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Cell Sep 2016Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we...
Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplasmosis, yet most of their genes remain uncharacterized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines ∼200 previously uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investigated, revealing essential functions during infection of human cells. Secondary screens identify as an invasion factor the claudin-like apicomplexan microneme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory organelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicomplexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falciparum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antiparasitic interventions.
Topics: Apicomplexa; Cells, Cultured; Claudins; Clustered Regularly Interspaced Short Palindromic Repeats; Fibroblasts; Genome, Protozoan; Genome-Wide Association Study; Host-Parasite Interactions; Humans; Malaria, Falciparum; Plasmodium falciparum; Protozoan Proteins; Toxoplasma; Toxoplasmosis
PubMed: 27594426
DOI: 10.1016/j.cell.2016.08.019 -
Cell Host & Microbe Apr 2023Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively...
Cryptosporidium is a leading cause of diarrheal disease in children and an important contributor to early childhood mortality. The parasite invades and extensively remodels intestinal epithelial cells, building an elaborate interface structure. How this occurs at the molecular level and the contributing parasite factors are largely unknown. Here, we generated a whole-cell spatial proteome of the Cryptosporidium sporozoite and used genetic and cell biological experimentation to discover the Cryptosporidium-secreted effector proteome. These findings reveal multiple organelles, including an original secretory organelle, and generate numerous compartment markers by tagging native gene loci. We show that secreted proteins are delivered to the parasite-host interface, where they assemble into different structures including a ring that anchors the parasite into its unique epicellular niche. Cryptosporidium thus uses a complex set of secretion systems during and following invasion that act in concert to subjugate its host cell.
Topics: Child, Preschool; Child; Humans; Cryptosporidium; Proteome; Cryptosporidiosis; Organelles; Cryptosporidium parvum; Protozoan Proteins; Host-Parasite Interactions
PubMed: 36958336
DOI: 10.1016/j.chom.2023.03.001 -
Life (Basel, Switzerland) Mar 2021The host cell invasion process of apicomplexan parasites like is facilitated by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis...
The host cell invasion process of apicomplexan parasites like is facilitated by sequential exocytosis of the microneme, rhoptry and dense granule organelles. Exocytosis is facilitated by a double C2 domain (DOC2) protein family. This class of C2 domains is derived from an ancestral calcium (Ca) binding archetype, although this feature is optional in extant C2 domains. DOC2 domains provide combinatorial power to the C2 domain, which is further enhanced in ferlins that harbor 5-7 C2 domains. Ca conditionally engages the C2 domain with lipids, membranes, and/or proteins to facilitating vesicular trafficking and membrane fusion. The widely conserved ferlins 1 (FER1) and 2 (FER2) are responsible for microneme and rhoptry exocytosis, respectively, whereas an unconventional TgDOC2 is essential for microneme exocytosis. The general role of ferlins in endolysosmal pathways is consistent with the repurposed apicomplexan endosomal pathways in lineage specific secretory organelles. Ferlins can facilitate membrane fusion without SNAREs, again pertinent to the Apicomplexa. How temporal raises in Ca combined with spatiotemporally available membrane lipids and post-translational modifications mesh to facilitate sequential exocytosis events is discussed. In addition, new data on cross-talk between secretion events together with the identification of a new microneme protein, MIC21, is presented.
PubMed: 33803212
DOI: 10.3390/life11030217 -
Life (Basel, Switzerland) Sep 2021is a unicellular eukaryote with a very polarized secretory system composed of micronemes rhoptries and dense granules that are required for host cell invasion. , like... (Review)
Review
is a unicellular eukaryote with a very polarized secretory system composed of micronemes rhoptries and dense granules that are required for host cell invasion. , like its relative , uses the endolysosomal system to produce the secretory organelles and to ingest host cell proteins. The parasite also has an apicoplast, a secondary endosymbiotic organelle, which depends on vesicular trafficking for appropriate incorporation of nuclear-encoded proteins into the apicoplast. Recently, the central molecules responsible for sorting and trafficking in and have been characterized. From these studies, it is now evident that has repurposed the molecules of the endosomal system to the secretory pathway. Additionally, the sorting and vesicular trafficking mechanism seem to be conserved among apicomplexans. This review described the most recent findings on the molecular mechanisms of protein sorting and vesicular trafficking in and revealed that has an amazing secretory machinery that has been cleverly modified to its intracellular lifestyle.
