Contact of Entamoeba histolytica with baby hamster kidney-21 (BHK-21) cell line on cysteine proteinase activity
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Indian J Med Res 119, April 2004, pp 157-161
Contact of Entamoeba histolytica with baby hamster kidney-21
(BHK-21) cell line on cysteine proteinase activity
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
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 histolytica, an intestinal parasite steps of invasion and tissue damage3. Though, amoebic
responsible for amoebic dysentery and liver abscess, is CP has been implicated in the cytopathic effect of
a major cause of morbidity and mortality worldwide1. E. histolytica on cultured cell monolayer 6, partial
E. histolytica damages the colonic mucosa and other inhibition of CP gene by antisense had no effect on
tissues of the human host through direct contact, which cytopathic or haemolytic activities8. The sequence of
is mediated by Gal/GalNAc lectins2; inhibition of lectin events, which follow contact between E. histolytica
activity with N-acetyle galactosamine (GalNAc) blocks and target cells9 is not clearly understood. The aim of
contact-mediated cytotoxicity3. the present study was therefore to explore the events
following contact of E. histolytica with target cells.
Several virulence factors of E. histolytica have The effect of E. histolytica strain HM1: IMSS contact
been described such as Gal/Gal NAc inhibitable with baby hamster kidney cells (BHK-21) on CP and
lectins 4 , amoebapore 5 , and cysteine proteinase the consequences of this cell-to-cell interaction, were
(CP)6-8. CP of E. histolytica is involved in several studied.
157158 INDIAN J MED RES, APRIL 2004
Material & Methods incubated again at 37ºC in humidified 5 per cent CO2
atmosphere for 1 h. Plate was centrifuged and released
51
Maintenance of cell culture: Axenic E. histolytica Cr in 100 µl of supernatant was counted in a gamma
trophozoites strain HM1:IMSS clone 6 was maintained counter (Multigamma, LKB, Sweden). All experiments
in TYI-S-33 medium supplemented with penicillin (100U/ were done in triplicate and repeated five times. Per cent
ml), streptomycin (100 µg/ml) and 15 per cent adult cell cytotoxicity was calculated by the following formula
bovine serum (Biological Industries, Haemek, Israel), as
previously described10. total release-spontaneous release
% cell cytotoxicity = x100
BHK-21 cell line (National Center for Cell Sciences, experimental release–spontaneous release
Pune, India) was cultured in RPMI-1640 (Gibco BRL, Azocasein assay: The proteolytic activity of CP was
Rockville, USA) supplemented with 2 mM L-glutamine, assayed as described earlier 13,14. CP acts on the
25 mM 4-(2-Hydroxyethyl) piperazine-1-ethenesulfonic chomogenic substrate azocasein (Sigma, USA) to release
acid (HEPES) buffer, 0.2 per cent sodium bicarbonate, low molecular weight soluble peptides into the
50 µM 2-mercaptoehtanol (Sigma, USA) and 10 per cent supernatant, which give a colour reaction that is measured
foetal calf serum (FCS, Biological Industries, Israel) with at 440 nm. Azocaseine (250 µl, 0.2% w/v) in 100mM
antibiotics penicillin 100U/ml and streptomycin phosphate buffer, pH 6.5 was incubated with 150 µl crude
100 µg/ml (Gibco BRL, Rockville, USA). The culture lysate of E. histolytica at 37ºC for 30 min and phosphate
was seeded and grown in six-well and 96-well tissue buffer without crude lysate was used as control. The
culture plates (Nunc, Rosklde, Denmark) as required reaction was stopped by adding 10 per cent
for different experiments, at 37ºC in humidified 5 per trichloroacetic acid (TCA) for 15 min at room
cent CO2 atmosphere. temperature. Mixture was centrifuged at 15,000 g for 5
min at room temperature; 1.2 ml of supernatant was
Adhesion of trophozoites on BHK-21 monolayer: collected in a vial containing 1.4 ml 1 M NaOH, mixed
BHK-21 monolayer was washed with RPMI-1640 and absorbance read at 440 nm.
without FCS. E. histolytica trophozoites were incubated
with 0.5M or 1.0 M GalNAc (Sigma, USA) to block E. Haemoglobin assay: Haemoglobinase activity of CP
histolytica lectin and 200 µM of L-trans-epoxysuccinyl- releases soluble low molecular weight peptides into the
leucyl-amido-(4-guanidino) butane (E 64) (Sigma, USA) supernatant, which is measured at 280 nm (A280)15. In
a CP inhibitor11 to block CP activity. The trophozoites brief, 500 µl of haemoglobin (2% haemoglobin powder
were added to BHK-21 monolayer (1:10 E. with 5 M urea) was incubated with 150 µl crude amoebic
histolytica:BHK-21) and incubated at 37ºC in humidified lysate at 37ºC for 15 min; blank was prepared by
5 per cent CO2 atmosphere for 60 min. Each well was incubation of amoebic crude lysate with 10 per cent
washed with chilled phosphate buffered saline (PBS), TCA15. The reaction was stopped by adding 5 ml of 10
pH 7.2, to separate the trophozoites from BHK-21 per cent TCA. Mixture was centrifuged at 15,000 g for
monolayer. Trophozoites were sonicated and crude lysate 5 min at room temperature; supernatant was collected
was used for measuring CP activity. All experiments and absorbance read at 280 nm.
were done in triplicate and repeated three times.
