J . t u k . Micmhiol., 45(2), 1498 pp. 9S-i2S 0 1998 by the Socicty ol Protozoologists

Identification of Significant Variation in the Composition of Lipophosphoglycan-like Molecules of E. histolytica and E. dispar’

SUSAN MOODY,* STEVEN BECKER,* YAEL NUCHAMOWITZ“ and DAVID MIRELMAN*.’ *Department of Membrane Research and Biophysics, Weizmann Institute of Science, 76100 Rehovot, Isruel

ABSTRACT. The lipophosphoglycan-like (LPG-like) molecules of E. histolytica virulent strains are clearly distinct from those of the avirulent E. hisrol-pica and E. dispar strains. Abundant ‘LPG’ levels are apparently limited to virulent strains, while lipophosphopep- tidoglycans (‘LPPG’s) are common to both virulent and avirulent strains of E. histolytica and E. dispar. It is therefore conceivable that ‘LPPG’ performs a function that is essential to survival within the host, while the ‘LPG’ performs a more specific function related to virulence.

Supplementary key words. Adherence, amebiasis, cytotoxicity.

MEBIASIS annually afflicts tens of millions of people with A symptoms that range in severity from diarrhea, to internal abscesses (most commonly hepatic) and death if untreated [21]. Infection results predominantly from ingestion of food and wa- ter fecally contaminated with the protozoan parasite Entarnoeba histolytica. While it is clear that amebiasis is largely confined to regions of the world where there are sanitation limitations, it has not been possible to rapidly assess the risk of an infected individual developing invasive symptoms. Field identification has until recently relied on microscopic examination of fecal specimens. This approach is inadequate for diagnosis however, because E. histolytica can not be differentiated from the mor- phologically indistinguishable E. dispar, which is usually found in asymptomatic cyst passers [5]. Molecular differentiation is now possible between E. histolytica and E. dispar using specific antibodies to detect antigens in feces and sera, and using poly- merase chain reaction (PCR) detection of species specific gene sequences (reviewed by Jackson and Ravdin [12]). Despite these advances, field evaluation of individuals simultaneously infected with both E. histolytica and E. dispar continues to pose a major challenge.

Determination of the relative virulence of E. histolytica strains is another important criteria that is currently difficult to evaluate in the field. Factors that influence the virulence status of individual E. histolytica strains remain ill-defined and mo- lecular markers of virulence have not been well characterized. Information of this nature is critical for ensuring correct patient diagnosis, for the prescription of appropriate medication and for evaluating the extent of the world’s population at risk from developing invasive symptoms due to infection with virulent E. histolytica strains.

Infiltration of host tissue is a contact-dependent event and the parasite molecules that facilitate this process are likely markers of amebic virulence. The most thoroughly studied molecular participants in this host infection process include: the galactose binding surface lectin or adhesin, a pore-forming peptide in- volved in host cell lysis and cysteine proteinase(s) that degrade the host’s extracellular matrix. Identification of species specific epitopes of the adhesion in E. histolytica and E. dispar strains currently provides the most reliable means of field differentia- tion.

A role for amebic lipophosphoglycan-like (LPG-like) mole-

I This paper was originally presented as part of the symposium on “Molecular and Cellular Aspects of the Interaction between Amebae and Colonic Environments,” August 18, 1996, Jerusalem, Israel. This symposium was co-chaired by Dr. Tsutomu Takeuchi and Prof. David Mirelman and held by the Medical Microbiology Interdisciplinary Com- mission (MEMIC), a commission created by the International Union of Microbiological Societies.

* To whom correspondence should be addressed. Telephone: 972-8- 9343 160; Fax: 972- 8-9468256; Email: bfmirelm@weizmann.weizmann.ac.il

cules as ligands for host-cell receptors has also been suggested by Stanley et a1 [ZO] who were able to inhibit cytolysis of CHO cells by the virulent E. histolytica strain HM-1:IMSS using a monoclonal antibody directed toward these amebic molecules. A number of reports suggest that these molecules are ubiquitous in E. histolytica strains, although heterogeneous with regard to molecular weight [ l , 11, 191. In this review we describe sig- nificant variation in the LPG-like molecules from both virulent and avirulent E. histolytica and as well as from E. dispar. We also discuss the potential biological significance of these dif- ferences and the possibility of using these molecules as markers of amebic virulence.

