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Characterization of bile salt uptake by Giardia lamblia
International
Journalfor
Parasilology,
Pergamon 0020-7519(95)00029-l
Vol. 25, No. 9, pp. 1089-1097, 1995 Australian Society for Parasitology Elsevier Science Ltd Printed in Great Britain 020%7519/95 $9.50 + 0.00
Characterization of Bile Salt Uptake by Giurdia lamblia C. E. W. HALLIDAY,
P. M. G. INGE
and M. J. G. FARTHING*
Digestive Diseases Research Centre, The Medical College of St Bartholomew’s Charterhouse Square, London ECIM SBQ, U.K.
Hospital,
(Received 21 November 1994; accepted 7 April 1995)
Abstract-Ha&lay C. E. W., Inge P. M. G. & Fart&g M. J. G. 1995. Characterization of bUe salt uptake by Giardia lamb&. Z&m&onal JownaZfor Parasit~y 25: 10894097. We have ahown prtviavmly that G~~~Wres~~eoajlleatedbileslltinritro,uBhrveltow~thelllecbuhppbyrrbichtbhs oeaus.U~eofsodipmta~rte(Tc)pad~~~(cC)rnith~tothae~dpninibffl exponential wmpment followed by a hear component, con&tent with a combi~tion of botb active and passive trxnsport procesws. The~ofm~ve~~pnmsswPsfivteersPgportcdbyexperimeats which &owed that bile salt uptake: (i) wax coeceutratioa depemknt (pppueat cs for TC amd GC were 0.21 and 0.63 mM, reap&h@); (ii) was competitively inhibits& (iii) was reduced by the metnbolic inhibitor sodium Rmwide (50 mM) and low temperature (4°C). Rile salt was not taken up by glutaraId&yde-fixed parasites, indkaling that bile salt was not merely being adsorbed on to the paraeite sorface. DiRerential contvifug8tion of lyz.e4iparrsiQs following exponure to radiolabelled GC, showed that the majority of bile salt was lacated in the cytoaol fraction (76%) with a relatively minor -poeeat aasochted witb cell membnne, imikating that bile salt bad been internal&&. Rile salt analysis of extracts of parasitea aml culture medium indicated that GC had not been metaboliwd by Ghfk. Thus, like the mammalian ileum, Giur& appears to take up comjugated bile salts by active and passive transport procewss Conjugated bile saltx are hwn to promote ~ystatioo and thw t&e uptake mei%hns may constitute an important survival meclmhm for the parasite enabling it to complete its life cycle. Key worak Giardia lamblti, bile salts; pathogenesis of malabsorption. INTRODUCTION
The pathogenesis of fat malabsorptionin giardiasis
is one of the most common protozoan pathogensof the human intestinal tract. The
is not well understood
*To whom correspondence should be addressed at: Department of Gastrcenterology, St Bartholomew’s Hospital, London EClA 7BE, U.K., fax 0171-982-6121.
up bile salt from a complex,
Giardia
lamblia
although
mucosal
damage
(Takano 8~ Yardley, 1964; Hoskins et al., 1967; parasite is distributed widely throughout the indusWright & Tomkins, 1977;Levinson & Nastro, 1978; trialized and developingworld and is an important Hartong et al., 1979;Yardley et al., 1964),bacterial cause of acute and chronic diarrhoea (Wolfe, 1978). overgrowth (Tomkins et al., 1978),bile salt deconjuUp to 50% of symptomaticindividuals have clinical gation (Tandon et al., 1977) and inhibition of lipolysis and biochemical evidence of fat malabsorption (Akimova et al., 1978;Smithet al., 1981)haveall been (Farthing, 1989)which may be an important factor proposedas possiblemechanisms.We have shown in impairing energy balance and growth in infants that Giardia avidly takes up conjugatedbile saltsin and children with borderline undernutrition (Farth- vitro (Farthing et al., 1985;Halliday et al., 1988)and therefore designed experiments to quantify and ing et al., 1986;Farthing, 1984). determinethe mechanismsby which Giardia takes lipid-containing
culture
medium such as would be found in the habitat in which Giardiu exists in the proximal
small intestine.
C. E. W. Halliday et al.
1090
MATERIALS
AND METHODS of Giardia trophoroites. Giardia lamblia (Portland 1 strain) were cultivated in modified TYI-S medium without bovine bile and harvested as described previously (Farthing et al., 1985). In the present study, new cultures were initiated by the addition of 1.5 2.5 x lo5 trophozoites per 16 ml borosilicate glass culture tube. Numbers of trophozoites per tube were determined by counting an aliquot in a hemacytometer after chilling on ice for 15 min to dislodge adherent trophozoites. When large numbers of parasites were required trophozoites were mass cultured by a roller bottle technique described previously (Farthing et al., 1982). Uptake of bile salts by Giardia. Native bile salt (GC or TC) (Sigma Chemical Co. Ltd, Poole, Dorset) with tracer amounts of the appropriate [14C] radiolabelled bile salt (1 uCi/culture tube) (Amersham International) diluted in TYIS medium was added to Giardia trophozoites in either logphase (24 h culture) or stationary-phase (72 h culture) of growth (Farthing et al., 1985; Farthing et al., 1983). Cultures were incubated at 37°C for variable time periods and trophozoites numbers were determined at the end of the incubation. Trophozoites were sedimented by centrifugation (500 g for 15 min) and washed three times in 10 mM phosphate buffered sodium chloride (PBS). The tinal pellet was solubilized in 100 ul Soluene (Packard Instrument Co., Inc., Illinois) and prepared for liquid scintillation spectroscopy by the addition of 4.5 ml scintillation mixture. Control tubes without parasites were similarly treated to determine non-specific binding. Radioactivity in the samples was determined in an Ultrabeta 1210 liquid scintillation spectrometer (LKB-Produkter AB, Sweden). Bile uptake was expressed as nmol/lO* trophozoites/min. Normalization of bile salt uptake to unit number of trophozoites takes into account the possibility that trophozoite multiplication may occur during the course of the experiment, particularly during the longer incubations. Efect of time on bile salt uptake. Giardia trophozoites were incubated at 37°C with GC (0.5 or 5.0 mM) or TC (0.5 mM) for periods of 5-240 min. Uptake at each time point was determined in triplicate. Data were analysed using a weighted non-linear least squares regression program on a Hewlett Packard desktop microcomputer (HP-85). The effect of time on bile salt uptake is given by expression 1: (Baulieu & Raynaud, 1970) Cultivation
and harvesting
Ut = U,,,.(l - eeklt) + kzt
(1)
where Ut is the trophozoite uptake in mnol/108 trophozoites at time t; U,,, is the maximal uptake in nmol/108 trophozoites; and k, and kz are constants. The first exponential term describes the initial uptake of bile salt and approaches U,,, as time increases. The second term describes a straight line. U, continues to increase linearly with time after U,, has been reached. Eflect of bile salt concentration on bile salt uptake. Uptake of CC or TC by Giardia trophozoites was determined over the bile salt concentration range 0.1-10 mM during 30 min incubations at 37°C using log-phase and stationary-phase trophozoites. Experiments were performed in triplicate. Kinetic data were analysed using a similar weighted non-
