Babesia bovis: effects of cysteine protease inhibitors on in vitro growth.

Abstract

In the present study, we examined the effects of four kinds of cysteine protease inhibitors (E64, E64d, leupeptin, and ALLN) on the in vitro asexual growth of Babesia bovis. Of these, only the lipophilic inhibitors, E64d and ALLN, were found to effectively inhibit the growth of B. bovis. In further experiments, E64d, but not ALLN, significantly suppressed the parasite's invasion of host erythrocytes, while both chemicals, especially ALLN, inhibited the parasite's replication within the infected erythrocytes. These data suggested the presence of cysteine protease(s) derived from B. bovis, in which the protease(s) would play important roles in the erythrocyte invasion and/or replication processes of the parasite.

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帯広畜産大学学術情報リポジトリOAK:Obihiro university Archives of Knowledge
' '
Title
Babesi a bovi s: Effects of cystei ne protease
i nhi bi tors on i n vi tro grow th
Author(s)
O kubo, Kazuhi ro, Yokoyam a, N aoaki , Govi nd,
Yadav, Al hassan, Andy, Igarashi , Ikuo
CitationExperi m ental Parasi tol ogy
Issue Date2007-05
URLhttp: //i r. obi hi ro. ac. j p/dspace/handl e/10322/1041
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Title
Babesia bovis: Effects of Cysteine Protease Inhibitors on In Vitro Growth

Authors
Kazuhiro Okubo, Naoaki Yokoyama,* Yadav Govind,† Andy Alhassan, and Ikuo Igarashi

Address
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary
Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan

*Corresponding Author
Naoaki Yokoyama: National Research Center for Protozoan Diseases, Obihiro University of
Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan
TEL.: +81-155-49-5649; FAX: +81-155-49-5643; E-mail: yokoyama@obihiro.ac.jp

†Present address
Department of Epidemiology and Preventive Medicine, College of Veterinary Science, Mathura UP. Pt.
Deendayal Upadhaya Veterinary University, Mathura-281001(U.P), India
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Abstract
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In the present study, we examined the effects of four kinds of cysteine protease inhibitors (E64,
E64d, leupeptin, and ALLN) on the in vitro asexual growth of Babesia bovis. Of these, only the
lipophilic inhibitors, E64d and ALLN, were found to effectively inhibit the growth of B. bovis. In
further experiments, E64d, but not ALLN, significantly suppressed the parasite’s invasion of host
erythrocytes, while both chemicals, especially ALLN, inhibited the parasite’s replication within the
infected erythrocytes. These data suggested the presence of cysteine protease(s) derived from B. bovis,
in which the protease(s) would play important roles in the erythrocyte invasion and/or replication
processes of the parasite.

