INFECTION AND IMMUNITY, Apr. 1976, p. 1103-1109 Copyright © 1976 American Society for Microbiology

Vol. 13, No. 4 Printed in U.SA.

Interaction of Chlamydia trachomatis Organisms and HeLa 229 Cells CHO-CHOU KUO* AND J. THOMAS GRAYSTON Department ofPathobiology* and the Department ofEpidemiology and International Health, School ofPublic Health and Community Medicine, University of Washington, Seattle, Washington 98195 Received for publication 15 October 1975

The infection of HeLa 229 cells in monolayer culture with trachoma (B/TW-5/ OT) and lymphogranuloma venereum (LGV) (L2/434/Bu) organisms was studied in terms of two parameters: radioactivity counts of cell-associated tritium labeled organisms at the initial stage of inoculation for measurement of attachment, and inclusion counts of infection cells after incubation for measurement of growth. Factors affecting attachment and inclusion formation and correlation of the two are presented. It was shown that attachment is an important initial step in infection by Chlamydia trachomatis. The rate of attachment was temperature dependent. The attachment of LGV organisms was affected more profoundly by temperature than was that of trachoma organisms. Attachment and inclusion formation of trachoma and LGV organisms were inhibited by heparin. Diethylaminoethyl-dextran was again shown to enhance attachment and inclusion formation of trachoma but not LGV organisms. NaF had no effect on attachment, but inhibited inclusion formation of both trachoma and LGV organisms. Both attachment and inclusion formation of trachoma organisms were strongly enhanced by centrifugation of the inoculum onto the cell monolayer. Although inclusion formation of trachoma organisms was much greater in susceptible cells (HeLa 229) than relatively insusceptible cells (fetal tonsil), attachment was only slightly greater. The results based on the test of two cell lines suggested that attachment probably is not a critical factor in determining a cell line's susceptibility to infection with trachoma organisms. There have been reports both by us and others of factors that enhance or inhibit the infectivity of Chlamydia trachomatis organisms in cell cultures (8, 12-14, 16, 21). Weiss and Dressler were the first to note that infection of cell culture by trachoma and psittacosis organisms was enhanced by centrifugation of inoculum onto cell monolayer (21). Since their discovery, this has been a standard procedure for cell culture isolation of trachoma organisms (7, 14). Enhancement of cell culture infection with psittacosis and trachoma organisms by the polycation diethylaminoethyl (DEAE)-dextran has been documented (8, 12, 13, 16). We reported that DEAE-dextran enhanced infection of trachoma but not lymphogranuloma venereum (LGV) organisms, which led us to propose the use of this criterion for differentiation between these two organisms (12). The polyanion dextran sulfate has been found to inhibit the infection of both trachoma and LGV organisms (13). These studies were based on inclusion counts, which are the final results of the infection process of attachment, penetration, and maturation of the organisms.

The use of radioisotope-labeled chlamydial organisms for studies of host-parasite interaction is now feasible since labeled organisms free from labeled host cell materials can be prepared. Chlamydial organisms are selectively labeled by incorporation of radioactive amino acids in the culture medium in the presence of cycloheximide (2) or emetine (3), which inhibit protein synthesis of the host cells without affecting that of the parasites. A standard purification method, such as density gradient centrifugation, removes the small amounts of contaminating labeled host materials. Using this technique, Friis has studied '4C-amino acid-labeled C. psittaci strain meningopneumonitis in L cell suspension cultures (6). He found that adsorption was not a significant factor in the process of uptake and that the efficiency of infection was directly proportional to the multiplicity of infection. A preliminary report on studies using '4C-amino acid-labeled C. trachomatis strain was reported by Becker et al. (4). The T'ang strain was used. This strain is a "fast-killing strain," serologically identical to LGV type 2 (20). The report presented the kinetics of ad-

1103

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INFECT. IMMUN.