MATERIALS AND METHODS

Parasites. All Entamoeba trophozoites were grown in TYI- S-33 medium and harvested in the logarithmic phase of growth [7]. E. histolytica virulent strains HM- 1:IMSS clone 6 (c16) [16], and HK-9 [lo] were cultured axenically. BNI:0591 tro- phozoites (isolated from a liver abscess of a patient by Dr. G. Burchard, Bernhard-Nocht Institute, Hamburg, Germany [3]) were co-cultured with bacterial flora. The avirulent strain Rah- man [8] was cultured axenically. Strain virulence refers to the reported capability of producing hepatic lesions in hamsters [4] or the rapid destruction of tissue cultured cell monolayers [ 2 ] . The E. dispar strains SAW 1734R clAR [17] and SAW 760RR clA (documented by Clark and Diamond; [6]) were grown mono-xenically together with C. fasciculata in the absence of bacteria.

Isolation and analysis of LPG-like molecules. LPG-like molecules were isolated from harvested amebic trophozoites es- sentially as described for the purification of Leishmania major promastigote LPG [13, 141 with the modifications that have been described in detail by Moody et a1 [ 181. Briefly, amebae (in batches of 3 X lo8 cells) were extracted with c h l o r o f o d methanovwater (1:2:0.8, v/v) to remove neutral lipids, phos- pholipids, glycolipids and low molecular weight metabolites. LPG-like molecules were isolated from the delipidated amebae by repeated extraction with 1-butanol-saturated water and pu- rified by chromatography on a 5 ml column of octyl-Sepharose (Pharmacia) equilibrated in 0.1M ammonium acetate, contain- ing 5% /-propanol and eluted in a linear gradient of Z-propanol (5-60%). The percentage of I-propanol in each fraction was determined by refractometry. The major carbohydrate compo- nents eluted from the column at 28% 1-propanol, as is char- acteristic of the LPGs of Leishmania. Subsequent anion ex- change (Fast-flow DEAE-Sepharose, Sigma) chromatography in the presence of 30% I-propanol and eluting with an ammonium acetate gradient, was essential for purification of the LPG-like molecules that were isolated from the virulent E. histolytica strains [ 18 1.

Carbohydrate containing fractions were detected by spotting aliquots onto silica HPTLC plates and spraying with orcinol

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10s J. EUK. MICROBIOL., VOL. 45, NO. 2, MARCH-APRIL 1998

Table 1. Molecular characteristics of the ‘LPG’ and ‘LPPG’ from E. histolyrica and E. dispar strains that were eluted trom octyl-Scpharose at 28% 1-propanol and subsequently fractionated using DEAE anion exchange chromatography.

Isolate

Weakly charged molecules (‘LPG’*) Highly charged molecules (‘LPPG.9

kg/lOx Anion exchange &lox Anion exchange Strain MWt (kDa) trophozoites elution peakh MWt (kDa) trophozoites elution peakh

E. hisiolvtica Virulent HM-I:IMSS c16 4s 98 1 lOmM 160 272 2SOmM

BNI:OS91 (xenic) 45 103 90mM I60 322 2SOinM HK-9 65 87 130mM 12s 37 2SOmM

Avirulcnt Rahman - - - 65 385 400mM

E. dispcir SAW l734R clAR - - - 16 354 SOOmM - - 93 1 so NA SAW 760RR CIA’ -

* Molecular weights determined by SDS-PAGE migration. I’ Buffers used were ammonium acetate in 30% I-propanol.

Fractionation of the glycoconjugates of SAW 760RR CIA were not attempted (NA)

reagent (20 mg/ml orcinol monohydrate in ethanoYConc. H,SO,/water, 75:10:5, by volume) and heating for 5 rnin at 110” C. Carbohydrate content was determined using the method of Dubois et al., [9] and protein content was assayed using BCA reagent (Pierce). The presence of phosphatidylinositol linkages was identified by susceptibility to Bacillus thuringiensis Phos- phatidylinositol-Specific Phospholipase C (PI-PLC; Oxford GlycoSystems Ltd., UK); hexose-I-phosphate linkages were detected by sensitivity to mild acid (40 mM Trifluoroacetic acid [TFA], 8min, 100” C; [15]). Amino acids were derivatized with o-phthalaldehyde (OPA) and analyzed by reverse phase chro- matography on a Hewlett Packard Amino Quant system.