linear least squares regression program. Tropbozoite bile salt uptake, U, is given by expression 2, the MichaelisMenten equation: u
=
Pmaa)w
C+K, where U is the trophozoite uptake in mnol/lOx trophozones/30 min; U, is the maximal trophozoite uptake in nmol@ trophozoites/30 min; C is the bile salt concentration (mM) in the incubation medium and K, is the Michaelis constant (mM). This expression describes a rectangular hyperbola. Efiect of TC on GC uptake. Competitive inhibition of bile salt uptake was examined by observing the effect of increasing concentrations of TC (O-2 mM) on GC uptake over a 30 min period at 37°C from medium containing 0.5 and 2 mM/GC. Experiments were performed iu duplicate and GC uptake in competition experiments was expressed as nmol/108 trophozoites/30 min. Data were again analysed using a weighted non-linear least squares regression program. The effect of TC on GC uptake is described by expression 3: (Holford & Sheiner, 1981)
05nadC) u = u,,, - ____ EH)+C where U is the GC uptake by trophozoites in nmol/lO* trophozoites/JO min; U, is the maximal GC uptake (when TC concentration is zero) in mnol/lOs trophotites/M mm; E,- is the maximum inhibitory effect in nmol/Iti trophozoites/30 min; C is the TC concentration (mM) arid Em is the concentration at which a 50% inhibitory effect is seeu (mM). Effect of metabolic inhibitors on bile saltuptake. Bile salt uptake by Giardia was assessed in the presence of a variety of inhibitors of cellular metabolism including sodium fluoride (50 mM) (Mahler & Cordes, 1966), the microfilament inhibitor cytochalasin B (10 &ml) (Feely & Erlandsen, 1982) and the anti-giardial drug, metrcmidazole (0.1 mM) (Jokipii & Jokipii, 1980, Farthing & Inge, 1986). Giardia trophozoites were exposed to each of these a$ems in modified TYI-S medium for 60 min at 37°C. 2 mM GC and [*4C]-labelled GC in tracer amounts (1 uCi/cuhure tube) were added to the culture tubes and incubated far a further 30 min at 37°C. GC uptake was assessed as described previously. To determine the effect of low temperature on GC uptake, Giardia were prechilled on ice for 30 min following which GC uptake was determined after a further 30 min incubation at 4°C. Finally, uptake of GC hy glutaraldehyde-tixed parasites was determined. Following exposure to 1% buffered glutaraldehyde for I h, trophozoites were washed in 100 mM glycine and resuspended in modified TYI-S medium for determination of GC uptake after a 30 min incubation at 37°C. Following exposure to inhibitors of cellular metabolism, low temperature and glutaraldehyde fixation, trophozoite viability was assessed by direct microscopic observation of 8agella motility and Trypan Blue (0.04%) exclusion, as we have described previously (Farthing & Inge, 1986; Inge & Farthing, 1987). Localization of bile salts in Giardia tropkozohs. Apptoximately 2.5 x lo* trophozoites were mass cultur& in roller bottles (Farthing et al., 1982) in modified TYI-S medium
Bile salt uptake by Giardia lamblia supplemented with 0.1 mM GC and tracer amounts of [i4C]GC (30 uCi/roller bottle). After washing 3 times in PBS, trophozoites were lyzed in a hypotonic buffer containing 1 mM EDTA, 2 mM HEPES buffer, pH 8.0, and 0.005% (w/v) phenylmethylsulphonyl fluoride (PMSF) using twenty strokes of a Dounce homogenizer. Phase contrast microscopy confirmed r99% disruption of trophozoites. After centrifugation (2000 g for I5 min) at 4°C to separate cytoplasm (Fraction Si) from cytoskeleton and adherent cell membrane, the resulting pellet (Fraction Pi) was washed twice in solution containing 2 mM EDTA, 2 mM dithiothreitol (DTT), 2 mM MgS04, 150 mM KCl, 0.005% PMSF and 10 mM Tris buffer (pH 6.9). The cytoplasmic fraction was then centrifuged again (50,000 g for 60 min) at 4°C to separate membrane vesicles (Fraction Ps) from soluble cytoplasmic components (Fraction SZ). Aliquots of each fraction were prepared for liquid scintillation [14c] spectroscopy and radioactivity present expressed as a percentage of that in the hypotonic lysate before differential centrifugation. Deconjugation of bile salts by Giardia. Giardia trophozoites were cultivated at 37°C for 72 h in 16 ml culture tubes containing modified TYI-S medium supplemented with 2.0 mM GC together with tracer amounts of [‘4C]-GC (1 uCi/ tube). After 72 h incubation, tubes were cooled on ice for 15 min. Giardia trophozoites were then sedimented by centrifugation and were washed 3 times in PBS. Samples of incubation medium and trophozoites were stored at -25°C prior to bile salt analysis by thin layer chromatography (TLC). Control incubations without Giardia were treated identically. Samples of radiolabelled bile salt-supplemented medium (0.5 ml) or trophozoites (- 107) were initially prepared by addition of 0.1 mM sodium hydroxide and incubating at 64°C for 20 min. Bile salts were extracted from these aqueous preparations by passing diluted samples through Sep-Pak Cis cartridges (Setchell & Worthington, 1982) and evaporating the methanol eluates to dryness. The residues were redissolved in methanol and subjected to TLC (Hofmann, 1962) on 0.5 mm silica gel G plates using Amylacetate : Propionic acid : Propan-l-01 : Water; 4 : 3 : 2 : 1 as the developing solvent (Gregg, 1966). Pure bile salt standards (GC, GDC, TC, TDC, C and DC) were run with each experimental extract. Standards were stained with phosphomolybdic acid and corresponding areas of experimental plates were scraped off and subjected to liquid scintillation spectroscopy. Statistics. Mann-Whitney U-test was used to evaluate differences between observations. RJWUJLTS
Time courseof bile salt uptake The effect of time on bile salt uptake by Giardia trophozoites is shown in Fig. 1. When trophozoites wereincubatedat 37°Cin GC (0.5 or 5.0 mM) or TC (0.5 mM) containing TYI-S medium the uptake of bile salt describedan exponential component with respect to time. However, a linear component of uptake was also quantitatively important. The slope of the linear portion of the curve for GC (0.5 mM)
1091
was 0.03 nmol/108 trophozoites/min and for TC (0.5 mM) was 0.26 nmol-’ IO8 min-‘. When the
linear component was subtracted from the experimental values an exponential curve was obtained (Fig. 1, broken lines).U,,, (maximaluptake) for GC (0.5 and 5.0 mM) and for TC (0.5 mM) were9.8,23.3 and
36.7 nmol-’
lo* min-‘,
respectively.
