Overexpression of rhoptry kinase alters the length of Eimeria tenella life cycle

International audienceEimeria tenella is a deadly and contagious apicomplexan protozoan parasite, which is responsible for avian coccidiosis. This pathology induces major economic losses for poultry industry worldwide. E. tenella invades the digestive epithelial cells, causing intestinal lesions that can lead to death. Coccidiostatic drugs and vaccination are necessary to control coccidiosis. However, with the apparition of resistance against anticoccidian molecules in parasite field strains and the high cost of vaccines, it appears necessary to improve the means of control of this parasite. Our research is focused on the understanding of E. tenella rhoptry protein kinase (ROPK) functions. It is well known that invasion of apicomplexan parasite is orchestrated by protein secretion. Among the proteins secreted, ROPKs are well described in Toxoplasma gondii as key virulence factors. ROPKs are involved in the modulation of numerous cellular functions andpathways allowing parasite development. The knowledge about the functions of E. tenella ROPK is limited. E. tenella kinome is predicted to contain 28 putative ROP kinases. Among them, two predicted kinases were identified in the rhoptry proteome of E. tenella sporozoites. In this context, our research is focused onunderstanding the mode of action of E. tenella ROPK. We previously established that the first kinase, EtROP1, is active and phosphorylates the cellular p53, inhibiting the apoptosis of parasited cells and thus promoting the parasite development. The second kinase, EtROP2, is an active kinase localized in the rhoptry compartment and is early expressed during the parasite life cycle. Interestingly, we show that the overexpression of EtROP2 could speed up the parasitic lifecycle, favoring the excretion of oocysts 1-2 days earlier than a wild-type strain. Understanding the role of EtROP2 in the host-parasite interaction may reveal this kinase as a good candidate in the race of improvement of the means of control for coccidiosis

EtROP2 is an early expressed kinase localisated in the rhoptry compartment of Eimeria tenella

International audienceCoccidia are obligate intracellular parasites responsible for human and veterinary diseases. Eimeria tenella is an apicomplexan protozoan which is responsible for avian coccidiosis. This pathology induces a major economic loss for poultry industry worldwide. The parasite invades the digestive epithelial cells, which cause intestinal lesion that can lead to death. Until today, prophylaxis permitted to fight against coccidiosis by using chemotherapy and vaccination. But, with the apparition of resistance against anticoccidian molecules and with the high cost of vaccines, it appears necessary to improve these means of control.During invasion, the parasite releases the content of rhoptries, apicomplexan specific secretory organelles. Toxoplasma gondii rhoptry protein repertoire includes kinases that are key virulence factors. Kinases are involved in the molecular dialogue between the parasite and host cell. This involvement allows them to modulate cellular functions and pathways allowing T. gondii development. E. tenella kinome is predicted to contain 28 putative ROP kinases. Among them, two predicted kinases were identified in the rhoptry proteome of E. tenella sporozoites. The first kinase EtROP1 is described as an active kinase that phosphorylates the p53 inhibiting the apoptosis of parasited cells and thus promoting the parasite development. The second kinase EtROP2 is an active kinase whose functions need to be characterized. This work shows the apical localization of EtROP2 by microscopy techniques and his early expression in the parasite life cycle. The EtROP2 study may reveal it as a good candidate in the race for the improvement of the means of coccidiosis control

EtROP2, une ROP kinase localisée au sein du compartiment des rhoptries et exprimée durant les stades précoces du cycle parasitaire d’<em>Eimeria tenella</em>

National audienceLes coccidies sont des parasites intracellulaires obligatoire responsables de maladies humaines et vétérinaires. Eimeria tenella est un protozoaire apicomplexe responsable de coccidiose aviaire. Cette pathologie est à l’origine de pertes économiques majeures au sein de l’industrie aviaire à travers le monde. L’infection par E. tenella se caractérise par un envahissement des cellules épithéliales du tube digestif, provoquant ainsi des lésions intestinales pouvant mener au décès de l’animal. Jusqu’à présent, les méthodes de prophylaxie telle que la vaccination et l’utilisation de molécules chimiques, permettaient de contrôler les coccidioses. Pourtant, l’apparition de résistances vis-à-vis des molécules anticoccidiennes ainsi que le coût élevé des vaccins, rendent nécessaire l’amélioration des moyens de lutte contre les coccidioses. Durant la phase d’invasion, le parasite libère le contenu de ses rhoptries, organelles de sécrétion spécifiques des Apicomplexes. Le répertoire protéique des rhoptries exprimé chez Toxoplasma gondii inclut des kinases, reconnues comme étant des facteurs de virulence clés. Ces kinases sont impliquées dans le dialogue moléculaire entre le parasite et la cellule hôte ce qui leur permet de moduler les voies et fonctions cellulaires favorisant ainsi le développement de T. gondii. Le kinome d’E. tenella est prédit comme contenant 28 ROP kinases putatives. Parmi elles, 2 kinases prédites comme étant actives ont été identifiées au sein du protéome des rhoptries du stade sporozoïte. La première kinase, EtROP1, est décrite comme étant une kinase active capable de phosphoryler la p53 inhibant ainsi l’apoptose des cellules parasitées et favorisant le développement du parasite. La seconde kinase EtROP2, est également une kinase active dont les fonctions doivent être caractérisées. Ce travail montre les premiers résultats de la caractérisation d’EtROP2. Il met en évidence sa localisation au pôle apical du parasite via des techniques de microscopie, ainsi que son expression précoce au cours du cycle parasitaire. L’étude de cette seconde kinase, EtROP2, pourrait révéler qu’il s’agit d’un bon candidat dans la course à l’amélioration des moyens de contrôle des coccidioses

