Amoebiasis

Background & objectives: Entamoeba histolytica, the causative agent of amoebiasis and amoebic liver abscess, lyses host cells by direct contact using surface lectins and releases cysteine proteinase (CP). Virulence of E. histolytica is directly related to activity of its CP. The relationship of CP activity and cytotoxicity has not been established. The present study was carried out to explore the events following contact of E. histolytica with target cells.

Methods: Protease activity of E. histolytica was measured by azocaseine and haemoglobin assays, and cysteine proteinase activity was assessed by substrate gel electrophoresis. Target cell lysis was measured by chromium release assay.

Results: Protease activity of E. histolytica was increased 2.5-fold following contact with BHK-21 cell line. CP activity of trophozoites alone was visualized at position 56, 35 and 29 kDa in substrate gel electrophoresis. Contact of trophozoites with target cells augmented the cytotoxic activity of amoebic CP. The increase in CP activity seen by substrate gel electrophoresis and cytotoxicity assay was blocked by pretreatment with E 64, a specific CP inhibitor and GalNAc, a contact inhibitor.

Interpretation & conclusion: The present data showed the involvement of amoebic CP in cytotoxicity and that the CP activity was enhanced on lectin-mediated contact of E. histolytica to the target cells. Further studies need to be done to understand the mechanism at the molecular level.

Key words BHK-21 - Entamoeba histolytica - GalNAc - lectin - virulence

Entamoeba histolylica, an intestinal parasite responsible for amoebic dysentery and liver abscess, is a major cause of morbidity and mortality worldwide1. E. histolytica damages the colonie mucosa and other tissues of the human host through direct contact, which is mediated by Gal/GalNAc lectins2; inhibition of lectin activity with N-acetyle galactosamine (GalNAc) blocks contact-mediated cytotoxicity3.

Several virulence factors of E. histolytica have been described such as Gal/Gal NAc inhibitable lectins4, amoebapore5, and cysteine proteinase (CP)6-8. CP of E. histolytica is involved in several steps of invasion and tissue damage3. Though, amoebic CP has been implicated in the cytopathic effect of E. histolytica on cultured cell monolayer6, partial inhibition of CP gene by antisense had no effect on cytopathic or haemolytic activities8. The sequence of events, which follow contact between E. histolytica and target cells9 is not clearly understood. The aim of the present study was therefore to explore the events following contact of E. histolytica with target cells. The effect of E. histolytica strain HM1 : IMSS contact with baby hamster kidney cells (BHK-21) on CP and the consequences of this cell-to-cell interaction, were studied.

Material & Methods

Maintenance of cell culture: Axenic E. hislolytica trophozoites strain HM1 :IMSS clone 6 was maintained in TYI-S-33 medium supplemented with penicillin (10OU/ ml), streptomycin (100 µg/ml) and 15 per cent adult bovine serum (Biological Industries, Haemek, Israel), as previously described10.

BHK-21 cell line (National Center for Cell Sciences, Pune, India) was cultured in RPMI-1640 (Gibco BRL, Rockville, USA) supplemented with 2 mM L-glutamine, 25 mM4-(2-Hydroxyethyl) piperazine-1-ethenesulfonic acid (MEPES) buffer, 0.2 per cent sodium bicarbonate, 50 nM2-mercaptoehtanol (Sigma, USA) and 10 percent foetal calf serum (FCS, Biological Industries, Israel) with antibiotics penicillin 100U/ml and streptomycin 100 µg/ml (Gibco BRL, Rockville, USA). The culture was seeded and grown in six-well and 96-well tissue culture plates (Nunc, Rosklde, Denmark) as required for different experiments, at 37°C in humidified 5 per cent CO2 atmosphere.

Adhesion of trophozoiles on BHK-21 monolayer: BHK-21 monolayer was washed with RPMI-1640 without FCS. E. hislolytica trophozoites were incubated with 0.5Mor 1.0 MGaINAc (Sigma, USA) to block E. histolytica lectin and 200 µM of L-trans-epoxysuccinyl-leucyl-amido-(4-guanidino) butane (E 64) (Sigma, USA) a CP inhibitor11 to block CP activity. The trophozoites were added to BHK-21 monolayer (1:10 E. hiatolytica: BHK-21) and incubated at 37°C in humidified 5 per cent CO2 atmosphere for 60 min. Each well was washed with chilled phosphate buffered saline (PBS), pH 7.2, to separate the trophozoites from BHK-21 monolayer. Trophozoites were sonicated and crude lysate was used for measuring CP activity. all experiments were done in triplicate and repeated three times.

Haemoglobin assay: Hacmoglobinase aelivity of CP releases soluble low molecular weight peplides into the supernatant, which is measured at 280 nm (A^sub 280^)15. In brief, 500 µl of haemoglobin (2% haemoglobin powder with 5 Murea) was incubated with 150 µl crude amoebic lysate at 37°C for 15 min; blank was prepared by incubation of amoebic crude lysate with 10 per cent TCA15. The reaction was stopped by adding 5 ml of 10 per cent TCA. Mixture was centrifugea at 15,000 g for 5 min at room temperature; supernatant was collected and absorbance read at 280 nm.