sorption of T'ang strain organisms to BSC, cells N.J.), and centrifuged at 45,500 x g for 90 min. The at 37 C and showed inhibition of attachment by band of infectious particles at the middle of the gradient was removed. They were suspended with heparin. and pelleted. The pellet was washed once For this report, trachoma and LGV orga- HBSS HBSS and resuspended in sucrose-phosphatewith nisms were labeled with tritiated amino acids glutamate buffer (SPG; sucrose, 75 g; KH2PO4, 0.52 in order to assist in studies of infection of HeLa g; Na2HPO4, 1.22 g; glutamic acid, 0.72 g; water to 1 229 cells in monolayer culture. The initial stage liter; pH 7.4 to 7.6), 1 ml per 32-oz (960-ml) culture of infection (attachment) was tneasured by ra- bottle, distributed to 0.5-ml aliquots, and frozen at dioactivity counts of cell-associated organisms. -65 C. Based on a study using the same purification Growth after incubation was measured by in- method of a mixture of unlabeled organisms and clusion counts. The purpose of this investiga- radioisotope-labeled noninfected HeLa cells, it can estimated that the final product after purification tion was to study factors affecting attachment be was contaminated with less than 0.1% host mateand inclusion formation and the correlation of rial. The final product usually contained 109 EB/ml the two. as enumerated by an electronmicroscopic method MATERIALS AND METHODS C. trachomatis strains. One trachoma and one LGV strain were used. The trachoma strain was B/ TW-5/OT (1). This strain was isolated in embryonated chicken egg from the eye of a trachoma patient and was subsequently adapted to grow in HeLa 229 cell culture (11). The LGV strain, L,/434/Bu, was obtained from Julius Schacter, University of California, San Francisco (18). This strain was isolated in egg from the bubo of an LGV patient and was also passed in HeLa 229 cells. Cell culture. HeLa 229 cells (11) and fetal tonsil (FT) cells, less than 20 passages, obtained from Marion Cooney, University of Washington, Seattle, were maintained in Eagle minimum essential medium with 10% heat-inactivated fetal calf serum without antibiotics (MEM). Preparation of tritium-labeled organisms. A dayold HeLa 229 cell layer in a 32-oz (960-ml) prescription bottle was inoculated with adequate TW-5 or 434 to obtain 90 to 100% infectivity. After 2 to 2.5 h of absorption, MEM was added and the bottle was incubated at 35 C (TW-5) or 37 C (434). About 18 h after infection, MEM was replaced with low-leucine MEM (1/10 concentration of leucine) in which 1 ,ug of emetine per ml (Sigma Chemical Co., St. Louis, Mo.) was added to inhibit more than 98% of the host protein synthesis without affecting that of the parasite (3). Two hours after the addition of emetine, 1 ,uCi each of 3H-labeled amino acid mixture and leucine, 30 to 50 Ci/mmol (New England Nuclear Corp., Boston, Mass.) per ml was added. The infected cell layers were harvested on day 3. The supernatant fluid was discarded, and the infected cell layers were rolled off from the glass with glass beads in 30 ml of Hanks balanced salt solution (HBSS). Cell suspensions were sonicated lightly for 20 s. The cell debris was removed by centrifugation at 500 x g for 10 min, and the elementary body particles (EB) were pelleted at 30,000 x g for 20 min. The pellets from two culture bottles were resuspended in 6 ml of HBSS, layered on top of 24 ml of 30% sucrose in 0.03 M tris(hydroxymethyl)aminomethane-buffered saline, and centrifuged at 8.000 x g for 60 min with a swinging-bucket rotor SW25.1 in a Spinco model L ultracentrifuge. Each pellet was resuspended in 1.5 ml of HBSS, layered on a 30-ml linear gradient of 30 to 65% renografin (Squibb and Sons Inc., Princeton,