Genome-Wide Expression Patterns of Rhoptry Kinases during the <i>Eimeria tenella</i> Life-Cycle

Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Apicomplexa specific kinases, such as rhoptry kinases (ROPK). Ropk is a gene family that is known to play important roles in host–pathogen interaction in Toxoplasma gondii but is still poorly described in Eimeria tenella, the parasite responsible for avian coccidiosis worldwide. In the E. tenella genome, 28 ropk genes are predicted and could be classified as active (n = 7), inactive (incomplete catalytic triad, n = 12), and non-canonical kinases (active kinase with a modified catalytic triad, n = 9). We characterized the ropk gene expression patterns by real-time quantitative RT-PCR, normalized by parasite housekeeping genes, during the E. tenella life-cycle. Analyzed stages were: non-sporulated oocysts, sporulated oocysts, extracellular and intracellular sporozoites, immature and mature schizonts I, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of intracellular stages were closely related to each other, suggesting a probable common role of ROPKs in hijacking signaling pathways and immune responses in infected cells. These results provide a solid basis for future functional analysis of ROPK from E. tenella

Incorporation of apolipoprotein E into HBV–HCV subviral envelope particles to improve the hepatitis vaccine strategy

International audienceAbstract Hepatitis C is a major threat to public health for which an effective treatment is available, but a prophylactic vaccine is still needed to control this disease. We designed a vaccine based on chimeric HBV–HCV envelope proteins forming subviral particles (SVPs) that induce neutralizing antibodies against HCV in vitro. Here, we aimed to increase the neutralizing potential of those antibodies, by using HBV–HCV SVPs bearing apolipoprotein E (apoE). These particles were produced by cultured stable mammalian cell clones, purified and characterized. We found that apoE was able to interact with both chimeric HBV–HCV (E1-S and E2-S) proteins, and with the wild-type HBV S protein. ApoE was also detected on the surface of purified SVPs and improved the folding of HCV envelope proteins, but its presence lowered the incorporation of E2-S protein. Immunization of New Zealand rabbits resulted in similar anti-S responses for all rabbits, whereas anti-E1/-E2 antibody titers varied according to the presence or absence of apoE. Regarding the neutralizing potential of these anti-E1/-E2 antibodies, it was higher in rabbits immunized with apoE-bearing particles. In conclusion, the association of apoE with HCV envelope proteins may be a good strategy for improving HCV vaccines based on viral envelope proteins

Defining the role of the Streptococcus agalactiae Sht-family proteins in zinc acquisition and complement evasion

International audienceStreptococcus agalactiae is part of the human intestinal and uro-genital microbiota, but also a leading cause of septicemia and meningitis in neonates. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including the transition metal ion zinc. The primary zinc acquisition system of the pathogen is the Adc/Lmb ABC permease, which is essential for viability in zinc-restricted environments. Here, we show that in addition to the AdcCB transporter, and the three zinc-binding proteins, Lmb, AdcA and AdcAII, S. agalactiae zinc homeostasis also involves two streptococcal histidine triad (Sht) proteins. Sht and ShtII are required for zinc uptake via the Lmb and AdcAII proteins with apparent overlapping functionality and specificity. Both Sht-family proteins possess five histidine triad motifs with similar hierarchies of importance for Zn homeostasis. Independent of its contribution to zinc homeostasis, Sht has previously been reported to bind factor H leading to predictions of a contribution to complement evasion. Here, we investigated ShtII to ascertain whether it had similar properties. Analysis of recombinant Sht and ShtII reveals that both proteins have similar affinities for factor H binding. However, neither protein aided in resistance to complement in human blood. These findings challenge prior inferences regarding the in vivo role of the Sht proteins in resisting complement-mediated clearance.IMPORTANCE This work examined the role of the two streptococcal histidine triad (Sht) proteins of Streptococcus agalactiae in zinc homeostasis and complement resistance. We showed that Sht and ShtII facilitate zinc homeostasis in conjunction with the metal-binding proteins Lmb and AdcAII. Here, we show that the Sht-family proteins are functionally redundant with overlapping roles in zinc uptake. Further, this work reveals that although the Sht-family proteins bind to factor H in vitro this did not influence survival in human blood