PubMed: 34575086
DOI: 10.3390/life11090937 -
Genes Jun 2020is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant...
is an apicomplexan protozoan parasite that invades the liver and bile duct epithelial cells in rabbits and causes severe hepatic coccidiosis, resulting in significant economic losses in the domestic rabbit industry. Hepatic coccidiosis lacks the typical clinical symptoms and there is a lack of effective premortem tools to timely diagnose this disease. Therefore, in the present study we cloned and expressed the two microneme proteins i.e., microneme protein 1 () and microneme protein 3 () from and used them as recombinant antigens to develop a serodiagnostic method for an effective diagnosis of hepatic coccidiosis. The cDNAs encoding and were cloned and the mRNA expression levels of these two genes at different developmental stages of were determined by quantitative real-time PCR analysis (qRT-PCR). The immunoreactivity of recombinant EsMIC1 (rEsMIC1) and EsMIC3 (rEsMIC3) proteins were detected by Western blotting, and indirect enzyme-linked immunosorbent assays (ELISAs) based on these two recombinant antigens were established to evaluate their serodiagnostic potential. Our results showed that the proteins encoded by the ORFs of (711 bp) and (891 bp) were approximately 25.89 and 32.39 kDa in predicted molecular weight, respectively. Both and showed the highest mRNA expression levels in the merozoites stage of . Western blotting analysis revealed that both recombinant proteins were recognized by positive sera, and the indirect ELISAs using rEsMIC1 and rEsMIC3 were developed based on their good immunoreactivity, with 100% (48/48) sensitivity and 97.9% (47/48) specificity for rEsMIC1 with 100% (48/48) sensitivity and 100% (48/48) specificity for rEsMIC3, respectively. Moreover, rEsMIC1- and rEsMIC3-based indirect ELISA were able to detect corresponding antibodies in sera at days 6, 8, and 10 post infection, with the highest positive diagnostic rate (62.5% (30/48) for rEsMIC1 and 66.7% (32/48) for rEsMIC3) observed at day 10 post infection. Therefore, both and can be used as potential serodiagnostic candidate antigens for hepatic coccidiosis caused by .
Topics: Animals; Antibodies, Protozoan; Antigens, Protozoan; Coccidiosis; Eimeria; Protozoan Proteins; RNA, Messenger; Rabbits; Sensitivity and Specificity; Serologic Tests
PubMed: 32610686
DOI: 10.3390/genes11070725 -
Tropical Parasitology 2017spp. was first described in mice in 1907. The first human case was reported in an acquired immune deficiency syndrome patient after which it gained importance. It is... (Review)
Review
spp. was first described in mice in 1907. The first human case was reported in an acquired immune deficiency syndrome patient after which it gained importance. It is one of the emerging protozoan parasites according to the Centre of Disease Control and Prevention. The special structure which is present in them such as rhoptries and micronemes are responsible for their virulence and pathogenicity. They can be transmitted from animals, human to human, water, food, and tends to cause waterborne outbreaks. The clinical manifestation in immunocompetent patient is self-limiting when compared to immunocompromised individual where it causes chronic diarrhea not responding to treatment. Hence, it is necessary to diagnose them early to prevent any complication in these patients. There are many investigations currently available such as stool microscopy after Sheather's concentration technique, rapid test targeting specific antigen, molecular methods, and imaging techniques.
PubMed: 29114483
DOI: 10.4103/tp.TP_25_17 -
Advances in Experimental Medicine and... 2011Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of... (Review)
Review
Cysteine proteases are important for the growth and survival of apicomplexan parasites that infect humans. The apicomplexan Toxoplasma gondii expresses five members of the C1 family of cysteine proteases, including one cathepsin L-like (TgCPL), one cathepsin B-like (TgCPB) and three cathepsin C-like (TgCPC1, 2 and 3) proteases. Recent genetic, biochemical and structural studies reveal that cathepsins function in microneme and rhoptry protein maturation, host cell invasion, replication and nutrient acquisition. here, we review the key features and roles of T. gondii cathepsins and discuss the therapeutic potential for specific inhibitor development.
Topics: Amino Acid Sequence; Cathepsins; Humans; Molecular Sequence Data; Phylogeny; Protein Transport; Toxoplasma
PubMed: 21660658
DOI: 10.1007/978-1-4419-8414-2_4 -
PLoS Pathogens Mar 2023Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the...