(19). By comparing the EB count with inclusion counts after single-cycle cell culture growth, it was determined that the ratio of inclusion-forming units to physical particles was from 0.6 to 1.6 with TW-5 and 1.0 to 2.3 with 434. These ratios were obtained in what we consider the most effective cell culture growth system as detailed below, including HeLa 229 cell monolayers, centrifugation of inoculum, and pretreatment of cells with DEAE-dextran (the last for TW-5 only). Method of studying attachment of C. trachomatis to HeLa cell monolayers. The cell culture tube used was a disposable flat-bottomed, 1-dram (4-ml) glass vial shell (outside diameter, 15 mm; height, 45 mm) with a no. 0 rubber stopper. A 1-ml suspension of 2 x 105 HeLa cells was inoculated into each tube and incubated overnight to obtain a confluent cell layer. Then the MEM was removed and the cell layer was washed twice with HBSS. One-tenth milliliter of 3H-labeled organisms was inoculated per tube. The number of EB per HeLa cell was about 1,000. Tubes were stored at 0 C, room temperature (RT) (20 to 22 C), or 37 C. After different time intervals of absorption, three tubes were studied. Inoculum was removed and the cell layer was washed five times with HBSS. Cell layers were observed microscopically both before inoculation and after five washes. One milliliter of NCS tissue solubilizer (Amersham/Searle, Arlington Heights, Ill.) was added, and the tubes were sealed with parafilm. On the next day, the digested cell suspension was dissolved in 10 ml of scintillation fluid 15 g of PPO (2,5diphenyloxazole) and 0.5 g of dimethyl POPOP [2,2p-phenylenebis(5-phenyloxazole] per liter of toluene} (Permablend TM1, Packard Instrument Co. Inc., Downers Grove, Ill.) and transferred to a scintillation vial. The radioactivity was counted with a Packard Tricarb scintillation spectrophotometer. The background count, determined from uninoculated tubes treated in the same manner, was subtracted. The radioactivity count was expressed as count per minute of the average of triplicate tubes. Total radioactivity count in the inoculum was also obtained, and the percentage of attachment was calculated. The radioactivity counts in the triplicate tubes were usually within 15% of the average. Tests were repeated at least once. Due to a technical difficulty, the radioactivity of EB in the washings was not determined.

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C. TRACHOMATIS AND HELA CELL INTERACTION

Measurement of infection of HeLa cells. Oneday-old HeLa cells were grown in the same culture tubes with a round cover slip. Unlabeled organisms were used in most of the experiments. The inoculum was diluted to obtain optimal inclusion counts. Inoculation and absorption were the same as described above. Infected tubes were incubated at 35 C for 3 days (TW-5) or 37 C for 2 days (434). After incubation, the cover slips were fixed with methanol and stained with May-Greenwald-Giemsa. Inclusions were counted in 30 fields at x400 magnification with the aid of a micrometer. The average of counts in three or four tubes were determined. The result was expressed as number of inclusions per 30 fields. Infectivity was expressed as inclusion-forming units per milliliter of inoculum after adjusting for dilution, magnification, and area counted. Various treatments used in the study. (i) Heat inactivation of the organisms. The organisms were completely inactivated at 56 C for 30 min in a water bath. (ii) DEAE-dextran treatment of cell monolayers. Before inoculation, HeLa cell monolayers were washed twice with HBSS containing 30 ,ug DEAEdextran (molecular weight, 2 x 106; Pharmacia, Uppsala, Sweden) per ml. (iii) Heparin treatment. The inoculum was resuspended in SPG containing 100 U of heparin (Lipohepin, heparin sodium injection aqueous; Riker Laboratories, Northridge, Calif.) per ml, and the control was suspended in SPG only. (iv) Sodium fluoride treatment. Before inoculation of organisms, HeLa cell monolayers were incubated with MEM containing 2 x 10-2 M NaF for 30 min. Then the fluid was removed and the cell monolayers were washed twice with HBSS containing 2 x 10-2 M NaF and finally inoculated with the organisms suspended in SPG containing 2 x 10-2 M NaF. (iv) Centrifugation of inoculum onto cell monolayers. At the time of inoculation of organisms, the tubes were centrifuged at 900 x g for 60 min at 20 C.

