Small-Intestinal Factors Promote Encystation of Giardia lamblia In Vitro

Small-Intestinal Factors Promote Encystation of Giardia lamblia In Vitro
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  INFECTION AND IMMUNITY, Mar. 1988, p. 705-707 Vol. 56, No. 3 0019-9567/88/030705-03 02.00/0 CopyrightX 1988, American Society for Microbiology Small-Intestinal Factors Promote Encystation of Giardia lambliaInVitro FRANCES D. GILLIN,* DAVID S. REINER, AND SHAYNE E. BOUCHER Department of Pathology, University of California San Diego, San Diego, California 92103 Received 8 June 1987/Accepted 18 November 1987 Bile salts and fatty acids stimulated differentiation ofcultured Giardia lamblia trophozoites into water-re- sistant cysts at the slightly alkaline pH of the small intestinal lumen. Maximum encystation occurred at pH 7.8. Thus, specificsmall-intestinalfactors may influence encystation in vivoas well as invitro. We recently induced encystation of Giardia lamblia tro- phozoites in vitrofor the first time  8 . Since in a previousstudy of encystation in the suckling mousemodel (10), we found cysts in the upper and middle portions of the small intestine (8), we proposed thatspecificsmall-intestinal con- ditions might promote encystation. Initially, we observed that the exposure of trophozoites to primary bilesalts led to >20-fold increases in the numbers of oval, refractile cells that reactedstrongly with cyst-specific antisera  8 . How- ever, few of these cells resisted exposure to hypotonic conditions, which is a hallmarkof fecalcysts (1, 2) and a rigorous criterionfor quantitation of encystation invitro. To better understand the differentiation process and to increasethe number of water-resistant cysts, we have now investi- gated in depth the roles of small-intestinal physiologic con- ditions in encystation. Our studies demonstrate that the slightly alkaline pH,which is characteristic of much of the small intestine (6), greatly increases the ability of bile salts and fatty acids to induce differentiation of G. lamblia tro- phozoites into water-resistant cysts. A typical water-resis- tant cyst is shown in Fig. 1. G.lamblia WB  ATCC 30957) trophozoites were grown to late log phase in TYI-S-33 medium (4) with bovine bile (5, 13) and serum  pH -7.1) as previously described (14). Parasites were harvested by chilling cultures to detach trophozoites,centrifuging, and washing once; the trophozoites were then suspended in Hanks balanced salt solution. If not otherwise indicated,the following protocol was used. Experiments were carried out in TYI-S-33 medium which hadbeen brought to pH 7.8 with NaOH and lacked bovine bile. Individual bile salts  sodium salts) and fatty acids  Sigma Chemical Co.) were added as specified in each experiment. Aqueous suspensions of fatty acids were warmed with stirring forthe minimum time needed to disperse themand immediatelyadded to the encystation medium. Piperacillin (500 ,ug/ml; Lederle Laboratories) andamikacin (125 jig/ml; Bristol Laboratories) whichdo not affect G. tamblia growth or differentiation (7), were addedbecause the bilesalt-fatty acid dispersions were not filter sterilized. Experiments were initiated by the addition of 106 washed trophozoites in a final volume of4 ml in 1-dram (4.8 ml capacity), glass screw-capped vials. Afterincubation for 4 days at 37°C, which yielded the greatest numbers of cysts in initial experiments, the para- sites were chilled,transferred to 15-ml conical centrifuge tubes, and sedimented (833 x g, 10°C). To lyse the tropho- zoites andany cysts with incomplete walls,parasites were * Corresponding author. washed twice and incubated overnight at 4°C in 15 ml of double-distilled water. The parasites were then suspended, sedimented, and washed again in cold double-distilled water. Most of the supernatant was removed, and thecysts were suspended in the remaining water. Washing removed most trophozoite ghosts and debris of water-lysed cells. Samples were counted in hemacytometerchambersby phase-contrast microscopy. The exact volume of each cyst suspension was measured and used to calculatethe total number of cysts per vial. Each value is the mean of five counts. Statistical significance was determined by the Student t test. Each experiment shown was typical of at least two repetitions. Since the pH of the intestinal fluid may be as high as 