Invasion of host cells by apicomplexan parasites such as Toxoplasma and Plasmodium spp requires the sequential secretion of the parasite apical organelles, the micronemes and the rhoptries. The claudin-like apicomplexan microneme protein (CLAMP) is a conserved protein that plays an essential role during invasion by Toxoplasma gondii tachyzoites and in Plasmodium falciparum asexual blood stages. CLAMP is also expressed in Plasmodium sporozoites, the mosquito-transmitted forms of the malaria parasite, but its role in this stage is still unknown. CLAMP is essential for Plasmodium blood stage growth and is refractory to conventional gene deletion. To circumvent this obstacle and study the function of CLAMP in sporozoites, we used a conditional genome editing strategy based on the dimerisable Cre recombinase in the rodent malaria model parasite P. berghei. We successfully deleted clamp gene in P. berghei transmission stages and analyzed the functional consequences on sporozoite infectivity. In mosquitoes, sporozoite development and egress from oocysts was not affected in conditional mutants. However, invasion of the mosquito salivary glands was dramatically reduced upon deletion of clamp gene. In addition, CLAMP-deficient sporozoites were impaired in cell traversal and productive invasion of mammalian hepatocytes. This severe phenotype was associated with major defects in gliding motility and with reduced shedding of the sporozoite adhesin TRAP. Expansion microscopy revealed partial colocalization of CLAMP and TRAP in a subset of micronemes, and a distinct accumulation of CLAMP at the apical tip of sporozoites. Collectively, these results demonstrate that CLAMP is essential across invasive stages of the malaria parasite, and support a role of the protein upstream of host cell invasion, possibly by regulating the secretion or function of adhesins in Plasmodium sporozoites.
Topics: Animals; Sporozoites; Microneme; Plasmodium berghei; Protozoan Proteins; Culicidae; Mammals; Malaria
PubMed: 36928686
DOI: 10.1371/journal.ppat.1011261 -
Sub-cellular Biochemistry 2008Microneme secretion supports several key cellular processes including gliding motility, active cell invasion and migration through cells, biological barriers, and... (Review)
Review
Microneme secretion supports several key cellular processes including gliding motility, active cell invasion and migration through cells, biological barriers, and tissues. The modular design of microneme proteins enables these molecules to assist each other in folding and passage through the quality control system, accurately target to the micronemes, oligimerizing with other parasite proteins, and engaging a variety of host receptors for migration and cell invasion. Structural and biochemical analyses of MIC domains is providing new perspectives on how adhesion is regulated and the potentially distinct roles MICs might play in long or short range interactions during parasite attachment and entry. New access to complete genome sequences and ongoing advances in genetic manipulation should provide fertile ground for refining current models and defining exciting new roles for MICs in apicomplexan biology.
Topics: Animals; Apicomplexa; Cryptosporidium; Eimeria; Models, Biological; Protein Structure, Tertiary; Proteomics; Protozoan Proteins; Toxoplasma
PubMed: 18512339
DOI: 10.1007/978-0-387-78267-6_2 -
Pathogens (Basel, Switzerland) Feb 2021, is the etiological agent of neosporosis, an infection that causes abortions in cattle and nervous system dysfunction in dogs. Invasion and egress are the key steps of...
BACKGROUND
, is the etiological agent of neosporosis, an infection that causes abortions in cattle and nervous system dysfunction in dogs. Invasion and egress are the key steps of the pathogenesis of infection. Microneme proteins (MICs) play important roles in the recognition, adhesion, and invasion of host cells in other apicomplexan parasites. However, some MICs and their functions in infection have rarely been reported.
METHODS
The homologous recombination strategy was used to investigate the function of MIC6 in infection.
RESULTS
ΔNcMIC6 showed a smaller plaque size and weakened capacities of invasion and egress than Nc1. Transcription levels of the egress-related genes CDPK1, PLP1, and AMA1 of ΔNcMIC6 were downregulated. Due to the lack of NcMIC6, virulence of the pathogen in the infected mouse was weakened. The subcellular localization of NcMIC1 and NcMIC4 in ΔNcMIC6, however, did not change. Nevertheless, the transcription levels of MIC1 and MIC4 in ΔNcMIC6 were downregulated, and the expression and secretion of MIC1 and MIC4 in ΔNcMIC6 were reduced compared with that in Nc1. Furthermore, the absence of NcMIC6 weakened the virulence in mice and lower parasite load detected in mice brains.
CONCLUSIONS
NcMIC6 is involved in host cell invasion and egress in and may work synergistically with other MICs to regulate the virulence of the pathogen. These data lay a foundation for further research into the function and application of NcMIC6.
PubMed: 33668497
DOI: 10.3390/pathogens10020201