RESULTS Attachment of trachoma and LGV organisms to HeLa 229 cells. The kinetics of the attachment of trachoma TW-5 and LGV 434 organisms to HeLa 229 cells during the first 2 h after inoculation at three different absorption temperatures (O C, RT, and 37 C) are shown in Fig. 1 and 2. With TW-5, attachment efficiency measured by radioactive counts increased as temperature increased from 0 to 37 C (Fig. 1). The most rapid attachment occurred early in the absorption period. Two hours post-inoculation, 7% of the organisms were attached at 0 C, 12% were attached at RT, and 14% were attached at 37 C. Heat-inactivated organisms at RT showed only 1.5% attachment. After 2 h of absorption the efficiency of attachment, compared with that at RT, was 58% at 0 C and 115% at 37 C. Attachment of LGV organisms also increased as absorption temperature increased

14$-D

-

-

-

-

-

1105

-oRT-INACT

60 90 15 30 120 MINUTES ABSORPTION FIG. 1. Rate ofattachment of 3H-labeled trachoma strain TW-5 to HeLa 229 cell monolayers at different temperatures during the first 2 h after inoculation. CPM, Counts per minute; RT, room temperature (20 to 22 C); INACT, inactivated organisms.

L2/434/Bu

(.3

60 90 120 15 30 MINUTES ABSORPTION FIG. 2. Rate of attachment of 3H-labeled LGV strain 434 to HeLa 229 cell monolayers at different temperatures during the first 2 h after inoculation. Abbreviations are as in Fig. 1.

(Fig. 2). However, with LGV the effect of temperature was greater. Two hours post-inoculation, 2% of the organisms were attached at 0 C, 9% were attached at RT, and 17% were attached at 37 C. Only 1.7% of inactivated organisms appeared to be attached. After 2 h of absorption the efficiency of attachment, compared with that at RT, was 22% at 0 C and 190O at 37 C. Effect of absorption temperature on inclusion formation. The effect of absorption temperature on both TW-5 and 434 organisms measured by inclusion counts in HeLa 229 cells is shown in Table 1. The findings parallel those

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KUO AND GRAYSTON

with radioactive counts and result in similar 3. Attachment was not affected by DEAE-dexefficiency ratios when 0 and 37 C are compared tran and inclusion formation was not enhanced, but in one of the duplicate tests it was with RT. Factors affecting attachment and growth inhibited. Heparin strongly inhibited both at(inclusion formation). (i) DEAE-dextran and tachment and inclusion formation. (ii) NaF. Since NaF has been known to inheparin. We have previously shown that polycations enhanced inclusion formation of tra- hibit phagocytosis of mammalian cells (10, 17), choma but not LGV organisms (13). The effect its effect on the attachment and inclusion forof these substances on the attachment of TW-5 mation of TW-5 and 434 organisms was studied. and 434 organisms was studied. Table 2 shows NaF at 2 x 10-2 M concentration was found to results of duplicate experiments indicating that inhibit inclusion formation but not attachment DEAE-dextran enhanced attachment and in- of both TW-5 and 434 (Tables 2 and 3). (iii) Centrifugation. With TW-5 organisms, clusion formation of TW-5, whereas heparin inhibited attachment and almost completely pre- centrifugation (900 x g for 60 min at 20 C) vented inclusion formation. Results of similar increased attachment by 8-fold and inclutreatment of LGV strain 434 are shown in Table sion formation by 10-fold. Effect of centrifugaTABLE 1. Effect ofdifferent absorption temperatures on inclusion formation of trachoma TW-5 and LGV 434 organisms in HeLa 229 cells Inclusion counts (no. of inclusions/30 fields)

Straina

Ratio

Test no.