One unit of enzyme activity was defined as the amount
Chromium release assay 12 : Culture medium was of enzyme required to cause a unit increase in absorbance
aspirated from the wells of 96-well plate seeded with BHK across a 1 cm path length, at 440 nm for azocaseine and
21 cells followed by addition of 20 µl each of foetal bovine 280 nm for haemoglobin assay15.
serum and 51Cr labeled sodium chromate (50 µCi/ml)
(BARC, India). Plate was incubated at 37ºC in humidified Substrate gel electrophoresis: Crude lysate of control
5 per cent CO2 atmosphere for 2 h, and washed twice to and experimental groups (10 µg protein) was
remove unincorporated 51Cr sodium chromate. E. electrophoresed at 50V at 4ºC on a 10 per cent
histolytica trophozoites were added to the BHK-21 polyacrylamide gel co-polymerized with 0.2 per cent
monolayer (1:10 :: E. histolytica: BHK-21) and plate was gelatin (Sigma, USA). Rainbow molecular weightSINGH et al : CYSTEINE PROTEINASE OF E. HISTOLYTICA ON BHK-21 159 markers (Invitrogen, Carlsbad, USA) were used for of trophozoites (2.7±1.7 and 2.7±0.9 U/mg protein) detection of correct size of cysteine proteinase of (P
160 INDIAN J MED RES, APRIL 2004
Discussion activity at 56-66 kDa increased after passage of
E. histolytica through hamster liver, which was related
The role of CP of E. histolytica in the degradation to increase in virulence of E. histolytica21.
of extracellular matrix, in vitro destruction of monolayer
and tissue damage 2 has been well established. Drugs in current use for amoebiasis have problems of
E. histolytica is believed to degrade target cells through toxicity and drug resistance. Cysteine proteinase is an
Gal/GalNAc mediated contact4 but factors responsible attractive potential target for new anti-amoebic drug
for activating amoebic CP are not clear. Proteases which development. However, better understanding of its
are present inside cells in inactive form are converted to mechanism of action is required to exploit its full potential.
active form by unknown signals. Similarly amoebic In conclusion, the present findings suggest that direct
cysteine proteases are synthesized as precursor proteins contact of E. histolytica with target cells through GalNAc
with predomains, prodomains and catalytic domains. results in increased CP activity. Further studies are required
Prepro enzymes are subsequently processed to mature to elucidate the molecular basis for this observation.
enzyme. The signal which activates conversion of
inactive enzyme to mature active enzyme is not known17. Acknowledgment
The findings of the present study showed that CP activity
of E. histolytica was dependent on Gal/GalNAc Authors acknowledge Dr Alok Bhattacharya, Jawaharlal Nehru
mediated contact with target cell. University, New Delhi, India, for providing Entamoeba histolytica
trophozoites strain HM1:IMSS clone 6.
The target cell cytotoxicity was inhibited by pre-
treatment of E. histolytica trophozoites with the CP References
specific inhibitor E 64 or GalNAc. It has been previously
shown that blocking of amoebic lectins with Gal/GalNAc 1. WHO/Pan America Health Organization. Expert Consultation
on Amoebiasis: Amoebiasis. WHO Wkly Epidemiol Rec 1997;
reduces both adherence and cytotoxicity3,18; however,
72 : 97-100.
the cytotoxicity was not completely abolished, as is the
case in our study also. Our results suggested that 2. Ravdin JI, Croft BY, Guerrant RL. Cytopathogenic mechanisms
inhibitable contact between E. histolytica and BHK-21 of Entamoeba histolytica. J Exp Med 1980; 152 : 377-90.
was important for the cytotoxicity, which was mediated 3. Ravdin JI, Gurraht RI. Role of adherence in cytopathogenic
by CP, since the effects were inhibited by E 64. However, mechanism of Entamoeba histolytica. J Clin Invest 1981; 68 :
other factors are also involved in the cytotoxicity. 1305-13.