These
observations are consistent with there being both an active transport and a diffusional componentfor bile salt uptake by Giardia. In addition, there would appear to be differences in the way in which individual bile saltsare handled,TC being taken up more avidly than GC. Effect of bile salt concentrationandgrowth phaseon bile salt uptake Figure 2 showsthe effect of bile salt concentration on bile salt uptake by Giardia trophozoites. Uptake of GC and TC was a concentration dependentand saturableprocess.Using expression(2), the Michaelis-Menten equation, estimatesof K, and U,,, for both bile saltsweredetermined.The K, for TC (0.21 mM) waslower than that for GC (0.63mM) and the U,,, was higher for TC than GC, being 64.6 and 24.5 nmol-’ log/30 mm-‘, respectively. Growth phase(active or stationary) had no effect on bile salt uptake. Using stationary phasetrophozoites, K,,, for GC was 0.53 mM and I-J,,,, 21.8 nmol-’ log/30 min-‘, values which were not different from those obtained with log-phaseorganisms(Fig. 2). Effect of TC on GC uptake Figure 3 showsthat competitive inhibition of GC uptake by Giardia trophozoites occurred when increasing concentrations of TC were added to TYI-S mediumcontaining either 0.5 or 2.0 mM GC during a 30min incubation. Uptake of GC from a 0.5 mM solution wascompletelyabolishedby increasing TC concentration (U,, = I%,,,,). However, uptake from a 2 mM solution was only partially inhibited by the addition of TC (U, 30.4 and E,, 31.2nmol-’ lo*/30 mine1 . Es,, concentrations were 0.07 and 0.18 mM TC for experimentsusing0.5 and 2.0 mM GC,
respectively). Effect of metabolicinhibitorson bile salt uptake Bile salt uptake was unaffected by preincubation with metronidazole or cytochalasin B (Table 1) neither of which reducedparasiteviability asassessed by Trypan Blue exclusion, and only metronidazole reducedflagellamotility. In contrast sodiumfluoride and low temperature(4°C) significantly inhibited GC uptake. Giardia, which lacks mitochondria is known to be dependenton glycolysisfor energy metabolism (Lindmark,
1980). Sodium fluoride inhibits glycolysis
C.
1092
E. W. Halliday et al. U max
= 36.7nmo1/10a/min 0.5mM
TC
K z = 0.26nmol/10B/min
KI
0
60
120
(4 Time
U max=
0 (W
9.8
180
240
180
240
(min)
nmol/lOa/min
K,
= 0.06nmol/10a
K2
= 0.03nmo1/10*/min
60
= O.O2nmot/lO~/min
/min
120 Time
(min)
Fig. la,
suggestingthat bile salt uptake by Giardia is energy dependent. Sodium fluoride also reduced parasite viability and flagella motility. Inhibition of microfilament function (cytochalasin B) had no effect on GC uptake, indicating that endocytosis was not involved. Bile salt uptake did not occur after glutaraldehyde fixation, making it unlikely that uptake was due to non-specificadsorption of bile salt on to the cell surface.
b
Localization
of bile salt within G&x&a
Fractionation of G&m&a foBowing incubation with tracer [14C’J-CXshowed that 78.5OAof total uptake was located in cytmol with only 1.3% associatedwith a rmnbrax Era&m. Total recovery of radioactive label was 87.6!& These&&gs are consistent with the view that bik salt is not merely absorbed into surface membrane but is transported intraceB~arly.
Bile salt uptake by Giardia
U max
K, ------
-.-.-
- ---_------____--_-___ _._.A.-.
_.C.
.-‘C.-’
= 23.2nmol/108/min
12
5.0mM
GC
= 0.14nmol/10*/min --,.--: _.C. _,/.-.
K, = 0.09nmo1/108/min
60
120
(4
1093
lamblia
Time
180
240
(min)
Fig. 1. Effect of time on conjugated bile salt uptake by Giardia trophozoites in vitro. Uptake curves are plotted for 0.5 mM TC (la), 0.5 mM GC (lb) and 5 mM GC (Ic) plotted as mean (S.E.M.). Results are derived from 3 separate experiments each performed in triplicate. Using the expression Ut = U-(1
- eKlt) + Krt (seetext)
bile salt uptake by Giardia can be resolved into exponential (- - - -) and linear (- . -. -) components, the former consistent with an active transport process and the latter with simple diffusion.
Deconjugationof bile salt by Giardia TLC of extracts of Giardia trophozoites or TYI-S culture medium following incubation for 72 h with radiolabelled bile salt (GC 2 mM) failed to show evidence of bile salt deconjugation. Although trace amounts of contaminating bile salts (taurocholate, Table l-Effect of metabolic inhibitors, low temperature and glutaraldehyde fixation on sodium glycocholate (GC) uptake by Giardia Experimental conditions Control’ Metronidaxole (0.1 mM) Sodium fluoride (50 mM) Cytochalasin B (10 w/ml) 4°C Glutaraldehyde (1%)
GC uptake (% control) mean f S.E.M.
flagella motilityb
100 101.3+11.2
+++ +
Trypan Blue exclusion (%) 98 90
37.7 +4.4+*
-
50
108.7 + 10.7
+++
97
3.5* 1.3** 19.1+3.3**
+ -
98 0
Giardia
aGiardia trophoxoites (- 5 x 106) incubated in TYI-S culture medium containing GC (2 mM). bResults are the mean of 3 experiments. +++ ~-90% motile; + < 15% motile; - all trophozoites non-motile. **PcO.Ol compared to control incubation.
taurodeoxycholate,glycocholate, glycodeoxycholate, cholateand deoxycholate;total < 6%) werefound in the native radiolabelled bile salt, these did not increaseduring experimentalor control incubations. DISCUSSION Previousstudieshave shownthat bile and bile salts not only have trophic effectswith respectto growth of Giardia both in vitro (Farthing et al., 1985; Farthing et al., 1983)and in vivo (Hegner& Eskridge, 1937;Bemrick, 1963),but are actually consumedby the parasiteduring in vitro culture (Farthing et al., 1985).Sinceintraluminal bile saltsare important for intestinal absorption of dietary fat (Carey et al., 1983) and steatorrhea is a common occurrence in symptomatic patients with giardiasis, we have performedexperimentsto characterizeand quantify the processby which Giardia trophozoites take up bile salts. The presentstudy indicatesthat Giardia takes up conjugatedbile saltsby a mechanismwhich appears to involve a combination of active and passive transport processes.Kinetic analysis of bile salt uptake with time, resolved uptake into an initial exponential phaseconsistentwith active transport, followed by a linear phase indicative of passive diffusion; this pattern of bile salt uptake is similar to
C. E. W. Halliday et at.
094 75
50
GC (active)
I
25
-1
GC (stationary)
_
d
I
0
u
2
4
6
8
Bile salt Concentration,
IO
mM
Fig. 2. The effect of conjugated bile salt concentration on bib salt uptake by Giardia trophoaoites. Results are plotted as mean (S.E.M.) and are derived from 4 separate experiments performad in triplicate. Uptake of TC and GC was a saturable process consistent with their wg a &&ed number of binding sites or transporters within the cell membrane. The apparent K,‘s for TC and GC were 0.21 and 0.63 mM, respectively. U, for TC was 64.6 and for GC 24.5 mnol/tOB trophozoites/30 min, respectively. Actively multiplying trophozoites (log phase growth) and stationary phase organisms took up GC similarly.