Antibody production. Guinea pig polyclonal antibodies di- rected toward HM- 1:IMSS c16 ‘LPG’ and subsequently affinity purified against the same antigen were prepared [IS].

Cytolysis assay. A semiquantative assay for determination of mammalian cell destruction was adapted from the method of Bracha and Mirelman [2]. Briefly, HeLa cells were cultured in 24-well tissue culture plates under a 5% CO, atmosphere at 36“ C, until reaching a confluent monolayer. Destruction was mon- itored following the addition of trophozoites (los) at a ratio of approximately 1:2 HeLa: trophozoites, in the presence or ab- sence of monospecific antibodies. When the wells containing the control amebic trophozoites had destroyed approximately 70% of the monolayer, as determined by light microscopy (40- 60 min), the plates were cooled to 4” C (10 min) and amebae were carefully removed by repeated washing with cold saline. The remaining HeLa were then fixed in 4% formaldehyde (10 rnin), washed and stained with methylene blue (0.1 M in borate buffer, pH 8.7, 10 min). Excess dye was removed by repeated washing with 0.01 M in borate buffer, then the dye incorporated into the HeLa cells was extracted with 0.1M HCI (37” C, 30 min) and the intensity of the dye in each well was measured at 660 nm. The level of destruction was monitored relative to wells containing the control HeLa monolayers, to which ame- bae had not been added.

RESULTS The LPG-like glycoconjugates of virulent and avirulent E.

histolyrica and E. &par are clearly distinct (Table 1). Virulent E. histolytica strains contain two major LPG-like glycoconju- gates that differ in molecular charge and weight. In contrast, the avirulent E. histolytica strain Rahman and the E. dispar strains SAW 1734R clAR and SAW 760RR clA contain only single major LPG-like molecules that are highly charged. All these molecules were identified to contain carbohvdrate. hex-

acid) and a hydrophobic anchor that could be released by PI- PLC hydrolysis. Molecules with a minor protein component (4% weight proteidweight glycoconjugate) were defined as ‘li- pophosphoglycans’ (‘LPG’s) and were identified in significant levels only in the virulent E. histolytica strains. Molecules with a significant amino acid content (12-15% weight proteidweight glycoconjugate) were identified in all strains and have been defined as lipophosphopeptidoglycans (‘LPPG’s).

Monospecific polyclonal antibodies raised and affinity se- lected against ‘LPG’ of the virulent E. histolyrica strain HM-1: IMSS c16 also identified the ‘LPPG’ component of this and other E. histolytica strains by Western blot analysis. The

SAW 760RR CIA

Fluorescence Intensity

Fig. 1. Flow cytometric analysis of ‘LPGILPPG’ in E. hi.vtdytic.cr strains using a monospecific guinea pig sera directed against the ’LPG’ of the virulent strain HM-1:IMSS c16 (Peak 2). Binding of the second- ary antibody alone, FITC conjugated goat anti-guinea pig to the amebae are included as controls (Peak 1). A. Virulent E. hisrolyfictr strains HM- 1:IMSS c16, BNI:OS91 (xenic) and HK-9. B. Avirulent Rahman. C. E.

ose-i-phosphate linkages (as suggested by sensitivity to mild dispar strains SAW 1734R clAR and SAW 760RR CIA.