0 Cb

RT

37 C

0 C/RT

37 C/RT

TW-5

1 2

169 376

246 523

298 731

0.69 0.72

1.21 1.40

434

1 2

64 74

111 146

230 253

0.58 0.51

2.07 1.73

3H-labeled organisms. bAbsorption temperature; 2-h absorption.

a

TABLE 2. Effect of several different agents on the attachment and inclusion formation of trachoma TW-5 organisms in HeLa 229 cells Treatment

DEAE-dextran (30 ,g/ml)

0.44

+

378

0.43

+ -

1,569 1,424 738

0.91

+

730

0.99

+ -

399 3,432 698

8.6

+

5,357

7.7

+ -

1,390 822 1,009 608

1

+ -

1 h, RT

1

I h, 37 C

1

1 h, 20 C

1 2

Heat resistance (37 C 2 h)

1,191 526 877

1 h, RT

2

Centrifugation (900 x g)

+ -

Test no.

2

NaF (2 x 10-2 M)

2.2

+

546 1,200 748 1,036

Absorption

2

Heparin (100 U/ml)

Attachment (counts/ min)

Control/test

2 h, RT

1 2

+

Ratio (+/-)

1.4

0.59 0.60

Inclusion formation (no. of inclusions/30 fields)

83 264 209 387 193 7 114 4 154 66 328 132 31 310 29 284

262 112 268 121

Ratio (+/-)

3.2

1.9 0.04 0.04 0.43

0.40 10.0

9.8 0.43 0.45

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VOL. 13, 1976

TABLE 3. Effect of several different agents on the attachment and inclusion formation ofLGV 434 organisms in HeLa 229 cells Treatment

Absorption

Test no.

Control/test onol'st

Attachment (counts/

Ratio (1)

min)

DEAE-dextran (30 Ag/ml)

Heparin (100 U/ml)

1 h, RlP

1 2

1 h, RT

1 2

NaF (2 x 10-2 M)

lh, 37 C

1 2

+

699 795 377 434

+ +

865 24 629 21

+ -

641 622 567 619

+ -

+

Heat resistance (37 C, 2 h)

2 h, RT

1 2

+ +

1,322 877 727 502

1.1 1.2

Inclusion formation (no. of inclusions/30 fields)

114 62 77 87

Ratio

(+1-

0.54 1.13

87 0.03

7

0.08

0.03

157 8

0.05

126 0.97

54

0.43

1.09

217 85

0.39

0.66

165 47 104 24

0.29

0.69

0.23

a RT: Room temperature, 20 to 22 C.

ent inhibitors depends on the metabolic source tion on 434 organisms was not studied. (iv) Heat resistance. Exposure of the orga- of cellular energy, whether oxidative or glyconisms to 37 C for 2 h before inoculation de- lytic (10). In this study a glycolytic inhibitor, creased attachment of TW-5 about 40% and 434 NaF, was shown to inhibit inclusion formation about 30%. Inclusion formation of TW-5 was but not to affect attachment. We have also tried reduced about 55% and of 434 about 75% (Tables an oxidative inhibitor, dinitrophenol. This material caused HeLa cells to detach from the 2 and 3). Attachment and inclusion formation of glass wall, thus preventing reliable results. TW-5 organisms in HeLa 229 and FT cells. Friis has studied interaction of L cells and C. Two mammalian cell lines, one susceptible psittaci with radioisotope-labeled meningo(HeLa 229) and the other relatively insuscepti- pneumonitis organisms (6). His study showed ble (FT) (5), were compared for their ability to that absorption at 0 C resulted in nearly impersupport attachment and growth of TW-5 orga- ceptible attachment with very low plating effinisms. Whereas attachment was only 27% less cience in a plaque assay, indicating that attachin FT than in HeLa 229 cells, growth of the ment is not a significant function in the process organisms as shown by inclusion formation was of infection, which is in contrast with C. trachomatis. 84% less (Table 4). The rate of attachment of C. trachomatis is DISCUSSION temperature dependent. However, the decrease It has been shown that entry of chlamydial of rate of attachment as the temperature was organisms into the mammalian cells is by reduced seemed to be much greater than would phagocytosis (4, 6, 9). The present study sug- be explained by the effect of temperature on the gests that attachment is an important initial Brownian motion of collision frequency (15), step in infection by C. trachomatis. When hep- which indicated that some specific mechanism arin was used to inhibit attachment, inclusion is involved in the attachment. The attachment formation was also inhibited. Phagocytosis (en- of LGV organisms was more sensitive to low docytosis) by mammalian cells can be inhibited temperature than was that of trachoma orgaat low temperature and requires energy (10). nisms. This is an interesting finding since we As shown in this study, absorption at 0 C to have previously demonstrated that sialic acid stop phagocytosis failed to completely inhibit receptors appear to be necessary for attachment inclusion formation. Some inhibitors of phago- of trachoma but not LGV organisms (13). Studcytosis have been available. The effect of differ- ies with viruses have shown that some viruses