E max
= 31.2nmolil
E 5”
=
OS imin
0.18mM
2mM GC
I E max
= 11.8nmol/l
E,
=
OS /min
0.07mM
1
Taurocholate
concentration,
mM
over active transport as the major mechanism of bile salt uptake, a process which is not snsxqtibk cixnpetitive inhibition.
to
Bile salt uptake by Giardia lamblia
that which occursin human ileum (Krag & Phillips, 1974;Stigsby& Krag, 1984).The apparent K,,, of TC for this processin Giardia was0.21 mM which is of the same order of magnitude reported for TC following kinetic analysisof human ileal perfusion studies with this bile salt (0.2-0.6 mM) (Krag & Phillips, 1974; Stigsby & Krag, 1984). Again the apparent K,‘s for GC and TC reported in the presentstudy with Giardia are very similar to those obtained by othersusinga variety of preparationsof rat ileum including intestinal perfusion (Schiff et al., 1972),intestinal sheets(Thomson, 1983)and isolated intestinal epithelial cells (Wilson & Treanor, 1975). The study by Reiner et al. (1993) demonstrating sodium dependencyof bile salt uptake by Giardia would also support an active, carrier-mediated transport process. Further evidencefor active transport of bile salts by Giardia is provided in the present study by experimentssuggestingthat bile salt uptake is an energy dependentprocessinhibited by low temperature and sodiumfluoride, an inhibitor of glycolysis. Sodium fluoride also inhibits the activity of protein phosphatasesand thus, additional effects on other aspects of cell metabolism cannot be excluded. Metronidazole, the drug of choice for treatment of Giardia infectionsin humanshad no effect on bile salt uptake after 60 min incubation, eventhough many of the parasiteshad beenrenderednon-motile. Metronidazole is thought to act by inhibiting nucleic acid synthesisand thus might not be expected to impair membranetransport processes after a relatively brief exposure. Our demonstration of competitive inhibition of GC uptake by the closely related primary bile salt TC, suggests that a limited numberof absorptivesites are presentin the parasiteplasmamembrane,raising the possibility that a specificreceptor siteor bile salt transport protein is present in this organism. Our observationsthat bile salt uptake by Giardia is at least in part energy dependentand may involve a specifictransport protein, is again similarto thoseof previous studiesof bile salt transport by mammalian epithelial cells(Wilson & Treanor, 1975). Fractionation of trophozoites after incubation with bile salt confirmed that a substantial proportion of bile salt was presentin the cytosol and was not simply bound to cell membranes,supporting the view that bile salt is internalized. However, further confirmation of this is required since cell disruption itself can result in changes in the intracellular distribution of exogenous substances as bile salts. The role of bile salts in the intracellular metabolism of Giardia is not known. We have suggested
1095
previously (Farthing et al., 1985) that bile salts enhancethe delivery of preformed membranephospholipids, a potentially important role sinceGiardia is unable to synthesizeits own phospholipid(Jarroll et al., 1981).Whether the parasiteusesbile salt asa metabolic substrateis as yet unclear, but unconjugated bile salt concentrations used in the present study stimulate parasite growth (Farthing et al., 1985, 1983).There is someevidenceto suggestthat Giardia may enter the gallbladder and bile ducts (Lyon & Swalm, 1925;Calder & Rigdon, 1935;De Muro, 1939;Goldsteinet al., 1978)whereconcentrations of bile saltswould be substantiallyhigher than thosefound in the proximal smallintestineor in any of the experimentsdescribedin the present study. More recentwork indicatesthat conjugatedbile salts at or above their critical micellarconcentrationsmay protect the parasite from toxic lipolytic products commonlyfound in the smallintestine(Gillin, 1987) and in vitro studiessuggestthat conjugatedbile salts may have a role in promoting encystation(Gillin et al., 1987, 1989),a vital step in Giardia’s life cycle which enablesthe parasiteto survive outsideits host. Thus, the uptake of host bile saltsby Giardia appears to offer an important survival advantage to the parasiteby promoting its growth, while at the same time acting as a trigger to encystation, ensuring completion of its life cycle. Further studies are required to elucidatethe mechanisms by which bile saltsinfluenceintracellular metabolismand to assess the impact of this parasite on bile salt homeostasis during humaninfection, in vivo. Acknowledgement-This work was supported by the Wellcome Trust and the Joint Research Board of St Bartholomew’s Hospital. REFERENCES
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Jarroll E. L., Muller P. .I., Meyer E. A. &c Morse S. A. 1981. Lipid and carbohydrate metabolism of Giardta lamhlia. Molecular Biochemistry & Parasitology 2: 187..196. Jokipii L. & Jokipii A. M. 1980. In vitro susceptibility of Giardia lamblia trophozoites to metronidazole and tinidazole. Journal of Infectious Diseases 141: 3 17-325. Krag E. & Phillips S. F. 1974. Active and passive bile acid absorption in man. Perfusion studies of the ileum and jejunum. Journal of Clinical Investigation 53: 16861694. Levinson J. D. & Nastro L. J. 1978. Giardiasis with total villous atrophy. Gastroenterology 14: 271-275. Lindmark D. G. 1980. Energy metabolism of the anaerobic protozoan Giardia lamblia. Moleculur Biochemistr,v di Parasitology 1: l-12. Lyon B. B. V. & Swalm W. A. 1925. Giardiasrs: Its frequency, recognition, treatment and certain clinical factors. American
Journal
of Medical
Science
170: 348.. 364.
Mahler H. R. & Cordes E. H. 1966. Biological Chemistry. pp. 256, 528 and 595. Harper 8c Row: New York. Munoz M. de L.. Weinback E. C., Wiedet S. i‘., Claggett C. E. & Levenbrook L. 1987. Giardia lamblia: Detection and characterisation of calmodulin. Expermwntat Parasitology 63: 4248. Porter A. 1916. An enumerative study of the cysts of Giurdiu (lamblia) intestinalis in human dysentenc faeces. hncet 1: 11661169. Reiner D. S., Hetsko M. L., Das S., Ward Ii. D.. McCaffery M. & Gillin F. D. 1993. Giardia famblia: Absence of cyst antigens and reduced secretory vesicle formation and bile salt uptake in an encystation-deficient suhline. Esperimental
Parasitology
17: 462-472.
Schiff E. R., Small N. C. & Dietschy J. M. 1972. Characterization of the kinetics of the passive and active transport mechanisms for bile acid absorption in the small intestine and colon of the rat. Journal of Chnicai Investigation 51: 1351m-1362. Setchell K. D. R. & Worthington J. 1982. PI rapid method for quantitative extraction of bile acids and their conjugates from serum using commerciaby available reverse phase octadecyl-silane bounded silica cartridges. Clinica Chimica Actu 125: 135144. Smith P. D., Horsburgh C. R. & Brown W. R. 1981. In virro studies on bile acid deconjugation and lipolysis inhibition hy Giardia
70@ 704.
lamblia.
