;Hackstadt & Caldwell, 1985;Su et al, 1988). Adherence exclusive of ingestion is greatly diminished due to trypsin treatment of purified EB, regardless of the serovar, and such chlamydiae are incapable of interfering with the adherence of heterologous serovars.…”

Host Modification of the Adherence Properties of Chlamydia trachomatis

“…We had previously reported that the adherence of two LGV serovars, 434(L2) and 404(L3), to HeLa 229 was unaffected by WGA (Bose & Paul, 1982), while that of UW-31(K) was enhanced in the absence of the stimulator of adherence, DEAE-dextran (Bose et al, 1983). Data summarized in Table 1 show that the adherence of LGV440 grown in HeLa cells was stimulated by WGA.…”

“…For labelling with [5,6-3H]uridine (25 yCi ml-' ; 925 kBq ml-l), regular chlamydial growth medium was used. Reticulate body-free EBs were obtained by velocity sedimentation of filtered samples through a linear 10 to 40% sucrose gradient (Bose & Paul, 1982). After concentration by sedimentation of the EB-containing fractions, samples were quick-frozen and stored at -80 "C.…”