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KUO AND GRAYSTON

TABLE 4. Attachment oftritium-labeled and inclusion formation of nonlabeled trachoma TW-5 organisms in HeLa 229 and FT cells Attachment"

Test line no.no. CellCell (counts/mm) Test Acontthmint"

1 2 a

Ratio (FT/HeLa)

Inclusion formation" (no. of inclusions/30

~ ~ ~ ~ ~fields)

Ratio (FT/HeLa)

HeLa 229 FT

1,488 1,126

0.76

213 31

0.15

HeLa 229 FT

1,623 1,157

0.69

563 88

0.16

Absorption at room temperature for 2 h.

ACKNOWLEDGMENTS that recognize sialic acid-containing receptors This study was supported by Public Health Service reappear to attach rapidly to host cells and the search grant 5-Rol-Ey-00219 from the National Eye Instiattachment is not temperature sensitive, tute. less viruses attach efficiently whereas other The technical assistance of Heidi Nicol is gratefully acand the attachment is sensitive to reduced tem- knowledged. perature (15). The reason has been attributed to LITERATURE CITED the more abundant distribution of sialic acid 1. E. Alexander, R., S. P. Wang, and J. T. Grayston. 1967. the surface cell receptors on (15). Further classification of TRIC strains from ocular Attachment of heat-inactivated organisms trachoma and other sources by the mouse toxicity was much reduced, suggesting that viability of prevention test. Am. J. Ophthalmol. 63:1469-1478. the organisms is important for the attachment. 2. Alexander, J. J. 1968. Separation of protein synthesis in meningopneumonitis agent from that in L cells by We have previously demonstrated that differential susceptibility to cycloheximide. J. BacteDEAE-dextran, a positively charged polycatriol. 95:327-332. ion, enhanced inclusion formation of trachoma 3. Becker, Y., and Y. Asher. 1972. Synthesis of trachoma agent proteins in emetine-treated cells. J. Bacteriol. but not LGV organisms (12, 13). In this study it was found that DEAE-dextran enhanced both 4. 109:966-970. Becker, Y., E. Hochberg, and Z. Zakay-Rones. 1969. attachment and inclusion formation of traInteraction of trachoma elementary bodies with host choma TW-5 organisms. Since inclusion formacells. Isr. J. Med. Sci. 65:121-124. tion was enhanced more than attachment, 5. Croy, T. R., C. C. Kuo, and S. P. Wang. 1975. Comparative susceptibility of eleven mammalian cell lines to DEAE-dextran may enhance the penetration of infection with trachoma organisms. J. Clin. Microthe organisms into the cell. There is no good biol. 1:434-439. explanation of why attachment and inclusion 6. Friis, R. R. 1972. Interaction of L cells and Chlamydia formation of LGV organisms were not enpsittaci: entry of the parasite and host responses to its development. J. Bacteriol. 110:706-721. hanced and why inclusion formation was some7. Gordon, F. B., and A. L. Quan. 1965. Isolation of the times inhibited. trachoma agent in cell culture. Proc. Soc. Exp. Biol. It is well known that LGV organisms grow Med. 118:354-359. much better than trachoma organisms in cell 8. Harrison, M. J. 1970. Enhancing effect of DEAE-dextran on inclusion counts of an ovine Chlamydia (Bedculture. Since the attachment of LGV orgasonia) in cell culture. Aust. J. Exp. Biol. Med. Sci. nisms to the cell is not greater than that of 48:207-213. trachoma organisms, it must be that LGV orga- 9. Higashi, N., A. Tamura, and M. Iwanaga. 1962. Develnisms either penetrate or multiply better than opmental cycle and reproductive mechanism of the meningopneumonitis virus in strain L cells. Ann. trachoma organisms. Additional evidence N.Y. Acad. Sci. 98:100-121. against attachment as the reason why LGV 10. Korn, E. D. 1975. Biochemistry of endocytosis, p. 1-26. grows better in cell culture is that absorption at In C. F. Fox (ed.), Biochemistry series one, vol. 2, 0 C affected LGV attachment more than incluBiochemistry of cell walls and membranes. University Park Press, Baltimore. sion formation. It is also unlikely that the rea11. Kuo, C. C., G. E. Kenny, and S. P. Wang. 1971. Trason is a difference in resistance to heat inactichoma and psittacosis antigens in agar gel double vation. The experimental results showed that immunodiffusion, p. 113-123. In R. L. Nichols (ed.), the reduction in attachment and inclusion forTrachoma and related disorders caused by chlamydial agents. Excerptia Medica, Amsterdam. mation of the organisms after exposure to 37 C C. C., S. P. Wang, and J. T. Grayston. 1972. for 2 h before inoculation was about the same. 12. Kuo, Differentiation of TRIC and LGV organisms based on The experimental results with HeLa 229 and enhancement of infectivity by DEAE-dextran in cell FT cell lines suggest that attachment probably culture. J. Infect. Dis. 125:313-317. is not a critical factor in determining a cell 13. Kuo, C. C., S. P. Wang, and J. T. Grayston. 1973. Effect of polycations, polyanions, and neuraminidase on the line's susceptibility to infection with trachoma infectivity of trachoma-inclusion conjunctivitis and organisms. Additional cell lines and strains lymphogranuloma venereum organisms in HeLa should be tested to confirm this point. cells. Infect. Immun. 8:74-79.