Digestive
Diseases
& Sciences
26:
Bile salt uptake by Giardia lamblia Stigsby B. & Krag E. 1984. A computer model simulating the intestinal absorption of bile acids. Scandinavian Journal of Gastroenterology 19: 802-807. Takano J. & Yardley J. H. 1964. Jejunal lesions in patients with giardiasis and malabsorption. Bulletin of the Johns Hopkins Hospital 116: 413429. Tandon B. N., Tandon B. K., Satpathy B. K. & Shriniwas. 1977. Mechanism of malabsorption in giardiasis: A study of bacterial flora and bile salt deconjugation in upper jejunum. Gut 18: 176-181. Thomson A. B. R. 1983. Uptake of bile acids into rat intestine. Effect of diabetes mellitus. Diabetes 32: 900-907. Tomkins A. M., Wright S. G., Drasar B. S. & James W. P. T. 1978. Bacterial colonisation of jejunal mucosa in giardiasis. Transactions c# the Royal Society of Tropical Medicine & Hygiene 72: 33-36.
1097
Wilson F. A. & Treanor L. L. 1975. Characterisation of the passive and active transport mechanisms for bile acid uptake into rat isolated intestinal epithelial cells. Biochimica Biophysics Acta 406: 28&293. Wolfe M. S. 1978. Giardiasis. New England Journal of Medicine 2% 3 19-32 1. Wright S. G. & Tomkins A. M. 1977. Quantification of the lymphocytic infiltrate in jejunal epithelium in giardiasis. Clinical Experimental Immunology 29: 408412. Yardley J. H., Takano J. & Hendrix T. R. 1964. Epithelial and other mucosal lesions of the jejunum in giardiasis. Jejunal biopsy studies. Bulletin of the Johns Hopkins Hospital 115: 389406.
Journalfor
Parasilology,
Pergamon 0020-7519(95)00029-l
Vol. 25, No. 9, pp. 1089-1097, 1995 Australian Society for Parasitology Elsevier Science Ltd Printed in Great Britain 020%7519/95 $9.50 + 0.00
Characterization of Bile Salt Uptake by Giurdia lamblia C. E. W. HALLIDAY,
P. M. G. INGE
and M. J. G. FARTHING*
Digestive Diseases Research Centre, The Medical College of St Bartholomew’s Charterhouse Square, London ECIM SBQ, U.K.
Hospital,
(Received 21 November 1994; accepted 7 April 1995)
Abstract-Ha&lay C. E. W., Inge P. M. G. & Fart&g M. J. G. 1995. Characterization of bUe salt uptake by Giardia lamb&. Z&m&onal JownaZfor Parasit~y 25: 10894097. We have ahown prtviavmly that G~~~Wres~~eoajlleatedbileslltinritro,uBhrveltow~thelllecbuhppbyrrbichtbhs oeaus.U~eofsodipmta~rte(Tc)pad~~~(cC)rnith~tothae~dpninibffl exponential wmpment followed by a hear component, con&tent with a combi~tion of botb active and passive trxnsport procesws. The~ofm~ve~~pnmsswPsfivteersPgportcdbyexperimeats which &owed that bile salt uptake: (i) wax coeceutratioa depemknt (pppueat cs for TC amd GC were 0.21 and 0.63 mM, reap&h@); (ii) was competitively inhibits& (iii) was reduced by the metnbolic inhibitor sodium Rmwide (50 mM) and low temperature (4°C). Rile salt was not taken up by glutaraId&yde-fixed parasites, indkaling that bile salt was not merely being adsorbed on to the paraeite sorface. DiRerential contvifug8tion of lyz.e4iparrsiQs following exponure to radiolabelled GC, showed that the majority of bile salt was lacated in the cytoaol fraction (76%) with a relatively minor -poeeat aasochted witb cell membnne, imikating that bile salt bad been internal&&. Rile salt analysis of extracts of parasitea aml culture medium indicated that GC had not been metaboliwd by Ghfk. Thus, like the mammalian ileum, Giur& appears to take up comjugated bile salts by active and passive transport procewss Conjugated bile saltx are hwn to promote ~ystatioo and thw t&e uptake mei%hns may constitute an important survival meclmhm for the parasite enabling it to complete its life cycle. Key worak Giardia lamblti, bile salts; pathogenesis of malabsorption. INTRODUCTION
The pathogenesis of fat malabsorptionin giardiasis
is one of the most common protozoan pathogensof the human intestinal tract. The
is not well understood
*To whom correspondence should be addressed at: Department of Gastrcenterology, St Bartholomew’s Hospital, London EClA 7BE, U.K., fax 0171-982-6121.
up bile salt from a complex,
Giardia
lamblia
although
mucosal
damage
(Takano 8~ Yardley, 1964; Hoskins et al., 1967; parasite is distributed widely throughout the indusWright & Tomkins, 1977;Levinson & Nastro, 1978; trialized and developingworld and is an important Hartong et al., 1979;Yardley et al., 1964),bacterial cause of acute and chronic diarrhoea (Wolfe, 1978). overgrowth (Tomkins et al., 1978),bile salt deconjuUp to 50% of symptomaticindividuals have clinical gation (Tandon et al., 1977) and inhibition of lipolysis and biochemical evidence of fat malabsorption (Akimova et al., 1978;Smithet al., 1981)haveall been (Farthing, 1989)which may be an important factor proposedas possiblemechanisms.We have shown in impairing energy balance and growth in infants that Giardia avidly takes up conjugatedbile saltsin and children with borderline undernutrition (Farth- vitro (Farthing et al., 1985;Halliday et al., 1988)and therefore designed experiments to quantify and ing et al., 1986;Farthing, 1984). determinethe mechanismsby which Giardia takes lipid-containing
culture
medium such as would be found in the habitat in which Giardiu exists in the proximal
small intestine.