Overexpression of Eimeria tenella Rhoptry Kinase 2 Induces Early Production of Schizonts

International audienceEimeria tenella is an obligate intracellular parasite responsible for avian coccidiosis. Like other apicomplexan parasites, such as Toxoplasma gondii, cell invasion and intracellular development rely on apical organelle content discharge, named micronemes and rhoptries. Some rhoptry (ROP) kinases (ROPK) are key virulence factors in T. gondii. To date, among the 28 ropk genes carried by E. tenella, only two to four were confirmed by proteomic analysis or immunostaining to be expressed at the sporozoite stage. We have previously shown that EtROP1 is implicated in the inhibition of host cell apoptosis by interacting with the cellular p53. This work functionally described the second ROP kinase expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Its overexpression leads to the shortening of the prepatent period and to the early development of first-generation schizonts. Conduction of RNA sequencing analysis and reverse transcriptase quantitative PCR (RT-qPCR) on the host cell allowed us to identify the mitogen-activated protein kinase (MAPK) pathway and the transcription factor cFos to be upregulated by EtROP2. We also showed by immunofluorescence assay that the active kinase EtROP2 is implicated in the p38 MAPK pathway activation. We established here that EtROP2 activates the p38 MAPK pathway through a direct or indirect phosphorylation, leading to the overexpression of the master transcription factor cFos known to be implicated in E. tenella development. IMPORTANCE Rhoptries are specialized secretory organelles found in zoite stages of apicomplexan parasites. In addition to well-conserved rhoptry neck proteins, their protein consists mostly of kinase proteins, highly divergent from eukaryotic kinases. Some of those kinases are described as major virulence factors in Toxoplasma gondii, secreted into the host cell to hijack signaling pathways. Most of those kinases remain to be characterized in Eimeria tenella. Deciphering their cellular function is a prerequisite to supporting their relevance as a druggable target in development of new means of Eimeria tenella control. Secreted divergent kinases that interact with host cell partners to modulate pathways are good candidates, as they coevolve with their host targets to ensure their function within the host and are less prone to mutations that would lead to drug resistance. The absence of any orthologous kinase in host cells makes these parasite kinases a promising drug target candidate

Eimeria tenella induces p38 MAPK signalling pathway

International audienceEimeria tenella is an obligate intracellular parasite responsible for avian coccidiosis. As for other apicomplexan parasites, cell invasion and intracellular development are based on apical organelles content discharge, named micronemes and rhoptries (ROP). Some ROP kinases (ROPK) are key virulence factors that hijack host cell signalling pathways. To date, among the 28 ropk encoded by E. tenella, only two proteins, EtROP1 et EtROP2, were identified at the sporozoite, the first infectious stage. We previously shown that EtROP1 was implicated in the inhibition of the host cell apoptosis by interacting with the cellular p53. Here, we functionally described the second ROPK expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Overexpression of EtROP2 by E. tenella modifies the first schizogony (asexual multiplication of the sporozoite) and reduces the prepatent period from 7 to 5,5 days. Molecular mechanism was investigated in transfected HEK293T cells, and may involve the activation of p38 MAPK signalling pathway, resulting in the overexpression of cFos transcription factor. Interestingly, cFos has already been described in E. tenella intracellular development. Collectively our results support the importance of EtROP2 in E. tenella life cycle and constitutes a new target for the development of highly specific therapeutics against coccidiosis

Eimeria tenella ROP kinase 2 induces p38 MAPK cellular pathway

National audienceEimeria tenella is an obligate intracellular parasite that causes avian coccidiosis. As for other apicomplexan parasites, such as Toxoplasma gondii, cell invasion and intracellular development rely on apical organelles content discharge, named micronemes and rhoptries. Some ROP kinases (ROPK) are key virulence factors in T. gondii. To date, among the 28 ropk encoded by E. tenella, only two were confirmed to be expressed at the sporozoite stage by proteomic analysis. We have previously shown that EtROP1 is implicated in the inhibition of the host cell apoptosis by interacting with the cellular p53. Here, we functionally characterized the second ROP kinase expressed at the sporozoite stage in E. tenella. EtROP2 is an active kinase that phosphorylates cell substrates of approximately 50 kDa. Its overexpression leads to the shortening of the prepatent period and to the early development of first generation schizonts. Conduction of RNA sequencing analysis and RT-qPCR on the host cell, allowed us to identified the MAPK pathway and the transcription factor cFos to be upregulated by EtROP2. We also showed by immunofluorescence assay that the active kinase EtROP2 is implicated in the p38 MAPK pathway activation. We established here, that EtROP2 activates the p38 MAPK pathway through a direct or indirect phosphorylation, leading to the overexpression of the master transcription factor cFos known to be implicated in E. tenella development

<em>Eimeria tenella</em> induit l’arrêt du cycle en G0/G1 et inhibe l’apoptose de la cellule hôte : rôle de la kinase EtROP1

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