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C. TRACHOMATIS AND HELA CELL INTERACTION

14. Kuo, C. C., S. P. Wang, B. B. Wentworth, and J. T. Grayston. 1972. Primary isolation of TRIC organisms in HeLa 229 cells treated with DEAE-dextran. J. Infect. Dis. 125:665-668. 15. Lonberg-Holm, K., and L. Philipson. 1974. Early interaction between animal viruses and cells. Monogr. Virol. 9:1-148. 16. Rota, T. R., and R. L. Nichols. 1971. Infection of cell cultures by trachoma agent: enhancement by DEAEdextran. J. Infect. Dis. 124:419-421. 17. Sbarra, A. J., and M. C. Karnovsky. 1959. The biochemical basis of phagocytosis. I. Metabolic changes during the ingestion of particles by PMN leukocytes. J. Biol. Chem. 234:1355-1362.

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18. Schachter, J., and K. F. Meyer. 1969. Lymphogranuloma venereum. II. Characterization of some recently isolated strains. J. Bacteriol. 99:636-638. 19. Wang, S. P., and J. T. Grayston. 1967. A potency test for trachoma vaccine utilizing the mouse toxicity prevention test. Am. J. Ophthalmol. 63:1443-1454. 20. Wang, S. P., and J. T. Grayston. 1971. Studies on the identity of the 'fast' egg-killing chlamydia strains, p. 322-336. In R. L. Nichols (ed.), Trachoma and related disorders caused by chlamydial agents. Excerpta Medica, Amsterdam. 21. Weiss, E., and H. R. Dressier. 1960. Centrifugation of rickettsia and viruses onto cells and its effect on infection. Proc. Soc. Exp. Biol. Med. 103:691-695.

Interaction of Chlamydia trachomatis organisms and HeLa 229 cells.

INFECTION AND IMMUNITY, Apr. 1976, p. 1103-1109 Copyright © 1976 American Society for Microbiology Vol. 13, No. 4 Printed in U.SA. Interaction of Ch...
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