C. E. W. Halliday et al.
1090
MATERIALS
AND METHODS of Giardia trophoroites. Giardia lamblia (Portland 1 strain) were cultivated in modified TYI-S medium without bovine bile and harvested as described previously (Farthing et al., 1985). In the present study, new cultures were initiated by the addition of 1.5 2.5 x lo5 trophozoites per 16 ml borosilicate glass culture tube. Numbers of trophozoites per tube were determined by counting an aliquot in a hemacytometer after chilling on ice for 15 min to dislodge adherent trophozoites. When large numbers of parasites were required trophozoites were mass cultured by a roller bottle technique described previously (Farthing et al., 1982). Uptake of bile salts by Giardia. Native bile salt (GC or TC) (Sigma Chemical Co. Ltd, Poole, Dorset) with tracer amounts of the appropriate [14C] radiolabelled bile salt (1 uCi/culture tube) (Amersham International) diluted in TYIS medium was added to Giardia trophozoites in either logphase (24 h culture) or stationary-phase (72 h culture) of growth (Farthing et al., 1985; Farthing et al., 1983). Cultures were incubated at 37°C for variable time periods and trophozoites numbers were determined at the end of the incubation. Trophozoites were sedimented by centrifugation (500 g for 15 min) and washed three times in 10 mM phosphate buffered sodium chloride (PBS). The tinal pellet was solubilized in 100 ul Soluene (Packard Instrument Co., Inc., Illinois) and prepared for liquid scintillation spectroscopy by the addition of 4.5 ml scintillation mixture. Control tubes without parasites were similarly treated to determine non-specific binding. Radioactivity in the samples was determined in an Ultrabeta 1210 liquid scintillation spectrometer (LKB-Produkter AB, Sweden). Bile uptake was expressed as nmol/lO* trophozoites/min. Normalization of bile salt uptake to unit number of trophozoites takes into account the possibility that trophozoite multiplication may occur during the course of the experiment, particularly during the longer incubations. Efect of time on bile salt uptake. Giardia trophozoites were incubated at 37°C with GC (0.5 or 5.0 mM) or TC (0.5 mM) for periods of 5-240 min. Uptake at each time point was determined in triplicate. Data were analysed using a weighted non-linear least squares regression program on a Hewlett Packard desktop microcomputer (HP-85). The effect of time on bile salt uptake is given by expression 1: (Baulieu & Raynaud, 1970) Cultivation
and harvesting
Ut = U,,,.(l - eeklt) + kzt
(1)
where Ut is the trophozoite uptake in mnol/108 trophozoites at time t; U,,, is the maximal uptake in nmol/108 trophozoites; and k, and kz are constants. The first exponential term describes the initial uptake of bile salt and approaches U,,, as time increases. The second term describes a straight line. U, continues to increase linearly with time after U,, has been reached. Eflect of bile salt concentration on bile salt uptake. Uptake of CC or TC by Giardia trophozoites was determined over the bile salt concentration range 0.1-10 mM during 30 min incubations at 37°C using log-phase and stationary-phase trophozoites. Experiments were performed in triplicate. Kinetic data were analysed using a similar weighted non-
linear least squares regression program. Tropbozoite bile salt uptake, U, is given by expression 2, the MichaelisMenten equation: u
=
Pmaa)w
C+K, where U is the trophozoite uptake in mnol/lOx trophozones/30 min; U, is the maximal trophozoite uptake in nmol@ trophozoites/30 min; C is the bile salt concentration (mM) in the incubation medium and K, is the Michaelis constant (mM). This expression describes a rectangular hyperbola. Efiect of TC on GC uptake. Competitive inhibition of bile salt uptake was examined by observing the effect of increasing concentrations of TC (O-2 mM) on GC uptake over a 30 min period at 37°C from medium containing 0.5 and 2 mM/GC. Experiments were performed iu duplicate and GC uptake in competition experiments was expressed as nmol/108 trophozoites/30 min. Data were again analysed using a weighted non-linear least squares regression program. The effect of TC on GC uptake is described by expression 3: (Holford & Sheiner, 1981)
05nadC) u = u,,, - ____ EH)+C where U is the GC uptake by trophozoites in nmol/lO* trophozoites/JO min; U, is the maximal GC uptake (when TC concentration is zero) in mnol/lOs trophotites/M mm; E,- is the maximum inhibitory effect in nmol/Iti trophozoites/30 min; C is the TC concentration (mM) arid Em is the concentration at which a 50% inhibitory effect is seeu (mM). Effect of metabolic inhibitors on bile saltuptake. Bile salt uptake by Giardia was assessed in the presence of a variety of inhibitors of cellular metabolism including sodium fluoride (50 mM) (Mahler & Cordes, 1966), the microfilament inhibitor cytochalasin B (10 &ml) (Feely & Erlandsen, 1982) and the anti-giardial drug, metrcmidazole (0.1 mM) (Jokipii & Jokipii, 1980, Farthing & Inge, 1986). Giardia trophozoites were exposed to each of these a$ems in modified TYI-S medium for 60 min at 37°C. 2 mM GC and [*4C]-labelled GC in tracer amounts (1 uCi/cuhure tube) were added to the culture tubes and incubated far a further 30 min at 37°C. GC uptake was assessed as described previously. To determine the effect of low temperature on GC uptake, Giardia were prechilled on ice for 30 min following which GC uptake was determined after a further 30 min incubation at 4°C. Finally, uptake of GC hy glutaraldehyde-tixed parasites was determined. Following exposure to 1% buffered glutaraldehyde for I h, trophozoites were washed in 100 mM glycine and resuspended in modified TYI-S medium for determination of GC uptake after a 30 min incubation at 37°C. Following exposure to inhibitors of cellular metabolism, low temperature and glutaraldehyde fixation, trophozoite viability was assessed by direct microscopic observation of 8agella motility and Trypan Blue (0.04%) exclusion, as we have described previously (Farthing & Inge, 1986; Inge & Farthing, 1987). Localization of bile salts in Giardia tropkozohs. Apptoximately 2.5 x lo* trophozoites were mass cultur& in roller bottles (Farthing et al., 1982) in modified TYI-S medium
Bile salt uptake by Giardia lamblia supplemented with 0.1 mM GC and tracer amounts of [i4C]GC (30 uCi/roller bottle). After washing 3 times in PBS, trophozoites were lyzed in a hypotonic buffer containing 1 mM EDTA, 2 mM HEPES buffer, pH 8.0, and 0.005% (w/v) phenylmethylsulphonyl fluoride (PMSF) using twenty strokes of a Dounce homogenizer. Phase contrast microscopy confirmed r99% disruption of trophozoites. After centrifugation (2000 g for I5 min) at 4°C to separate cytoplasm (Fraction Si) from cytoskeleton and adherent cell membrane, the resulting pellet (Fraction Pi) was washed twice in solution containing 2 mM EDTA, 2 mM dithiothreitol (DTT), 2 mM MgS04, 150 mM KCl, 0.005% PMSF and 10 mM Tris buffer (pH 6.9). The cytoplasmic fraction was then centrifuged again (50,000 g for 60 min) at 4°C to separate membrane vesicles (Fraction Ps) from soluble cytoplasmic components (Fraction SZ). Aliquots of each fraction were prepared for liquid scintillation [14c] spectroscopy and radioactivity present expressed as a percentage of that in the hypotonic lysate before differential centrifugation. Deconjugation of bile salts by Giardia. Giardia trophozoites were cultivated at 37°C for 72 h in 16 ml culture tubes containing modified TYI-S medium supplemented with 2.0 mM GC together with tracer amounts of [‘4C]-GC (1 uCi/ tube). After 72 h incubation, tubes were cooled on ice for 15 min. Giardia trophozoites were then sedimented by centrifugation and were washed 3 times in PBS. Samples of incubation medium and trophozoites were stored at -25°C prior to bile salt analysis by thin layer chromatography (TLC). Control incubations without Giardia were treated identically. Samples of radiolabelled bile salt-supplemented medium (0.5 ml) or trophozoites (- 107) were initially prepared by addition of 0.1 mM sodium hydroxide and incubating at 64°C for 20 min. Bile salts were extracted from these aqueous preparations by passing diluted samples through Sep-Pak Cis cartridges (Setchell & Worthington, 1982) and evaporating the methanol eluates to dryness. The residues were redissolved in methanol and subjected to TLC (Hofmann, 1962) on 0.5 mm silica gel G plates using Amylacetate : Propionic acid : Propan-l-01 : Water; 4 : 3 : 2 : 1 as the developing solvent (Gregg, 1966). Pure bile salt standards (GC, GDC, TC, TDC, C and DC) were run with each experimental extract. Standards were stained with phosphomolybdic acid and corresponding areas of experimental plates were scraped off and subjected to liquid scintillation spectroscopy. Statistics. Mann-Whitney U-test was used to evaluate differences between observations. RJWUJLTS
Time courseof bile salt uptake The effect of time on bile salt uptake by Giardia trophozoites is shown in Fig. 1. When trophozoites wereincubatedat 37°Cin GC (0.5 or 5.0 mM) or TC (0.5 mM) containing TYI-S medium the uptake of bile salt describedan exponential component with respect to time. However, a linear component of uptake was also quantitatively important. The slope of the linear portion of the curve for GC (0.5 mM)
1091
was 0.03 nmol/108 trophozoites/min and for TC (0.5 mM) was 0.26 nmol-’ IO8 min-‘. When the
linear component was subtracted from the experimental values an exponential curve was obtained (Fig. 1, broken lines).U,,, (maximaluptake) for GC (0.5 and 5.0 mM) and for TC (0.5 mM) were9.8,23.3 and
36.7 nmol-’
lo* min-‘,
respectively.