7.8 (6) and a major function of bile salts is to formmixed micelles with fattyacids, we examined the effects of pH on induction of encystation by glycocholate or myristic acid alone and in combination (Fig. 2 . Encystation increased with pH both in controls with no additions and in the presence of glyco- cholate alone or myristic acid alone. The stimulatory effects of glycocholate and myristic acid together were synergistic at pH 7.0 to 7.8 and were greatest at pH 7.8. Therefore, subsequentexperiments were carried out at pH 7.8. Ency- station decreased sharply above the optimum pH (Fig. 2 . We next asked whether thestimulation of encystation was a specific property of myristic acid or whether other fatty acids were effective. Each fatty acid tested significantly stimulated encystation both alone and with glycocholate (Table 1 . Again, the effects of fatty acid and glycocholate were synergistic. The effect of the concentrationsof two highly stimulatory fatty acids are shown in Table 2. In the presence of glycocholate, 0.5 to 1 mM myristic (C14) or pentadecanoic (C15)acid was optimal, and encystation de- FIG. 1. A water-resistant in vitro-derived cyst, photographed with Nomarski differential interferencecontrastoptics(x3,100). 705  706 NOTES 2.52.0 .-I   LI >- 1.5   z LI) L 1.0 r- 0.5 0.0 pHFIG. 2. Effects of pH on encystation ofG. lamblia in the presenceof 8 mM glycocholate alone (0), 0.5 mM myristicacid alone (A), or glycocholate and myristicacidtogether  0) or with no additions  O, control). The pH refersto the initial pH; the number of water-resistantcysts is per vial. creased at concentrations of 2 mM. In contrast, exposure of trophozoites to 2 mM myristicacid or pentadecanoic acid without bile salt led tolevels of encystation approximately two-thirds of the maximum. Higher concentrations of these fatty acidsdid not further increaseencystation. In bile, the ratio of primary to secondary bilesalts is about 3:1 (11). In our earlier studies  8 , we observed that the primary bile salt glycine and taurine conjugates lead to more-rapid encystation than do secondary bile salt conju- gates. Unconjugated bile salts were not tested because they are toxic to G. lamblia. In the present study, glycocholate (12 mM with myristicacidstimulated thehighest levels of encystation, 3 x 105 compared with 1.1 x 105 per vial TABLE 1. Effect of fatty acids on encystation Mean no. of water-resistant cysts/ vial (103 [SD]) withb: Fatty acid No 8 mM glycocholateglycocholate Expt la None 2.2 (0.9) 30.4 (5.3) C4 7.3 (4.8)b 40.2 (10.4)b C6 5.8 (2.2)d 64.6  6.8 d C8 7.7 (20)d 133.4  23.0 d C10 8.6 (5.7)C 81.1 (8.3)d C12 9.4 (4.6)d 125.9 (17.9)d C13 6.0 (1.0)d 93.6 (15.9)d C14 12.4  7.6 c 153 4 (31.4)d C15 8.2 (3.2)d 242.0 (35.3)d C16 6.6 (5 0)b 156.4 (30.1)d C18 36.4  5.6 d 153.7 (19.6)d Expt 2 None 7.2 (4.6)30.9 (8.3)Oleicacid, 0.1 mMe 8.1 (3.5) 92.6  13.2 d a 0.5 mM fattyacid wasused in experiment 1. b P   0.05,significantlygreater encystation than control without fatty acid.   0.01. d p   0.005. e Higher concentrations of oleic acid were toxic. TABLE 2. Effects of fatty acid concentration and glycocholate on encystation Mean no. of water-resistant cysts Fatty acid (103 [SD]) with: (concn, mM No glycocholate 8 mM glycocholate None 11.7 (5.4) 48.2 (8.8) Myristicacid (C14) 0.25 11.2 (6.3) 22.2 (9.5) 0.518.8 (7.9)a 277.4 (18.4)a 1.024.3  2.8 a 331.6 (34.7)- 2.0 253.3(30.6)a136.9 (18.2)a Pentadecanoic acid (C15) 0.1 16.1(8.2) 49.0 (12.1)0.5 1.3 (2.1) 352.4 (41.2)a2.0 192.0 (46.7)- 187.2 (35.3)a a p < 0.005 compared with controls lacking fattyacid. obtained with glycochenodeoxycholate (6 mM). The reasons for these differences between the two most-prevalent pri- mary bile salts are not understood. Since our studies support the hypothesis thatsmall-intes- tinal conditions mightbeimportant triggers of encystation  8 , it is important to understand theroles and interactions of these factors in the intestinal milieu. When acidic gastric chyme enters the duodenum, it is rapidly neutralized by secretedbicarbonate. While the fluid in the uppermost part of the duodenum tends to be somewhat acidic, the pH gradually increases distally and may be as high as 7.6to 8.0 in the lower jejunum (3, 6 . The common bile duct transports both biliary and pancreatic secretions, including bicarbonate, into the intestine at approximately the mid-duodenal