Identification of the Toxoplasma gondii mitochondrial ribosome, and characterisation of a protein essential for mitochondrial translation

Apicomplexan parasites cause diseases such as malaria and toxoplasmosis. The apicomplexan mitochondrion shows striking differences from common model organisms, including fundamental processes such as mitochondrial translation. Despite evidence that mitochondrial translation is essential for parasite survival, it is largely understudied. Progress has been restricted by the absence of functional assays to detect apicomplexan mitochondrial translation, a lack of knowledge of proteins involved in the process and the inability to identify and detect mitoribosomes. We report the localization of 12 new mitochondrial proteins, including 6 putative mitoribosomal proteins. We demonstrate the integration of three mitoribosomal proteins in macromolecular complexes, and provide evidence suggesting these are apicomplexan mitoribosomal subunits, detected here for the first time. Finally, a new analytical pipeline detected defects in mitochondrial translation upon depletion of the small subunit protein 35 (TgmS35), while other mitochondrial functions remain unaffected. Our work lays a foundation for the study of apicomplexan mitochondrial translation

Two essential Thioredoxins mediate apicoplast biogenesis, protein import, and gene expression in Toxoplasma gondii.

Apicomplexan parasites are global killers, being the causative agents of diseases like toxoplasmosis and malaria. These parasites are known to be hypersensitive to redox imbalance, yet little is understood about the cellular roles of their various redox regulators. The apicoplast, an essential plastid organelle, is a verified apicomplexan drug target. Nuclear-encoded apicoplast proteins traffic through the ER and multiple apicoplast sub-compartments to their place of function. We propose that thioredoxins contribute to the control of protein trafficking and of protein function within these apicoplast compartments. We studied the role of two Toxoplasma gondii apicoplast thioredoxins (TgATrx), both essential for parasite survival. By describing the cellular phenotypes of the conditional depletion of either of these redox regulated enzymes we show that each of them contributes to a different apicoplast biogenesis pathway. We provide evidence for TgATrx1's involvement in ER to apicoplast trafficking and TgATrx2 in the control of apicoplast gene expression components. Substrate pull-down further recognizes gene expression factors that interact with TgATrx2. We use genetic complementation to demonstrate that the function of both TgATrxs is dependent on their disulphide exchange activity. Finally, TgATrx2 is divergent from human thioredoxins. We demonstrate its activity in vitro thus providing scope for drug screening. Our study represents the first functional characterization of thioredoxins in Toxoplasma, highlights the importance of redox regulation of apicoplast functions and provides new tools to study redox biology in these parasites

Functional characterisation of cysteine cathepsins as new virulence factors in the apicomplexan parasite Cryptosporidium parvum

National audienceCryptosporidium parvum is an apicomplexan parasite responsible for a zoonotic disease affecting both human health and livestock. The parasite is transmitted through the fecal-oral route and develops in ileal epithelial cells. The resulting enteric disease leads to watery diarrhoea that can be life-threatening in individuals with an immature or a compromised immune system. The only approved treatments against cryptosporidiosis have limited efficacy in the total clearance of the parasite. Improving our understanding of the parasite biology and host-pathogen interactions, as well as identifying new drug targets, are key steps towards the development of much needed effective control strategies. Cysteine proteases play key roles in the pathogenesis of protozoan parasites, including cell or tissue penetration, hydrolysis of host or parasite proteins, and evasion or modulation of the host immune response, making them attractive chemotherapeutic and vaccine targets. For instance, papain-like proteases (clan CA, family C1) from protozoan parasites responsible for human or bovine trypanosomiasis (cruzipain, congopain 1) and malaria (falcipains 2) are already well characterised as druggable targets based on in vitro and in vivo models. Genes encoding cathepsin L-like proteases (clan CA, family C1) have been reported in the genome of C. parvum, but only one (ie. cryptopain-1) has been analysed biochemically 3 to date. By combining our knowledge and expertise from the parasitology and protease biochemistry areas, we propose to detect cysteine protease-dependent activities throughout the C. parvum life cycle and characterise new cryptopain-like proteases in order to improve our understanding of their biological functions and identify novel targets for chemotherapy

Etat des lieux de la recherche sur la cryptosporidiose au centre INRAE de Nouzilly (2021-09-22). Séminaire invité au Laboratoire Microorganismes: Génome Environnement, Clermont-Ferrand, France.