One unit of enzyme activity was defined as the amount of enzyme required to cause a unit increase in absorbance across a 1 cm path length, at 440 nm for azocascine and 280 nm for haemoglobin assay15.

Substrate gel electrophoresis: Crude lysate of control and experimental groups (10 µg protein) was electrophoresed at 50V at 4°C on a 10 per cent polyacrylamide gel co-polymerized with 0.2 per cent gelatin (Sigma, USA). Rainbow molecular weight markers (Invitrogen, Carlsbad, USA) were used for detection of correct size of cysteine proteinase of E. histolytica. Sodium dodecyl sulphate (SDS) was removed by two washing in 2.5 per cent triton X1OO for 30 min each at room temperature. The gel was incubated in developing buffer [20 mM dithioth threitol (DTT) and 1OO mM sodium acetate pH 4.2 with 1% triton Xl 00] at 37°C with continuous shaking for 3 h; and stained with coomassie blue for 30 min. Zone of protease activity was visualized as clear band against a blue background16.

Statistical analysis: Comparisons between groups were performed by Mann-Whitney sign test and P value

Results

Chromium release assay: CytotoxicityofBHK-21 targets mediated by E. histolytica alone was 34.5±3.7 per cent, while that of trophozoites pre-incubated with E 64, was 16.1±9.4 per cent (P

Cysteine proteinase activity: The protease activity of E. histolytica was 20.9±1.7 and 21.8±0.5 U/mg protein by azocasein and haemoglobin assays respectively. This activity was significantly inhibited by E 64 pre-treatment of trophozoites (2.7±1.7 and 2.7±0.9 U/mg protein) (P

Substrate gel electrophoresis: Crude lysates of E. histolytica with and without contact with BHK-21 monolayer were subjected to gelatin substrate gel electrophoresis. The bands depicting protease activity were more intense in lane containing crude lysate of E. histolytica, which had contact with BHK-21 monolayer; bands were visible at 29, 35 and 56 kDa. Highest activity was seen in the region of 56 kDa, which represents neutral CP. No gelatin degradation was seen in BKH-21 alone or E 64 treated E. histolytica (Fig. 2).

Discussion

The role of CP of E. histolytica in the degradation of extracellular matrix, in vitro destruction of monolayer and tissue damage2 has been well established. E. hislolytica is believed to degrade target cells through Gal/GalNAc mediated contact4 but factors responsible for activating amoebic CP are not clear. Proteases which are present inside eel Is in inactive form are converted to active form by unknown signals. Similarly amoebic cysteine proteases are synthesized as precursor proteins with predomains, prodomains and catalytic domains. Prepro enzymes are subsequently processed to mature enzyme. The signal which activates conversion of inactive enzyme to mature active enzyme is not known17. The findings of the present study showed that CP activity of E. histolytica was dependent on Gal/GalNAc mediated contact with target cell.

The target cell cytotoxicity was inhibited by pretreatment of E. histolytica trophozoites with the CP specific inhibitor E 64 or GaINAc. It has been previously shown that blocking of amoebic lectins with Gal/GalNAc reduces both adherence and cytotoxicity3,18; however, the cytotoxicity was not completely abolished, as is the case in our study also. Our results suggested that inhibitable contact between E. histolytica and BHK-21 was important for the cytotoxicity, which was mediated by CP, since the effects were inhibited by E 64. However, other factors are also involved in the cytotoxicity.

CP activity of E. histolytica, which had been in contact with BHK-21 monolayer, was greater than that of E. histolytica alone; pre-treatment of E. histolytica by GaINAc blocked the increase in CP activity suggesting that GaINAc mediated signals were required for conversion of prepro form of cysteine proteinase into active enzyme. CP activity was almost completely blocked by E 64 treated E. histolytica trophozoites.

Cysteine proteinase activity seen in substrate gel electrophoresis at 28, 35 and 56 kDa region suggests involvement of more than one cysteine proteinase. ACP3 (E. histolytica CPl ) has been shown to have two distinct activities at 27-30 kDa19, CP2 at 35 kDa and CP5 at 29 kDa16 and neutral CP has been reported to have proteinase activity at 56 kDa20. The increased CP activity following contact with monolayer therefore involved CP 1, 2, 5 and neutral CP. We have earlier shown that the activity at 56-66 kDa increased after passage of E. histolytica through hamster liver, which was related to increase in virulence of E. histolytica21.

Drugs in current use for amoebiasis have problems of toxicity and drug resistance. Cysteine proteinase is an attractive potential target for new anti-amoebic drug development. However, better understanding of its mechanism of action is required to exploit its full potential. In conclusion, the present findings suggest that direct contact of E. histolytica with target cells through GaINAc results in increased CP activity. Further studies are required to elucidate the molecular basis for this observation.

Acknowledgment

Authors acknowledge Dr Alok Bhattacharya, Jawaharlal Nehru University, New Delhi, India, for providing Entamoeba histolytica trophozoites strain HM1:IMSS clone 6.

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Divyendu Singh, S.R. Naik & Sita Naik*

Departments of Gastroenterology & * Immunology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India

Received September 9, 2003

Reprint requests : Dr Sita Naik, Professor & Head, Department of Immunology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Raebareli Road, Lucknow 226014, India

e-mail: sitanaik@sgpgi.ac.in

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