level. In the intestinal fluid and in vitro (11,15),bile salts tend to formmixed micelles with products of lipolysis such as fattyacids. In vitro, unsaturated fatty acids are toxic to G. lamblia (9, 14, 16; D. S. Reiner, F. D. Gillin, and A. Zenian, Am. Soc. Trop. Med.Hyg. 1984, 145), but saturated fatty acids are not (14). We had proposed that exposure to toxicfactors mightpromote differentiation of G. lamblia. However, saturated fatty acids stimulated encysta- tion at least as well as oleic acid. Moreover, the presence of bilesalts and alkaline pH, which greatly increase encysta- tion, reduced the toxicity of oleic acid (14). Increased pH promotesboth the ionization of fattyacids and their ten- dency to be incorporated into micelles with bile salts (12). The roles of bile saltsin encystation may be the result of their capacity to bind fatty acids rather than direct effects on G. lamblia. These studies suggest that local differences in pH and bile salt and fatty acid concentrations may alsoinfluence the efficiency of encystation of G. lamblia in vivo. We are very grateful to A. F. Hofmann for help in understanding the complexities of bile salt-fatty acid-pH interactions, to J. Sauch and C. Davis for their critiques of the manuscript, and to S. McFarlin for preparing the manuscript. These studies were supported by Environmental Protection Agency Cooperative Work Agreement CR811950-02-1 and by Public Health Service grants Al 19863 and AM 35108 from the National Institutes of Health. LITERATURE CITED 1. Bingham, A. K., E. L. Jarroll, E. A. Meyer, and S. Radulescu. 1979. Giardia sp.: physical factors of excystation in vitro and excystation vs eosinexclusion as determinants of viability. Exp. Parasitol. 47:284-291. INFECT. IMMUN.  VOL. 56, 1988 NOTES 707 2. Bingham, A. K., and E. A. Meyer. 1979. Giardia excystation can beinduced invitro in acidic solutions. Nature  London) 227:301-302. 3. Davenport, H. W. 1977. Physiology of the digestive tract. Year Book Medical Publishers, Chicago. 4. Diamond, L. S., D. Harlow, and C.C. Cunnick. 1978. A new medium for the axenic cultivation of Entamoeba histolytica and other Entamoeba. Trans.R. Soc. Trop. Med. Hyg. 72:431-432. 5. Farthing, M. J. G., S. R. Varon, andG. T. Keusch. 1983. Mammalian bile promotesgrowth of Giardia lamblia in axenic culture. Trans.R.Soc. Trop. Med. Hyg. 77:467-469. 6. Fordtran, J. S., and T. W. Locklear. 1966. Ionic constituents and osmolality of gastric and small-intestinal fluids aftereating. Am. J. Dig. Dis. 11:503-521. 7. Gault, M. J., D. S. Reiner, and F. D. Gillin. 1985. Tolerance of axenically cultivated Entamoeba histolytica and Giardialam- bliato a variety of antimicrobial agents. Trans.R. Soc. Trop. Med. Hyg. 79:60-62. 8. Gillin, F. D., D. S. Reiner, M. J. Gault, H. Douglas, S. Das, A. Wunderlich, and J. Sauch. 1987. Encystation and expression of cyst antigens by Giardia lamblia in vitro. Science235:1040- 1043. 9. Hernell, L., H. Ward, L. Blackberg, and M. E. A. Periera.1986. Killing of Giardialamblia by human milk lipases: an effect mediated by lipolysis ofmilk lipids. J. Infect. Dis. 153:715-720. 10.Hill, D. R., R. L. Guerrant,R.D. Pearson, and E.L. Hewlett. 1983. Giardia lamblia infection of suckling mice. J. Infect. Dis. 147:217-221. 11. Hofmann, A. F.1984. Chemistryand enterohepatic circulation of bile acids. Hepatology 4:4S-14S. 12. Hofmann, A. F., and A. Roda. 1984. Physicochemical properties of bile acids and their relationship to biological properties: an overview of the problem. J. LipidRes.25:1477-1489. 13. Keister, D. B. 1983. Axenic culture ofGiardia lamblia in TYI-S-33 medium supplemented with bile. Trans. R. Soc.Trop. Med. Hyg. 77:487-488. 14. Reiner, D. S., and F. D. Gillin. 1986. Human milk kills Giardia lamblia by generating toxic lipolytic products. J. Infect. Dis. 154:825-831. 15. Roda, A., A. F. Hofmann, and K. J. Mysels. 1983. The influence of bile salt structure on self-association in aqueous solutions. J. Biol. Chem. 258:6362-6370. 16. Rohrer, L., K. H. Winterhalter, J. Eckert and P. Kohler. 1986. Killing ofGiardialamblia by human milk is mediated by unsaturated fattyacids. Antimicrob. Agents Chemother. 30: 254-257. 17. Sjovall, J. 1959. On the concentrationof bile acids in the human intestine during absorption. Acta Physiol. Scand. 46:339-345.
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