Séminaire invité au Laboratoire Microorganismes: Génome Environnement, Clermont-Ferrand, France

Cryptosporidium parvum can subvert the host immune response through manipulation of CRAMP expression during neonatal infection

Due to the immaturity of their immune system, neonates are highly sensitive to intestinal infections. During the neonatal period, antimicrobial peptide (AMP) composition differs substantially from that of adults. This is the case in the small intestine for the cathelicidin-related antimicrobial peptide (CRAMP) expressed preferentially in the neonatal period while conversely other AMPs such as Reg3γ are expressed later in life. Among enteric neonatal diseases, Cryptosporidiosis is a zoonotic disease and is highly prevalent in children less than 5 years old in developing countries and in neonatal ruminants worldwide. Cryptosporidium parvum is the etiological agent of this diarrheal disease and infects exclusively epithelial cells. Innate immunity is important to control the acute phase of infection in neonates with dendritic cells and IFNγ playing a major role. Antimicrobial peptides are important contributors of innate immunity, but the role of CRAMP, which is elevated in the intestine of neonates has never been investigated during Cryptosporidiosis so far. In this work, we observed in the neonatal murine model of cryptosporidiosis that unlike other antimicrobial molecules such as Reg3 and Lysozyme, CRAMP expression was significantly reduced in the intestine during infection. By using different genetically modified mouse models, we demonstrated that the reduced CRAMP expression was independent of IFN , a pro-inflammatory cytokine strongly produced during infection, but also of Myd88, an adaptor molecule involved in innate immune signalling. We also excluded the role of gut flora in this response. When C. parvum infected neonatal mice orally received exogenous CRAMP to compensate the reduced expression of this AMP, the parasitic load of neonates was significantly decreased. In addition, when free parasites were in direct contact with CRAMP, this AMP affected the viability of sporozoites. All together, these data suggest that C. parvum induces the reduction of CRAMP expression to escape the anti-parasiticidal effect of CRAMP

<em>Cryptosporidium parvum</em> can subvert the host immune response through manipulation of CRAMP expression during neonatal infection

National audienceDue to the immaturity of their immune system, neonates are highly sensitive to intestinal infections. During the neonatal period, antimicrobial peptide (AMP) composition differs substantially from that of adults. This is the case in the small intestine for the cathelicidin-related antimicrobial peptide (CRAMP) expressed preferentially in the neonatal period while conversely other AMPs such as Reg3γ are expressed later in life. Among enteric neonatal diseases, Cryptosporidiosis is a zoonotic disease and is highly prevalent in children less than 5 years old in developing countries and in neonatal ruminants worldwide. Cryptosporidium parvum is the etiological agent of this diarrheal disease and infects exclusively epithelial cells. Innate immunity is important to control the acute phase of infection in neonates with dendritic cells and IFNγ playing a major role. Antimicrobial peptides are important contributors of innate immunity, but the role of CRAMP, which is elevated in the intestine of neonates has never been investigated during Cryptosporidiosis so far. In this work, we observed in the neonatal murine model of cryptosporidiosis that unlike other antimicrobial molecules such as Reg3 and Lysozyme, CRAMP expression was significantly reduced in the intestine during infection. By using different genetically modified mouse models, we demonstrated that the reduced CRAMP expression was independent of IFN , a pro-inflammatory cytokine strongly produced during infection, but also of Myd88, an adaptor molecule involved in innate immune signalling. We also excluded the role of gut flora in this response. When C. parvum infected neonatal mice orally received exogenous CRAMP to compensate the reduced expression of this AMP, the parasitic load of neonates was significantly decreased. In addition, when free parasites were in direct contact with CRAMP, this AMP affected the viability of sporozoites. All together, these data suggest that C. parvum induces the reduction of CRAMP expression to escape the anti-parasiticidal effect of CRAMP

Do human digestive physicochemical parameters contribute to children higher susceptibility to cryptosporidiosis ?

