Eimeripain, a Cathepsin B-Like Cysteine Protease, Expressed throughout Sporulation of the Apicomplexan Parasite Eimeria tenella

The invasion and replication of Eimeria tenella in the chicken intestine is responsible for avian coccidiosis, a disease that has major economic impacts on poultry industries worldwide. E. tenella is transmitted to naïve animals via shed unsporulated oocysts that need contact with air and humidity to form the infectious sporulated oocysts, which contain the first invasive form of the parasite, the sporozoite. Cysteine proteases (CPs) are major virulence factors expressed by protozoa. In this study, we show that E. tenella expresses five transcriptionally regulated genes encoding one cathepsin L, one cathepsin B and three cathepsin Cs. Biot-LC-LVG-CHN2, a cystatin derived probe, tagged eight polypeptides in unsporulated oocysts but only one in sporulated oocysts. CP-dependant activities were found against the fluorescent substrates, Z-FR-AMC and Z-LR-AMC, throughout the sporulation process. These activities corresponded to a cathepsin B-like enzyme since they were inhibited by CA-074, a specific cathepsin B inhibitor. A 3D model of the catalytic domain of the cathepsin B-like protease, based on its sequence homology with human cathepsin B, further confirmed its classification as a papain-like protease with similar characteristics to toxopain-1 from the related apicomplexan parasite, Toxoplasma gondii; we have, therefore, named the E. tenella cathepsin B, eimeripain. Following stable transfection of E. tenella sporozoites with a plasmid allowing the expression of eimeripain fused to the fluorescent protein mCherry, we demonstrated that eimeripain is detected throughout sporulation and has a punctate distribution in the bodies of extra- and intracellular parasites. Furthermore, CA-074 Me, the membrane-permeable derivative of CA-074, impairs invasion of epithelial MDBK cells by E. tenella sporozoites. This study represents the first characterization of CPs expressed by a parasite from the Eimeria genus. Moreover, it emphasizes the role of CPs in transmission and dissemination of exogenous stages of apicomplexan parasites

Cryptosporidiosis in domestic ruminants in France : molecular epidemiology and zoonotic potential

La cryptosporidiose est une infection du tube digestif, causée par un protozoaire du genre Cryptosporidium, à l'origine de diarrhées néonatales chez les jeunes ruminants. Cette affection revêt une importance en santé publique et en santé animale, ces deux aspects étant liés par l’existence d'espèces zoonotiques, la principale étant C. parvum. Les données sur l'épidémiologie moléculaire de Cryptosporidium spp chez les ruminants abondent au niveau international mais sont limitées pour la France. Ce travail a confirmé la forte prévalence et les forts niveaux d'excrétion en oocystes de Cryptosporidium chez les jeunes ruminants. L'espèce C. parvum a été identifiée aussi bien chez les veaux, les chevreaux que chez les agneaux. A l'inverse, les espèces C. xiaoi (chez le jeune) et C. ubiquitum (adultes en gestation) n'ont été retrouvées que chez les caprins et les espèces C. bovis et C. ryanae uniquement chez les veaux.Deux espèces zoonotiques ont été identifiées, C. ubiquitum et C. parvum. Tous les sous-types de C. parvum identifiés appartiennent à la famille zoonotique IIa. Le sous-type IIaA15G2R1 a été majoritairement retrouvé quelle que soit l'espèce hôte. Cette observation confirme ce qui a été observé dans d'autres pays à savoir le rôle de réservoir des jeunes ruminants dans la transmission d'isolats de C. parvum à l'homme. Enfin, ce travail a mis en évidence la complexité de l'épidémiologie de l'infection par Cryptosporidium spp avec une évolution de la prévalence et du niveau d'excrétion ainsi que de la distribution des espèces et sous-types de C. parvum d'une année sur l'autre au sein d'un même élevage.Cryptosporidiosis is an infection of the digestive tract due to protozoa of the genus Cryptosporidium, which cause neonatal diarrhoea in young ruminants. This infection has a real significance in public and animal health; in fact, both of these aspects are bound by the existence of zoonotic species including C. parvum, the predominant species. Molecular epidemiology data on Cryptosporidium spp are very numerous over the world; however, little information has been published in France.This work confirmed the high prevalence and high level of excretion of Cryptosporidium oocysts in young ruminants. C. parvum species was identified in calves, goat kids and also in lambs. By contrast, the two species, C. xiaoi (goat kids) and C. ubiquitum (pregnant goats) were only found in goats whereas C. bovis and C. ryanae were observed only in calves.Two zoonotic species have been identified: C. ubiquitum and C. parvum. All C. parvum subtypes belongs to the IIa zoonotic family. IIaA15G2R1 subtype was mainly found in all ruminants' species. This observation confirms the potential role of young ruminants in transmission of zoonotic isolates of C. parvum to humans which has already been observed in previous studies over the world. Finally, this work highlighted the complexity of the epidemiology of Cryptosporidium spp infection, in particular the evolution in the prevalence and level of excretion and in the distribution of Cryptosporidium spp species or C. parvum subtypes according to the year of sampling in the same livestock

Identification of two new proteases of the apicomplexan parasite Eimeria tenella as potential therapeutic targets of avian coccidiosis