These
observations are consistent with there being both an active transport and a diffusional componentfor bile salt uptake by Giardia. In addition, there would appear to be differences in the way in which individual bile saltsare handled,TC being taken up more avidly than GC. Effect of bile salt concentrationandgrowth phaseon bile salt uptake Figure 2 showsthe effect of bile salt concentration on bile salt uptake by Giardia trophozoites. Uptake of GC and TC was a concentration dependentand saturableprocess.Using expression(2), the Michaelis-Menten equation, estimatesof K, and U,,, for both bile saltsweredetermined.The K, for TC (0.21 mM) waslower than that for GC (0.63mM) and the U,,, was higher for TC than GC, being 64.6 and 24.5 nmol-’ log/30 mm-‘, respectively. Growth phase(active or stationary) had no effect on bile salt uptake. Using stationary phasetrophozoites, K,,, for GC was 0.53 mM and I-J,,,, 21.8 nmol-’ log/30 min-‘, values which were not different from those obtained with log-phaseorganisms(Fig. 2). Effect of TC on GC uptake Figure 3 showsthat competitive inhibition of GC uptake by Giardia trophozoites occurred when increasing concentrations of TC were added to TYI-S mediumcontaining either 0.5 or 2.0 mM GC during a 30min incubation. Uptake of GC from a 0.5 mM solution wascompletelyabolishedby increasing TC concentration (U,, = I%,,,,). However, uptake from a 2 mM solution was only partially inhibited by the addition of TC (U, 30.4 and E,, 31.2nmol-’ lo*/30 mine1 . Es,, concentrations were 0.07 and 0.18 mM TC for experimentsusing0.5 and 2.0 mM GC,
respectively). Effect of metabolicinhibitorson bile salt uptake Bile salt uptake was unaffected by preincubation with metronidazole or cytochalasin B (Table 1) neither of which reducedparasiteviability asassessed by Trypan Blue exclusion, and only metronidazole reducedflagellamotility. In contrast sodiumfluoride and low temperature(4°C) significantly inhibited GC uptake. Giardia, which lacks mitochondria is known to be dependenton glycolysisfor energy metabolism (Lindmark,
1980). Sodium fluoride inhibits glycolysis
C.
1092
E. W. Halliday et al. U max
= 36.7nmo1/10a/min 0.5mM
TC
K z = 0.26nmol/10B/min
KI
0
60
120
(4 Time
U max=
0 (W
9.8
180
240
180
240
(min)
nmol/lOa/min
K,
= 0.06nmol/10a
K2
= 0.03nmo1/10*/min
60
= O.O2nmot/lO~/min
/min
120 Time
(min)
Fig. la,
suggestingthat bile salt uptake by Giardia is energy dependent. Sodium fluoride also reduced parasite viability and flagella motility. Inhibition of microfilament function (cytochalasin B) had no effect on GC uptake, indicating that endocytosis was not involved. Bile salt uptake did not occur after glutaraldehyde fixation, making it unlikely that uptake was due to non-specificadsorption of bile salt on to the cell surface.
b
Localization
of bile salt within G&x&a
Fractionation of G&m&a foBowing incubation with tracer [14C’J-CXshowed that 78.5OAof total uptake was located in cytmol with only 1.3% associatedwith a rmnbrax Era&m. Total recovery of radioactive label was 87.6!& These&&gs are consistent with the view that bik salt is not merely absorbed into surface membrane but is transported intraceB~arly.
Bile salt uptake by Giardia
U max
K, ------
-.-.-
- ---_------____--_-___ _._.A.-.
_.C.
.-‘C.-’
= 23.2nmol/108/min
12
5.0mM
GC
= 0.14nmol/10*/min --,.--: _.C. _,/.-.
K, = 0.09nmo1/108/min
60
120
(4
1093
lamblia
Time
180
240
(min)
Fig. 1. Effect of time on conjugated bile salt uptake by Giardia trophozoites in vitro. Uptake curves are plotted for 0.5 mM TC (la), 0.5 mM GC (lb) and 5 mM GC (Ic) plotted as mean (S.E.M.). Results are derived from 3 separate experiments each performed in triplicate. Using the expression Ut = U-(1
- eKlt) + Krt (seetext)
bile salt uptake by Giardia can be resolved into exponential (- - - -) and linear (- . -. -) components, the former consistent with an active transport process and the latter with simple diffusion.
Deconjugationof bile salt by Giardia TLC of extracts of Giardia trophozoites or TYI-S culture medium following incubation for 72 h with radiolabelled bile salt (GC 2 mM) failed to show evidence of bile salt deconjugation. Although trace amounts of contaminating bile salts (taurocholate, Table l-Effect of metabolic inhibitors, low temperature and glutaraldehyde fixation on sodium glycocholate (GC) uptake by Giardia Experimental conditions Control’ Metronidaxole (0.1 mM) Sodium fluoride (50 mM) Cytochalasin B (10 w/ml) 4°C Glutaraldehyde (1%)
GC uptake (% control) mean f S.E.M.
flagella motilityb
100 101.3+11.2
+++ +
Trypan Blue exclusion (%) 98 90
37.7 +4.4+*
-
50
108.7 + 10.7
+++
97
3.5* 1.3** 19.1+3.3**
+ -
98 0
Giardia
aGiardia trophoxoites (- 5 x 106) incubated in TYI-S culture medium containing GC (2 mM). bResults are the mean of 3 experiments. +++ ~-90% motile; + < 15% motile; - all trophozoites non-motile. **PcO.Ol compared to control incubation.
