microorganisms Article Genome-Wide Expression Patterns of Rhoptry Kinases during the Eimeria tenella Life-Cycle Adeline Ribeiro E Silva † , Alix Sausset †, Françoise I. Bussière, Fabrice Laurent, Sonia Lacroix-Lamandé and Anne Silvestre * Institut National de Recherche pour L’agriculture, L’alimentation et L’environnement (INRAE), Université de Tours, ISP, 37380 Nouzilly, France; [email protected] (A.R.E.S.); [email protected] (A.S.); [email protected] (F.I.B.); [email protected] (F.L.); [email protected] (S.L.-L.) * Correspondence: [email protected]; Tel.: +33-2-4742-7300 † These two first authors contributed equally to the work. Abstract: Kinome from apicomplexan parasites is composed of eukaryotic protein kinases and Api- complexa 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 char- acterized 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, Citation: Ribeiro E Silva, A.; Sausset, first- and second-generation merozoites, and gametes. Transcription of all those predicted ropk was A.; Bussière, F.I.; Laurent, F.; Lacroix- Lamandé, S.; Silvestre, A. Genome- confirmed. The mean intensity of transcription was higher in extracellular stages and 7–9 ropk were Wide Expression Patterns of Rhoptry specifically transcribed in merozoites in comparison with sporozoites. Transcriptional profiles of Kinases during the Eimeria tenella intracellular stages were closely related to each other, suggesting a probable common role of ROPKs Life-Cycle. Microorganisms 2021, 9, in hijacking signaling pathways and immune responses in infected cells. These results provide a 1621. https://doi.org/10.3390/ solid basis for future functional analysis of ROPK from E. tenella. microorganisms9081621 Keywords: rhoptry kinases; gene expression; Eimeria tenella; life-cycle Academic Editors: Isabelle Florent, Iva Koláˇrová and Andrea Bard ˚unekValigurová 1. Introduction Received: 17 June 2021 The Apicomplexa phylum consists of a large group of parasitic protists of medical and Accepted: 26 July 2021 Published: 29 July 2021 veterinary importance (Plasmodium, Toxoplasma, Cryptosporidium, Eimeria, etc). The genus Eimeria contains more than 1000 parasitic species, which invade the epithelial cell lining in Eimeria spp Publisher’s Note: MDPI stays neutral the intestinal tract of their host, causing coccidiosis. All . are considered to be with regard to jurisdictional claims in highly host-specific. Avian coccidiosis is caused by seven Eimeria species, among which published maps and institutional affil- Eimeria tenella, E. maxima, and E. acervulina are the main species responsible for coccidiosis in iations. poultry, worldwide. Clinical coccidiosis is associated with a reduction in body-weight gain, a decrease in the feed conversion index, and may result in a high mortality rate. Clinical and sub-clinical infections have a huge economic impact on poultry production. To control coccidiosis, coccidiostats are used as a feed additive in broilers production [1]. Attenuated live vaccines, which are very efficient, but expensive to produce, are mostly used in layers Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. and labels with high economic importance in their production [2]. Natural alternative This article is an open access article strategies, such as probiotics and phytochemicals, are becoming more explored [3]. distributed under the terms and The life-cycle of Eimeria is monoxenous with one phase in the environment and conditions of the Creative Commons another in the host (asexual multiplication and sexual reproduction, respectively), with Attribution (CC BY) license (https:// several developmental stages, either intracellular or extracellular (Figure1), characterized creativecommons.org/licenses/by/ by asynchronicity. Free-living stages consist of oocysts, which are very resistant to en- 4.0/). vironmental conditions. After oral contamination of chickens with sporulated oocysts, Microorganisms 2021, 9, 1621. https://doi.org/10.3390/microorganisms9081621 https://www.mdpi.com/journal/microorganisms Microorganisms 2021, 9, x FOR PEER REVIEW 2 of 16 Microorganisms 2021, 9, 1621 several developmental stages, either intracellular or extracellular (Figure 1), characterized2 of 14 by asynchronicity. Free-living stages consist of oocysts, which are very resistant to envi- ronmental conditions. After oral contamination of chickens with sporulated oocysts, re- leased sporozoites invade intestinal epithelial cells and undergo three rounds of asexual released sporozoites invade intestinal epithelial cells and undergo three rounds of asexual multiplications (first-, second-, and third-schizogony), each generating a large number of multiplications (first-, second-, and third-schizogony), each generating a large number merozoites. Third-generation merozoites differentiate into micro- and macro-gametes. of merozoites. Third-generation merozoites differentiate into micro- and macro-gametes. GameteGamete fecundation results in thethe productionproduction ofof zygoteszygotes (gamogony).