Key words: Babesia bovis, cysteine protease, inhibitor, invasion, replication.
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Babesia bovis, an obligatory intraerythrocytic parasite of the phylum Apicomplexa, is a major
causative agent of bovine babesiosis, which causes severe clinical symptoms, such as fever, anemia,
and cerebral dysfunctions, in cattle due to their asexual growth (Homer et al., 2000). The disease often
results in great economic losses in the livestock industry worldwide (Brown and Palmer, 1999), and
effective strategies are desired for eradicating babesiosis.
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Cysteine proteases are known to play vital roles in the growth of several protozoan parasites. In
Trypanosoma spp., for example, the cysteine protease-specific inhibitors impair their host cell invasion
and arrest the intracellular development (Meirelles et al., 1992) or kill the cultured blood stream forms
of the parasites (Troeberg et al., 1999). In Plasmodium spp., several protozoan cysteine proteases are
involved in the erythrocyte invasion (Greenbaum et al., 2002), the degradations of hemoglobin and
erythrocyte cytoskeletal proteins (Sijwali and Rosenthal, 2004; Takakuwa, 2001), and the final
erythrocyte rupture for their egression (Wickham et al., 2003). Cysteine protease inhibitors have been
studied for the development of new chemotherapeutic measures for trypanosomiasis (Engel et al.,
1998), malaria (Olson et al., 1999), schistosomiasis (Wasilewski et al., 1996), and leishmaniasis (Das
et al., 2001). However, no cysteine protease has been reported in Babesia parasites, except for “cys1,”
which is a cathepsin-like cysteine protease found in Babesia (Theileria) equi (Eakin et al., 1990;
Holman et al., 2002; Mehlhorn and Schein, 1998).
In the present study, we examined the inhibitory effects of four commercial cysteine protease
inhibitors, E64 (trans-Epoxy-succinyl-L-leucylamido(4-guanidino)butane), E64d
((2S,3S)-trans-Epoxy-succinyl-L-leucylamido-3-methylbutane ethyl ester), leupeptin, and ALLN
(N-acetyl-leucine-leucine-norleucinal), on the in vitro asexual growth of cultured B. bovis. These
inhibitors are known to inhibit thiol proteases, such as calpain and cathepsin, but only leupeptin can
also inhibit serine proteases (Carole and Wang, 1998). Furthermore, E64d and ALLN have membrane
permeability, while E64 and leupeptin do not (Holman et al., 2002). These four inhibitors have been
widely used in the research of the cysteine proteases of many protozoa, including a Plasmodium
parasite (Dahl and Rosenthal, 2005), which is closely related to the Babesia parasite.
The Texas strain of B. bovis was cultured in purified bovine erythrocytes (RBCs) using a
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serum-free GIT medium (Wako Pure Chemical Industries, Ltd., Osaka, Japan) as described previously
(Bork et al., 2005). The growth-inhibition test of four cysteine protease inhibitors followed previously
described methods for measuring the drug activity (Okubo et al., 2006a; Bork et al., 2003) with some
modifications. Briefly, infected RBCs were diluted with non-infected RBCs to obtain 0.5%
parasitemia. Fifty μl of the infected RBC mixture was subsequently suspended in 450 μl of a culture
medium supplemented with 1 (or 20) - 220 μM of E64, E64d, leupeptin, or ALLN. The suspension
was placed in a 48-well culture plate (Nunc A/S, Roskilde, Denmark) and then incubated in a
humidified multigas water-jacketed incubator at 37°C in 5% CO
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2, 5% O2, and 90% N2 for 3 days. In
parallel, normal cultures, which were added with the same final volume of DMSO (solvent) instead of
each chemical, were prepared as the control. The culture medium was replaced daily with 450 μl of the
fresh medium containing the indicated concentration of each chemical.
Out of the four tested cysteine protease inhibitors, only the lipophilic inhibitors, E64d and ALLN,
significantly affected the asexual growth of B. bovis at the concentration of 50 μM (Fig. 1). On the
other hand, the hydrophilic inhibitors, E64 and leupeptin, did not affect the growth of the parasite in the
range of 20 - 200 μM (Fig. 1; data not shown). The IC50 values of E64d and ALLN were calculated to
be 29.9 and 25.1 μM, respectively, on the basis of the inhibitory rates (%) of E64d and ALLN at
various concentrations within 1 - 220 μM (Table 1). These data suggest that B. bovis has cysteine
protease(s). On the other hand, E64 and leupeptin did not show any effects on the asexual growth of B.
bovis in the present study. One possible explanation of the negative results is that the target cysteine
protease(s) might exist in the parasite’s body; therefore, the membrane non-permeable agents could not
reach it. Another possible explanation is that the E64 and leupeptin inhibited the target cysteine
protease(s) but the protease(s) did not play an essential role in the asexual growth of the parasite. In P.
falciparum, for example, targeted disruption of the cysteine protease, falcipain 1, did not influence their
asexual growth (Saliha et al., 2004).
Next, we investigated the effects of E64d and ALLN on the erythrocyte invasion of B. bovis by
an in vitro invasion test using high-voltage pulsing, as described previously (Okubo et al., 2006a;
Franssen et al., 2003), with some modifications. Briefly, after B. bovis-infected RBCs were suspended
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