taurodeoxycholate,glycocholate, glycodeoxycholate, cholateand deoxycholate;total < 6%) werefound in the native radiolabelled bile salt, these did not increaseduring experimentalor control incubations. DISCUSSION Previousstudieshave shownthat bile and bile salts not only have trophic effectswith respectto growth of Giardia both in vitro (Farthing et al., 1985; Farthing et al., 1983)and in vivo (Hegner& Eskridge, 1937;Bemrick, 1963),but are actually consumedby the parasiteduring in vitro culture (Farthing et al., 1985).Sinceintraluminal bile saltsare important for intestinal absorption of dietary fat (Carey et al., 1983) and steatorrhea is a common occurrence in symptomatic patients with giardiasis, we have performedexperimentsto characterizeand quantify the processby which Giardia trophozoites take up bile salts. The presentstudy indicatesthat Giardia takes up conjugatedbile saltsby a mechanismwhich appears to involve a combination of active and passive transport processes.Kinetic analysis of bile salt uptake with time, resolved uptake into an initial exponential phaseconsistentwith active transport, followed by a linear phase indicative of passive diffusion; this pattern of bile salt uptake is similar to
C. E. W. Halliday et at.
094 75
50
GC (active)
I
25
-1
GC (stationary)
_
d
I
0
u
2
4
6
8
Bile salt Concentration,
IO
mM
Fig. 2. The effect of conjugated bile salt concentration on bib salt uptake by Giardia trophoaoites. Results are plotted as mean (S.E.M.) and are derived from 4 separate experiments performad in triplicate. Uptake of TC and GC was a saturable process consistent with their wg a &&ed number of binding sites or transporters within the cell membrane. The apparent K,‘s for TC and GC were 0.21 and 0.63 mM, respectively. U, for TC was 64.6 and for GC 24.5 mnol/tOB trophozoites/30 min, respectively. Actively multiplying trophozoites (log phase growth) and stationary phase organisms took up GC similarly.
E max
= 31.2nmolil
E 5”
=
OS imin
0.18mM
2mM GC
I E max
= 11.8nmol/l
E,
=
OS /min
0.07mM
1
Taurocholate
concentration,
mM
over active transport as the major mechanism of bile salt uptake, a process which is not snsxqtibk cixnpetitive inhibition.
to
Bile salt uptake by Giardia lamblia
that which occursin human ileum (Krag & Phillips, 1974;Stigsby& Krag, 1984).The apparent K,,, of TC for this processin Giardia was0.21 mM which is of the same order of magnitude reported for TC following kinetic analysisof human ileal perfusion studies with this bile salt (0.2-0.6 mM) (Krag & Phillips, 1974; Stigsby & Krag, 1984). Again the apparent K,‘s for GC and TC reported in the presentstudy with Giardia are very similar to those obtained by othersusinga variety of preparationsof rat ileum including intestinal perfusion (Schiff et al., 1972),intestinal sheets(Thomson, 1983)and isolated intestinal epithelial cells (Wilson & Treanor, 1975). The study by Reiner et al. (1993) demonstrating sodium dependencyof bile salt uptake by Giardia would also support an active, carrier-mediated transport process. Further evidencefor active transport of bile salts by Giardia is provided in the present study by experimentssuggestingthat bile salt uptake is an energy dependentprocessinhibited by low temperature and sodiumfluoride, an inhibitor of glycolysis. Sodium fluoride also inhibits the activity of protein phosphatasesand thus, additional effects on other aspects of cell metabolism cannot be excluded. Metronidazole, the drug of choice for treatment of Giardia infectionsin humanshad no effect on bile salt uptake after 60 min incubation, eventhough many of the parasiteshad beenrenderednon-motile. Metronidazole is thought to act by inhibiting nucleic acid synthesisand thus might not be expected to impair membranetransport processes after a relatively brief exposure. Our demonstration of competitive inhibition of GC uptake by the closely related primary bile salt TC, suggests that a limited numberof absorptivesites are presentin the parasiteplasmamembrane,raising the possibility that a specificreceptor siteor bile salt transport protein is present in this organism. Our observationsthat bile salt uptake by Giardia is at least in part energy dependentand may involve a specifictransport protein, is again similarto thoseof previous studiesof bile salt transport by mammalian epithelial cells(Wilson & Treanor, 1975). Fractionation of trophozoites after incubation with bile salt confirmed that a substantial proportion of bile salt was presentin the cytosol and was not simply bound to cell membranes,supporting the view that bile salt is internalized. However, further confirmation of this is required since cell disruption itself can result in changes in the intracellular distribution of exogenous substances as bile salts. The role of bile salts in the intracellular metabolism of Giardia is not known. We have suggested
1095
previously (Farthing et al., 1985) that bile salts enhancethe delivery of preformed membranephospholipids, a potentially important role sinceGiardia is unable to synthesizeits own phospholipid(Jarroll et al., 1981).Whether the parasiteusesbile salt asa metabolic substrateis as yet unclear, but unconjugated bile salt concentrations used in the present study stimulate parasite growth (Farthing et al., 1985, 1983).There is someevidenceto suggestthat Giardia may enter the gallbladder and bile ducts (Lyon & Swalm, 1925;Calder & Rigdon, 1935;De Muro, 1939;Goldsteinet al., 1978)whereconcentrations of bile saltswould be substantiallyhigher than thosefound in the proximal smallintestineor in any of the experimentsdescribedin the present study. More recentwork indicatesthat conjugatedbile salts at or above their critical micellarconcentrationsmay protect the parasite from toxic lipolytic products commonlyfound in the smallintestine(Gillin, 1987) and in vitro studiessuggestthat conjugatedbile salts may have a role in promoting encystation(Gillin et al., 1987, 1989),a vital step in Giardia’s life cycle which enablesthe parasiteto survive outsideits host. Thus, the uptake of host bile saltsby Giardia appears to offer an important survival advantage to the parasiteby promoting its growth, while at the same time acting as a trigger to encystation, ensuring completion of its life cycle. Further studies are required to elucidatethe mechanisms by which bile saltsinfluenceintracellular metabolismand to assess the impact of this parasite on bile salt homeostasis during humaninfection, in vivo. Acknowledgement-This work was supported by the Wellcome Trust and the Joint Research Board of St Bartholomew’s Hospital. REFERENCES
Akimova R. F., Benediktov I. I. & Soloviev M. M. 1978. Study of fermentative hydrolysis of fat in the presence of Lumblia duo&n.& (in vitro). Parazitologia 12: 9-14. Baulieu E. E. & Raynaud J. P. 1970. A “proportion graph” method for measuring binding systems.European Journal of Biochemistry 13: 293-304. Bemrick W. J. 1963. The effect of bile flow on Giardia duodenalis race simoni in the intestine of a laboratory strain of Rattus norvegicus. Journal of Parasitology 49: 956-959. Calder R. M. & Rigdon R. H. 1935. Giardia infestation of gallbladder and intestinal tract. American Journal of Medical Science 190: 82-88. Carey M. C. 1982. The Enterohepatic Circulation. In: The Liver: Biology and Pathobioiogy (Edited by Arias I., Popper H., Schachter D. & Shafritz D. A.), pp. 429465. Raven Press: New York. Carey M. C., Small D. M. & Bliss C. M. 1983. Lipid digestion and absorption. Annual Reviews of Physiology 45: 651477.
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