Vol. 26, No. 2
INFETICON AND IMuNIT, Nov. 1979, p. 775-778
Isoelectric Focusing of Chlamydia trachomatis ROBERT J. KRAAIPOEL* AND ANS M. vAN DUIN Deparnent of Clinical Microbiology and Antimicrobial Therapy, Erasmus University, Rotterdam, T'he Netherlads Received for publication 3 August 1979
HeLa-229 cells and the elementary bodies of Chlamydia trachomatis had a net negative electrical surface charge at neutral pH when measured by isoelectric focusing. Inclusion-forming and non-inclusion-forming elementary bodies focused in one band at pI 4.64.
Chlamydia trachomnatis is an intracellular parasite that multiplies by means of a developmental cycle within vacuoles (inclusions) derived from inva-inations of host cell membrane during the act of phagocytosis. Death of the host cells infected with C. trachomatis is associated with the emergence of a new population of infectious elementary bodies (EB). However, only 30 to 60% of the EB, obtained after a single-cycle cell culture growth, are able to form inclusions in a HeLa-229 monolayer cell culture system (4). This result may be because some of the EB were not viable (or infective) at the time of inoculation. Techniques to increase the infective number of EB would be important in biochemical studies of EB. One such technique is isoelectric focusing (IEF). IEF has been succesflly used to separate the viable from the nonviable HeLa cells by their difference in net electrical surface charge (12). Means other than infection to distinguish between the inclusion-forming EB and the noninclusion-forming EB have not been described. Our study was designed to use IEF as a means of separating viable, inclusion-forming EB from non-inclusion-forming EB. The infection of HeLa-229 cells with C. trachomatis is enhanced by the positively charged polycation diethylaminoethyl (DEAE)-dextran and inhibited by polyanions. Therefore, it is assumed that repulsive electrical forces play a major role in the attachment of the EB to the cell (1, 5, 10). To check this hypothesis, we measured the isoelectric point and thus the net electrical surface charge of HeLa-229 cells and C. trachomatis EB. C. trachomatis organisms of the lymphogranuloma venereum type and serotype D were chosen for the experiments (13). These organisms were supplied by S. P. Wang, University of Wasington, Seattle. Monolayers of HeLa-229 cells were infected as previously described (7). On day 3 after infection, the supernatant fluid
was discarded, and the infected cell layer was removed from the bottle with glass beads in 30 ml of Hanks balanced salt solution. The cell suspension was sonicated for 20 s (MSE Ultrasonic Disintegrator, 100 W, 20 kilocycles/s, Measuring and Scientific Equipment Ltd, London, England). The cell debris was removed by centrifugation at 500 x g for 10 min, and the EB were pelleted at 25,000 x g for 20 min. The pellet was resuspended in Hanks balanced salt solution and layered on top of 30% sucrose in 30 mM 2hydroyethylpiperane -N'-ethanesulfonic N2acid (HEPES), pH 7.3, and centrifuged at 8,000 x g for 60 min in a swinging-bucket rotor. The final pellet was used in the IEF experiments. After formation of the pH gradient in the IEF column (9, 14) (see legend to Fig. 1), 4 ml of the gradient solution (pH 7) was withdrawn and mixed with 107 to 108 EB (8). This solution was pumped back into the column. Focusing was continued at 700 V (3 mA), and the negatively charged EB migrated toward the anode into the bottom of the column. After 3 to 4 h the EB formed a sharp, visible band where the net electrial charge was zero. This was the isoelectric point (pI). After removal of the electrode solution, the IEF column was emptied at a flow rate of 1.5 ml/min. About 30 fractions were collected. pH and absorbance at 280 nm and 420 nim were measured. Chlamydia (LGV-2) EB were found in fractions 5 to 10 at pI 4.25 (Fig. 1). C. trachomatis EB of serotype D were found in fractions 9 to 13 at pI 4.25 (Fig. 2). The number of EB in each fraction was estimated qualitatively by a method for total particle counts (8). This estimation corresponded well with the absorption peak at 420 nm. After washing, the focused EB still proved to be infectious to monolayers of HeLa-229 cells. To avoid possible artifacts in the pI value, Chlamydia EB were immediately removed from the column after reaching their isoelectric point.
00 i%; V
13 17 21 25 29 fraction number FIG. 1. Fractionation of C. trachomatis (LGV-2) EB byIEF. The 110-ml IEF column (LKB 8100-1, Bromma, Sweden) contained 1% (wt/vol) Ampholine (LKB) with a pH range of 3.5 to 10. The anode solution (bottom) and the cathode solution contained H3P04 and ethanolamine, respectively (9). A 3-mA current was maintained through the column, which was kept at 40C. After 24 h the pHgradient, stabilized by a linear sucrose gradient (50 to 5%), was established. The EB were loaded into the column as a zone at the pH 7 level. After 3 to 4 h of focusing, the column was elated as described in the text. Protein concentration was monitored at 280 nm (A), and both inclusion-forming and non-inclusion-forming EB were monitored at 420 nm (0). The open circles represent the pH at 40C. 5
Chlamydia (LGV-2) EB focused at a mean pI of 4.64 ± 0.14 standard deviation, N = 8. The same pI result was obtained if the Chlamydia organisms were inactivated at 500C for 30 min in a water bath. Because of the difference in pI found with the two methods, it was concluded that the EB sedimented during elution. Before loading the IEF column, as shown in Table 1, 54% of the EB formed inclusions. On day 1, half of a harvest was pumped into the column at the level of pH 8.2. After 4 h of focusing, the sharp, visible band at pH 4.70 was removed and quantitated. The total number of focused EB was similar to the total number of EB pumped into the column. The conclusion must therefore be that all EB, inclusion forming and non-inclusion forming, focused at the same
The remainder of the harvest was frozen at
-70oC for 24 h in phosphate-buffered saline. On day 2 the experiment was repeated. The freezethawing procedure resulted in marked loss of viable EB, as seen in Table 1. To estimate the surface charge of HeLa-229 cells, IEF of these cells was performed as described by Sherbet et al. (12). We found a pI value of 6.85 ± 0.24 standard deviation, N = 4, for viable cells and a pI of 5.47 ± 0.53 standard deviation, N = 4, for nonviable cells. Using HeLa cells, Sherbet et al. found pI values of 6.85 ± 0.11 for viable and 5.32 ± 0.11 for nonviable cells. Our results with HeLa-229 cells were thus comparable with the previously published values for HeLa cells. The results of our study showed that both the viable and the nonviable EB of C. trachomatis
VOL. 26, 1979
17 13 21 25 29 fraction number FIG. 2. Fractionation of C. trachomatis (serotype D) EB by IEF. Protein concentration was monitored at 280 nm (A), and both inclusion-forming and non-inclusion-forming EB were monitored at 420 nm (0). The open circles represent the pH at 40C.