4. Mann BJ. Structure and function of the Entamoeba histolytica
CP activity of E. histolytica, which had been in Gal/GalNAc lectin. Int Rev Cytol 2002; 216 : 59-80.
contact with BHK-21 monolayer, was greater than that
5. Leipee M. Amoebapores. Parasitol Today 1997; 13 : 179-84.
of E. histolytica alone; pre-treatment of E. histolytica
by GalNAc blocked the increase in CP activity 6. Keene WE, Hidalgo ME, Orozco E, McKerrow JH. Entamoeba
suggesting that GalNAc mediated signals were required histolytica: correlation of the cytopathic effect of virulent
for conversion of prepro form of cysteine proteinase into trophozoites with secretion of a cysteine proteinase. Exp
active enzyme. CP activity was almost completely Parasitol 1990; 71 : 199-206.
blocked by E 64 treated E. histolytica trophozoites. 7. Jacobs T, Bruchhaus I, Dandekar T, Tannich E, Leipee M.
Isolation and molecular characterization of a surface-bound
Cysteine proteinase activity seen in substrate gel proteinase of Entamoeba histolytica. Mol Microbiol 1998; 27 :
electrophoresis at 28, 35 and 56 kDa region suggests 269-76.
involvement of more than one cysteine proteinase. ACP3 8. Ankri S, Stolarsky T, Mirelman D. Antisense inhibition of
(E. histolytica CP1) has been shown to have two distinct expression of cysteine proteinases does not affect Entamoeba
activities at 27-30 kDa19, CP2 at 35 kDa and CP5 at 29 histolytica cytopathic or haemolytic activity but inhibits
kDa 16 and neutral CP has been reported to have phagocytosis. Mol Microbiol 1998; 28 : 777-85.
proteinase activity at 56 kDa20. The increased CP activity 9. Reed SL, Keen WE, McKerrow JH. Thiol proteinase expression
following contact with monolayer therefore involved CP correlates with pathogenicity of Entamoeba histolytica. J Clin
1, 2, 5 and neutral CP. We have earlier shown that the Microbiol 1989; 27 : 2772-7.SINGH et al : CYSTEINE PROTEINASE OF E. HISTOLYTICA ON BHK-21 161
10. Diamond LS, Harlow DR, Cunnick CC. A new medium for the enzymes: A practical approach. Oxford : IRL Press; 1989 p.
axenic cultivation of Entamoeba histolytica and other Entamoeba. 25-56.
Trans R Soc Trop Med Hyg 1978; 72 : 431-2.
16. Hellburg A, Leippe M, Bruchhaus I. Two major 'higher molecular
11. Barret AJ, Kembhavi AA, Brown MA, Kirschke H, Knight mass proteinase' of Entamoeba histolytica are identified as CP1
CG, Tamai M, et al. L-trans-epoxysuccinyl-leucyl-amido-(4- and CP2. Mol Biochem Parasitol 2000; 105 : 305-9.
guanidino) butan (E-64) and its analogues as inhibitor of cysteine
proteinases including Cathepsin B, H and L. Biochem J 1982; 17. Que X, Reed SL. Cysteine proteinases and the pathogenesis of
201 : 189-98. amebiasis. Clin Microbiol Rev 2000; 13 : 196-206.
12. Huston CD, Houpt ER, Mann BJ, Hahn CS, Petri WA 18. McCoy JJ, Mann B, Petri WA Jr. Adherence and cytotoxicity
Jr. Caspase 3-dependent killing of host cells by the of Entamoeba histolytica or how lectins let parasites stick
parasite Entamoeba histolytica. Cell Microbiol 2000; 2 : around. Infect Immun 1994; 62 : 3045-50.
617-25. 19. Luaces AL, Barrett AJ. Affinity purification and biochemical
13. Scholze H, Tannich E. Cysteine endopeptidases of Entamoeba characterization of histolysin, the major cysteine proteinase of
histolytica. In : Barrett AJ, editor. Methods in enzymology. Entamoeba histolytica. Biochem J 1988; 250 : 903-9.
New York : Academic Press; 1986 p. 512-23.
20. Keen WE, Petri MG, Allen S, Mc Kerrow JH. The major neutral
14. Gupta S, Ghose P, Naik S, Naik SR. Proteinase activity cysteine proteinase of E. histolytica. J Exp Med 1986; 163 :
& virulence of Entamoeba histolytica on passage 536-49.
through hamster liver. Indian J Med Res 1998; 107 :
21. Ghosh PK, Gupta S, Naik S, Ayyagari A, Naik SR. Effect of
173-7.
bacterial association on virulence of Entamoeba histolytica to
15. Sarath G, Motte de la RS, Wangner FW. Protease assay baby hamster kidney cell monolayers. Indian J Exp Biol 1998;
methods. In: Beynon RJ, Bond JS, editors. Proteolytic 36 : 911-5.
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.inYou can also read
