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To determine if the host-modulated adherence characteristics of the intracellular bacterial pathogen Chlamydia trachomatis were due to the acquisition of altered surface-exposed proteins, highly purified chlamydiae grown in two different host cells were analysed. Two serovars, L1 and E, were grown for multiple passages in both HeLa and McCoy host cells. Numerous protein differences in the chlamydial elementary bodies (EB) of each serovar grown in the two different hosts were detected by two-dimensional (2-D) gel electrophoresis and fluorography of radioactively labelled proteins. At least four to six serial passages in the alternative host were necessary before the changes were apparent. Iodination of suspensions of purified chlamydiae and 2-D electrophoresis revealed several surface proteins that were determined by the host cells in which the bacteria had replicated. These iodinated chlamydial proteins were removed by treatment of the iodinated EB with trypsin, indicating their location at the bacterial surface. Two of the major constituents of the outer-membrane complex, the cysteine- and methionine-rich 60 kDa and 40 kDa proteins, remained unchanged in both molecular mass and charge during the host adaptation. Several chlamydial proteins capable of binding iodinated host membrane preparations also exhibited host-dependent alterations. Immunoblotting experiments with a rabbit and a human polyclonal sera indicated that distinct host-specified chlamydial proteins were reactive with the two sera.

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 chlamydial 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.

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.

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.

Twelve mycobacillin-negative (My-) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. In whole-cell fermentations of some of these My- mutants a penta- and a nonapeptide accumulated; these peptides were also obtained in a cell-free system in which a new tripeptide was also detected. The amino acid composition, N- and C-terminal residues and amino acid sequence of these peptides agreed with those of equivalent segments of the mycobacillin molecule. The mycobacillin-synthesizing enzyme can be divided into three fractions that catalyse different steps in biosynthesis, and the defective enzyme fractions in the various mutant strains were identified by reconstitution experiments in vitro. The defects were further pin-pointed in mutant enzyme fractions by an ATP in equilibrium Pi exchange reaction and also by cell-free synthesis involving the use of membrane-bound enzyme. The defects so identified indicated the formation of tri-, penta- and nonapeptides as intermediates in the mycobacillin biosynthetic pathway.

Thirteen mycobacillin-negative (My-) mutants of Bacillus subtilis B3 were isolated from an auxotrophically tagged mycobacillin producer organism. The wild-type producer, three feeble producers and three strictly My- mutants did not accumulate any ninhydrin-positive peptide in the culture medium while the remaining seven My- mutants did accumulate ten such peptides whose amino acid composition indicated that there might be only three different peptides. The N-terminal and C-terminal amino acid residues implicated one of these peptides as a pentapeptide intermediate in mycobacillin synthesis; this was further confirmed by its molecular weight and sequence. Studies on cell-free synthesis showed that only the enzyme system from the wild-type strain synthesized mycobacillin while the defective ones from all the My- mutants synthesized one and the same pentapeptide as found in the culture broth of some of the mutants. Further studies in which the enzymes responsible for mycobacillin synthesis by cell-free extracts were separated into three fractions, A, B and C, showed that seven of the mutants were defective in fraction B whereas the three other mutants had defects in both fractions B and C. Thus the pentapeptide Pro----Asp----Glu----Tyr----Asp appears to be implicated in mycobacillin biosynthesis.

Dibutyryl cyclic GMP, but not dibutyryl cyclic AMP, derepresses sporulation and synthesis of mycobacillin and dipicolinic acid under conditions of glucose repression in Bacillus subtilis strain B34. Neither of these compounds appears to affect sporulation and synthesis of mycobacillin and dipicolinic acid in this strain under normal physiological conditions. Mutants insensitive to glucose repression were indifferent to the addition of either of the nucleotides both in the presence and in the absence of glucose. A role for dibutyryl cyclic GMP in annulling the repressing effect of glucose on sporulation and on synthesis of mycobacillin and dipicolinic acid is thus indicated.