TABLE 1. Quantitation of the total number of EB of C. trachomatis (LGV-2) and the percentage of inclusion-forming EB before and after IEF % Inclusion-forming __m _____Ttalno.ofB__Bb
Day Total no. of EB' Day
Loaded onto col-
pH at EB
using 7 x 105 8.2 1 31 4.70 3.5 X 105 2 6 7.8 4.60 The total number of EB (inclusion forming and non-inclusion forming) was estimated by a method for total counts (8). particle b The percentage of inclusion-forming EB is calculated from the ratio of the number of inclusions in a HeLa229 monolayer (3) to the total number of Chlamydia EB (8). column 7 x 108 4.3 x 105
focused in one band. Hence, the technique of LEF was not capable of separating the inclusionforming EB from the non-inclusion-forming EB of C. trachomatis. The positively charged polycation DEAE-dextran enhances the inclusion formation of certain
using 54 9
chlamydial strains (2, 5, 6, 11). It has been suggested that the positively charged DEAE-dextran diminishes the repulsive forces that may exist between the Chlamydia EB and the HeLa229 cells (1, 6, 10). The pI measurements showed that the net electrical surface charge of both the
EB and the HeLa-229 cells was negative at pH 7.3. The results of our IEF experiments confirmed the assumption that repulsive electrical forces exist between EB and HeLa cells. However, DEAE-dextran enhances the attachment and inclusion formation of trachoma but not of lymphogranuloma venereum organisms (4). According to the suggested mechanism of action of DEAE-dextran, a difference in net electrical surface charge between trachoma (serotype D) and lymphogranuloma venereum organisms (LGV2) might be expected. Our experiments showed, however, that both strains had a similar pI and thus a similar electrical surface charge. These findings lead us to the conclusion that the surface charge plays only a minor role in the mechanism of infectivity enhancement by DEAE-dextran. We thank R. C. Noble and Karin Reimann for review of the manuscript.
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INFECT. IMMUN. Infect. Immun. 13:1103-1109. 5. Kuo, C. C., S. P. Wang, and J. T. Grayston. 1972. Differentiation of TRIC and LGV organisms based on enhancement of infectivity by DEAE-dextran in cell culture. J. Infect. Dis. 125:313-317. 6. Kuo, C. C., S. P. Wang, and J. T. Grayston. 1973. Effect of polycations, polyanions, and neuraminidase on the infectivity of trachoma-inclusion conjunctivitis and lymphogranuloma venereum organisms in HeLa cells: sialic acid residues as possible receptors for trachomainclusion conjunctivitis. Infect. Immun. 8:74-79. 7. Kuo, C. C., S. P. Wang, and J. T. Grayston. 1977. Growth of trachoma organisms in HeLa 229 cell culture, p. 328-336. In D. Hobson and K. K. Holmes (ed.), Nongonococcal urethritis and related infections. American Society for Microbiology, Washington, D. C. 8. Reeve, P., and J. Taverne. 1962. A simple method for total particle counts of trachoma and inclusion blennorhoea viruses. Nature (London) 195:923-924. 9. Righetti, P. G., and J. W. Drysdale. 1976. Isoelectric focusing. In T. S. Work and E. Work (ed.), Laboratory techniques in biochemistry and molecular biology, vol. 5, part II. North-Holland Publishing Co., Amsterdam. 10. Ripa, K. T., and P. A. Mardh. 1977. Cultivation of Chlamydia trachomatis in cycloheximide treated McCoy cells. J. Clin. Microbiol. 6:328-331. 11. Rota, T. R., and R. L. Nichols. 1971. Infection of cell cultures by trachoma agent: enhancement by DEAEdextran. J. Infect. Dis. 124:419-421. 12. Sherbet, G. V., M. S. Lakshmi, and K. V. Rao. 1972. Characterisation of ionogenic groups and estimation of the net negative electric charge on the surface of celLs using natural pH gradients. Exp. Cell Res. 70:113-123. 13. Wang, S. P., and J. T. Grayston. 1970. Immunologic relationship between genital TRIC, lymphogranuloma venereum, and related organisms in a new microtiter indirect immunofluorescence test. Am. J. Ophthalmol. 70:367-374. 14. Winter, A., and C. Karlsson. 1976. Preparative electrofocusing in density gradients. Application note 219, LKB-produkter, Bromma, Sweden.