(gamogony). AfterAfter maturation,maturation, zygoteszygotes evolve intointo non-sporulatednon-sporulated oocystsoocysts that areare releasedreleased inin thethe environment.environment. InIn thethe environment,environment, the the sporogony sporogony will will result result in in the the production production of ofsporulated sporulated oocysts oocysts under under ap- appropriatepropriate hygrometric hygrometric and and temperature temperature conditions. conditions. The The most most pathogenic pathogenic phase phase for forthe thehost host is the is merogony, the merogony, during during which which successive successive intracellular intracellular multiplications multiplications induce induce diges- digestivetive mucosal mucosal lesions. lesions. Live attenuated Live attenuated vaccines vaccines correspond correspond to Eimeria to Eimeria strainsstrains that have that have only onlyone merogony, one merogony, leading leading to fewe to fewerr mucosal mucosal lesions lesions than thanwild wild-type-type (WT) (WT) strains. strains. FigureFigure 1.1. EimeriaEimeria tenellatenella life-cycle.life-cycle. SporogonySporogony occursoccurs inin thethe environment,environment, whereaswhereas schizogonyschizogony andand gamogonygamogony occuroccur inin thethe chickenchicken digestivedigestive tract.tract. For thethe wild-typewild-type strain,strain, threethree roundsrounds ofof schizogonyschizogony produceproduce first-,first-, second-, second- and, and third-generation third-generation merozoites. merozoite NSO,s. NSO non-sporulated, non-sporulated oocysts, oocysts, SO sporulatedSO sporu- oocysts,lated oocysts, eSPZextracellular eSPZ extracellular sporozoites, sporozoites, iSPZ intracellular iSPZ intracellular sporozoites, sporozoites, ImmSCHZ ImmSCHZ immature immature schizonts, schizonts, MatSCHZ mature schizonts, MI first-generation merozoites, MII second-generation mer- MatSCHZ mature schizonts, MI first-generation merozoites, MII second-generation merozoites, and ozoites, and MIII third-generation merozoites. MIII third-generation merozoites. TheThe k kinomeinome of of coccidia coccidia (Toxoplasma, (Toxoplasma, Neospora, Neospora and, and EimeriaEimeria) is) iscomposed composed of ofeukaryotic eukary- oticprotein protein kinases kinases (ePKs) (ePKs) [4,5] and [4,5 ]a andcoccidia a coccidia-specific-specific kinase kinasefamily family(rhoptry (rhoptry kinases, kinases, ROPK) ROPK)[6]. ROPK [6]. se ROPKquences sequences are highly are divergent highly divergent from ePK from sequences ePK sequences [7] and include [7] and active include ki- activenases (i.e. kinases, a kinase (i.e., adomain kinase organi domainzed organized in 12 subdomains in 12 subdomains that retain that all retain the key all residues the key residuesneeded for needed catalysis, for catalysis,as the catalytic as the triad catalytic KDD), triad pseudo KDD),-kinases pseudo-kinases (inactive enzymes, (inactive char- en- zymes,acterized characterized by the absence by of the a complete absence of catalytic a complete domain catalytic and/or domain a mutated and/or catalytic a mutated triad), catalyticand non- triad),canonical and kinases non-canonical (i.e., active kinases kinases (i.e., with active most kinases of the withresidues most necessary of the residues for ca- necessarytalysis, but for with catalysis, some differences but with some in the differences conserved in residues). the conserved residues). RopksRopks werewere amplifiedamplified from from an an ancestral ancestral gene. gene.T. T. gondii gondiitype type I, II,I, andII, and III containIII contain 37, 55,37, and55, and 38 ropk 38 ropk, respectively, respectively [6]. Individual[6]. Individualropk ropkdeletion deletion in a in type a type II T. II gondii T. gondiiresulted resulted in less in virulentless virulent strains strains for 16 forropk