National audienceCryptosporidium parvum is responsible for a zoonotic disease affecting both human health and livestock. The parasite infects its host through the oral route and develops in ileal epithelial cells, leading to acute and sometimes lethal diarrhoea. The severity of cryptosporidiosis is closely related to the immune status of its host, young ruminants, infants, and immunocompromised individuals being more susceptible. However, the impact of the gastrointestinal route the parasite takes before reaching its site of infection on the severity of the disease, has never been investigated. The in vitro computer-controlled TNO gastrointestinal model (TIM) was used for a comparative study of C. parvum survival and virulence under adult and child digestive conditions. Parasite survival and excystation kinetics in the in vitro digestive tract were determined by flow cytometry analysis while virulence was assessed after reinoculation of sporozoites onto HCT-8 cells. A luciferase reporter gene was also used to follow sporozoite activity throughout the digestive process. A global transcriptome analysis by RNA-Seq will be performed to identify differentially expressed parasite genes. Preliminary data show that the excystation rate is almost maximal in the duodenal compartment, one hour after the beginning of digestion in the TIM. However, a higher amount of parasites reaches the distal ileal compartment while protected in their oocyst shell upon simulation of child compared to adult digestive conditions. After three hours of digestion, the luciferase activity expressed by released sporozoites is significantly higher in the distal intestinal compartments of child compared to adult. Differences in digestive physicochemical parameters may partially explain why children are more susceptible to cryptosporidiosis than adults. This study is the first one exploring the impact of various digestive conditions on Cryptosporidium using a sophisticated gastrointestinal model

Metal-captured inhibition of pre-mRNA processing activity by CPSF3 controls very efficiently Cryptosporidium infection

Session : Vaccination, chemotherapy and controlInternational audienceCryptosporidium parvum is one of the most important diseases of young ruminant livestock, particularly neonatal calves. Infected animals may suffer from profuse watery diarrhoea, dehydration and in severe cases death can occur. Chemotherapy is very limited and there is a need for more effective treatments. Here we show inhibition of cleavage and polyadenylation specificity factor 3 (CPSF3) as a new strategy to control Cryptosporidium infection. Remarkably, we find that oxaborole-mediated inhibition of CPSF3 reduces intestinal parasite burden in both immunocompromised and neonatal mouse models with far better efficacy than nitazoxanide. We present crystal structures (1.6 to 2.0 Å) revealing an unprecedented mechanism of action, whereby the mRNA processing activity of CPSF3 is efficiently blocked by the binding of the oxaborole group at the metal-dependent catalytic center. Our data provide insights to accelerate the development of next-generation anti-Cryptosporidium therapeutics. Given the high structural similarity of the CPSF3 drug site of other parasites, it is very likely that the CPSF3 inhibition mechanism proposed here is shared across parasite species, such as Plasmodium, Toxoplasma and trypanosomatids

Metal-captured inhibition of pre-mRNA processing activity by CPSF3 controls Cryptosporidium infection

Cryptosporidium is an intestinal pathogen that causes severe but self-limiting diarrhea in healthy humans, yet it can turn into a life-threatening, unrelenting infection in immunocompromised patients and young children. Severe diarrhea is recognized as the leading cause of mortality for children below 5 years of age in developing countries. The only approved treatment against cryptosporidiosis, nitazoxanide, has limited efficacy in the most vulnerable patient populations, including malnourished children, and is ineffective in immunocompromised individuals. Here, we investigate inhibition of the parasitic cleavage and polyadenylation specificity factor 3 (CPSF3) as a strategy to control Cryptosporidium infection. We show that the oxaborole AN3661 selectively blocked Cryptosporidium growth in human HCT-8 cells, and oral treatment with AN3661 reduced intestinal parasite burden in both immunocompromised and neonatal mouse models of infection with greater efficacy than nitazoxanide. Furthermore, we present crystal structures of recombinantly produced Cryptosporidium CPSF3, revealing a mechanism of action whereby the mRNA processing activity of this enzyme is efficiently blocked by the binding of the oxaborole group at the metal-dependent catalytic center. Our data provide insights that may help accelerate the development of next-generation anti-Cryptosporidium therapeutics