International audienc

Characterisation of a cysteine protease expressed by Eimeria tenella and identification of its post-traductionnal regulator

Session Poster : Invasion and motilityCysteine proteases of the papain family are major virulent factors expressed by protozoa. They have been involved in many steps of parasites life cycle like cell invasion, intracellular replication, gametocyte formation and parasite differentiation. Their multiple roles in key steps of parasites biology make them attractive new therapeutic targets. Using BlastP, we identified 5 genes encoding for cysteine proteases in the genome of E. tenella. We named them Eimeripain, EtCPL, EtCPC1, EtCPC2 and EtCPC3 encoding respectively for one cathepsin B, one cathepsin L, and three cathepsin C. Complementary approaches of molecular biology and biochemistry revealed that most of these proteases are highly expressed and active in the unsporulated oocysts, suggesting a role in sporulation and/or gametogenesis. Eimeripain is the only activity that persists throughout the life cycle. We show that a specific inhibitor of Human cathepsin B, CA074-ME, inhibits Eimeripain and affects the capacity of sporozoites to invade MDBK cells. These data suggest that Eimeripain plays a central and pleiotropic role in Eimeria life cycle. Cysteine protease inhibitors from the Chagasin family are proteins expressed by protozoa that specifically bind to and inhibit cysteine cathepsins. As such, they participate to parasite pathogenesis. We identified a cysteine protease inhibitor, Eimestatine, expressed by E. tenella, which specifically inhibits the activity of Eimeripain in biochemical assays. Preliminary data suggest that Eimestatine forms a complex at each life stage, which may indicate a tight regulation of Eimeripain throughout the infectious process

Effect of CA-074 Me on MDBK cell invasion by <i>E. tenella</i> sporozoites.

<p>Purified sporozoites were incubated with 10 to 100 µM of CA-074ME, a specific cathepsin B inhibitor permeable to membranes or with 1% DMSO. Sporozoites were then washed and incubated with MDBK cells. Infected cells were fixed and intracellular parasites were detected by IF using specific sera against sporozoites, and counted. The data represent three independent experiments. * denotes significant differences at p<0.05 in the capacity of CA-074 Me- treated parasites to invade cells compared to the control.</p

Ribbon diagram superposition of the catalytic domains of the <i>E. tenella</i> cathepsin B and human cathepsin B.

<p>The X-ray crystallographic structure of the human cathepsin B (pdb 1 gmy) is colored in green and the proposed structure of homology-based model of the <i>E. tenella</i> cathepsin B is in orange. The catalytic triad residues (Cys266, His445 and Asn465) are depicted in the <i>ball-and-stick</i> representation. On the left panel, the occluding loop is represented in purple with the two adjacent histidine residues (His352, His353) in the <i>ball-and-stick</i> representation. On the right panel, the surface exposed loops specific to <i>E. tenella</i> cathepsin B are in red. The residues delimitating the loops: Asn334-Ser341, Glu377-Lys383 and Asp389-Thr395 are shown.</p

Expression profile of CPs in <i>E. tenella</i>.

<p>(A) Total RNAs from unsporulated oocysts (0), oocysts in sporulation for 6 h (6) and fully sporulated oocysts (48) were extracted and RT-PCR were performed using specific primers to <i>etcpb, etcpl, etcpc1, etcpc2</i> and <i>etcpc3</i>. (B) The single-copy <i>actin</i> gene was amplified in parallel as a control (<i>etactin</i>). The down regulation of <i>etactin</i> during sporulation has been previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031914#pone.0031914-Kinnaird1" target="_blank">[17]</a>. The RT-PCR products were resolved on a 0.7% agarose gel stained with ethidium bromide. The band observed at 0.8 kb with primers specific to <i>etcpc2</i> and total RNAs from unsporulated oocyst (0) is a nonspecific amplification product.</p

Biochemical activities of CPs throughout sporulation.

<p>(A) Activities detected on Z-FR-AMC. Lysates of oocysts (1 mg/ml) taken at 0, 6, 12, 24 and 48 h after the beginning of sporulation were incubated with the substrate Z-FR-AMC (10 µM). The morphology of oocysts (under light microscopy) throughout the course of sporulation is shown. The scale bar represents 2 µm. (B) Activities detected on Z-FR-AMC and Z-LR-AMC in presence of the global cysteine protease inhibitor E-64 or the human cathepsin B specific inhibitor CA-074. Lysates (1 mg/ml) of oocysts taken at 0 h (black bars) and 48 h (white bars) after commencement of sporulation were pre-incubated with the inhibitors before adding the substrates. The data represents two independent experiments.</p

Profile of active CPs throughout sporulation.

<p>(A) Lysates of unsporulated oocysts (0) and oocysts in sporulation for 6, 12, 24, 36 and 48 h were incubated with the probe Biot-LC-LVG-CHN₂. The complexes were revealed by Western blot using streptavidin peroxidase. (B) Lysates of oocysts obtained at 0 h or 48 h of sporulation time (St) were pre-incubated or not (−) with E-64 (28 µM) or CA-074 (100 µM) before addition of Biot-LC-LVG-CHN₂. The interaction between the probe and CP catalytic sites was detected by Western blot using streptavidin peroxidase. The arrowhead indicates the activity corresponding to the <i>E. tenella</i> cathepsin B (33 kDa).</p