MOODY ET AL.-ENTAMOEBA LIPOPHOSPHOGLYCAN

A

11s

B

Strains

100

90

80

70 h z (j(j 0

50 e g 40 n

30

20

10

0

c1

Treatment Fig. 2. A. Comparison of the level of HeLa monolayer destruction by a selection of Enramoeba strains: HM-I :IMSS c16; BNI:0591 (xenic):

Rahman and E. dispur SAW 1734R clAR. Percentage destruction was calculated as a decrease in the amount of dye incorporated into remaining HeLa cell monolayers after incubation and removal of the trophozoites. Each experiment was done in triplicate under the same conditions and thc graphs represent the average of each treatment. Error bars are not shown as the standard deviation for each treatment is too small to represent on this scale. B. Comparison of the levels of HeLa destruction by E. histulvticu strain HM-1:IMSS c16 in the absence and i n the presence of various concentrations of monospecific guinea-pig sera, or with non-immune guinea-pig sera. The column representing HeLa destruction by trophozoites i n the presence of non-immune guinea-pig sera was approximately equivalent to the level of destruction achieved in the presence o f amebae alone in these experiments

‘LPPG’ of the E. dispar strains SAW 1734R clAR and SAW 760RR CIA in contrast, were not identified by western blot anal- ysis when reacted with this polyclonal sera (data not shown).

Surface exposure of the LPG-like components of the virulent E. histolytica strain HM-1:IMSS c16, BNI:0591 xenic and HK- 9 (Fig. 1A) was identified by flow cytometric analysis, using the monospecific polyclonal antibodies. Importantly, the epi- topes identified by these antibodies were not accessible to a significant degree at the surface of either the avirulent E. his- tolytica strain Rahman (although well recognized by this anti- sera on western blots of total cell lysates; Fig. 1B) or the E. dispar strains SAW 1734R clAR and SAW 760RR CIA (Fig. 1 C). In addition, the polyclonal antibodies directed toward the LPG-like glycoconjugates from the virulent HM- 1 :IMSS c16 trophozoites were able to partially protect HeLa monolayers from cytolysis by trophozoites of the same strain (Fig. 2) .

DISCUSSION The LPG-like molecules of E. histolytica virulent strains are

clearly distinct from those of the avirulent E. histolytica and E. dispar strains. Abundant ‘LPG’ levels are apparently limited to virulent strains, while ‘LPPG’ is common to both virulent and avit-ulent strains of E. histolytica and E. dispar. It is therefore conceivable that ‘LPPG’ performs a function that is essential to survival within the host, while the ‘LPG’ performs a more specific function related to virulence.

Abundant surface exposure of these ‘LPG-like’ glycoconju- gates in the virulent strains of E. histolytica HM1:IMSS c16, BNI:0591 and HK-9 was identified by antibody detection. Im-

portantly, while the ‘LPPG’ levels of the E. histolytica avirulent strain Rahman are similar to those of the most virulent strains. and these molecules are well recognized by western blot anal- ysis of the total lysates (data not shown), surface exposure of these molecules was poorly identified by Flow cytometric anal- ysis. In addition, the ‘LPPGs’ of the E. dispar strains examined do not share epitopes that are detected by these antibodies using western blot (data not shown), or Flow cytometric analysis.

Surface exposure of these LPG-like glycoconjugates in the virulent E. histolytica strains would enable these molecules to interact with host-cells [20]. Consistent with this suggestion, is the ability of the polyclonal antibody directed toward these mol- ecules to offer a degree of protection to HeLa monolayers, from cytolysis by virulent amebae. A similar observation using a monoclonal antibody also directed toward these molecules was first recorded by Stanley et a1 [20]. In addition, the relatively poor capacity of the avirulent E. histolytica strain Rahman and the E. dispar strains to destroy HeLa monolayers also suggests a correlation between surface exposure of these molecules and virulence.

The biological significance of the apparent correlation be- tween ‘LPG’ abundance and virulence has not yet been inves- tigated. One may speculate however, that an ‘LPG’ specific re- agent may provide a diagnostic marker that would significantly improve field identification of strain virulence. An immuno- diagnostic kit could therefore be envisaged, that utilizes avail- able markers to differentiate E. histolytica and E. dispur, and has the added capacity of rapidly assessing strain virulence on the basis of the reactivity with an ‘LPG’-specific reagent.

12s J . ECK. MICROBIOL.. VOL. 45. NO. 2. MARCH-APRIL 1998

ACKNOWLEDGMENTS This research was supported in part by a grant f rom the Com-

mission of thc European Communities, Avicennc Program. and by the Center for Molecular Biology of Tropical Diseases. Weizmann Institute.

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