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Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (<30 min) in both B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis. [ABSTRACT FROM AUTHOR]

Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis.

Lankestrella (Apicomplexa; Haemococcidia) have been described from the blood of lizards distributed in American and Western European areas. In the present study, we characterized morphologically and molecularly the haemococcidian parasites (sporozoites) that infect the blood cells of Eremias persica and Ophisops elegans, two species of lacertid lizards from central Iran. By the microscopic examination of the smears, we identified parasites as the genus Lankestrella. In total, two positive samples of each species have been investigated on the basis of the partial 18S rRNA gene analyses. The result of similarity analysis with our sequences using the Basic Local Alignment Search Tool (BLAST) indicated a11 four parasites belonging to one haplotype of Lankestrella sp. The phylogenetic analysis revealed that the new Lankestrella sp is related to Lankestrella sp identified from two lizards belonging to Spain and the Socotra Archipelago. This is the first report of Lankestrella parasites in the new lizard's host. The present study provided additional information about the new host of Lankestrella species and added new knowledge to clarify the future phylogenetic relationship between these parasites. Our results emphasize the importance of screening haemococcidian parasites in Iran. [ABSTRACT FROM AUTHOR]

ABSTRACTThe growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata-infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC50) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata-infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCETheileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata, contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper’s significance in advancing veterinary science and improving livestock health globally.

ABSTRACTToxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma. The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that T. gondii CPDH (TgCPDH) potentially functions as an alternative enzyme to perform the same reaction as CPOX under low-oxygen conditions. In this study, we demonstrated that TgCPDH exhibits CPDH activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wild-type and heme-deficient parasites did not alter their intracellular growth under both ambient and low-oxygen conditions. This research marks the first report of a CPDH-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings revealed that it does not directly affect acute Toxoplasma infections in vitro and in vivo.IMPORTANCEToxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings.

ABSTRACT The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum, namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium. The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCECryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium-unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein.

Apicomplexan parasites mobilize ionic calcium (Ca 2+ ) from intracellular stores to promote microneme secretion and facilitate motile processes including gliding motility, invasion, and egress. Recently, a multipass transmembrane protein, ICM1, was found to be i mportant for c alcium m obilization in Plasmodium falciparum and P. berghei . Comparative genomics and phylogenetics have revealed putative ICM orthologs in Toxoplasma gondii and other apicomplexans. T. gondii possesses two ICM-like proteins, which we have named TgICM1-L (TGGT1&#95;305470) and TgICM2-L (TGGT1&#95;309910). TgICM1-L and TgICM2-L localized to undefined puncta within the parasite cytosol. TgICM1-L and TgICM2-L are individually dispensable in tachyzoites, suggesting a potential compensatory relationship between the two proteins may exist. Surprisingly, mutants lacking both TgICM1-L and TgICM2-L are fully viable, exhibiting no obvious defects in growth, microneme secretion, invasion, or egress. Furthermore, loss of TgICM1-L, TgICM2-L, or both does not impair the parasite's ability to mobilize Ca 2+ . These findings suggest that additional proteins may participate in Ca 2+ mobilization or import in Apicomplexa, reducing the dependence on ICM-like proteins in T. gondii . Collectively, these results highlight similar yet distinct mechanisms of Ca 2+ mobilization between T. gondii and Plasmodium .IMPORTANCECa 2+ signaling plays a crucial role in governing apicomplexan motility; yet, the mechanisms underlying Ca 2+ mobilization from intracellular stores in these parasites remain unclear. In Plasmodium , the necessity of ICM1 for Ca 2+ mobilization raises the question of whether this mechanism is conserved in other apicomplexans. Investigation into the orthologs of Plasmodium ICM1 in T. gondii revealed a differing requirement for ICM proteins between the two parasites. This study suggests that T. gondii employs ICM-independent mechanisms to regulate Ca 2+ homeostasis and mobilization. Proteins involved in Ca 2+ signaling in apicomplexans represent promising targets for therapeutic development., Competing Interests: The authors declare no conflict of interest.

The role of malate dehydrogenase (MDH) in the metabolism of various medically significant protozoan parasites is reviewed. MDH is an NADH-dependent oxidoreductase that catalyzes interconversion between oxaloacetate and malate, provides metabolic intermediates for both catabolic and anabolic pathways, and can contribute to NAD+/NADH balance in multiple cellular compartments. MDH is present in nearly all organisms; isoforms of MDH from apicomplexans (Plasmodium falciparum, Toxoplasma gondii, Cryptosporidium spp.), trypanosomatids (Trypanosoma brucei, T. cruzi) and anaerobic protozoans (Trichomonas vaginalis, Giardia duodenalis) are presented here. Many parasitic species have complex life cycles and depend on the environment of their hosts for carbon sources and other nutrients. Metabolic plasticity is crucial to parasite transition between host environments; thus, the regulation of metabolic processes is an important area to explore for therapeutic intervention. Common themes in protozoan parasite metabolism include emphasis on glycolytic catabolism, substrate-level phosphorylation, non-traditional uses of common pathways like tricarboxylic acid cycle and adapted or reduced mitochondria-like organelles. We describe the roles of MDH isoforms in these pathways, discuss unusual structural or functional features of these isoforms relevant to activity or drug targeting, and review current studies exploring the therapeutic potential of MDH and related genes. These studies show that MDH activity has important roles in many metabolic pathways, and thus in the metabolic transitions of protozoan parasites needed for success as pathogens., (© 2024 The Author(s).)

Babesia and Plasmodium pathogens, the causative agents of babesiosis and malaria, are vector-borne intraerythrocytic protozoan parasites, posing significant threats to both human and animal health. The widespread resistance exhibited by these pathogens to various classes of antiparasitic drugs underscores the need for the development of novel and more effective therapeutic strategies. Antifolates have long been recognized as attractive antiparasitic drugs as they target the folate pathway, which is essential for the biosynthesis of purines and pyrimidines, and thus is vital for the survival and proliferation of protozoan parasites. More efficacious and safer analogs within this class are needed to overcome challenges due to resistance to commonly used antifolates, such as pyrimethamine, and to address liabilities associated with the dihydrotriazines, WR99210 and JPC-2067. Here, we utilized an in vitro culture condition suitable for the continuous propagation of Babesia duncani, Babesia divergens, Babesia MO1, and Plasmodium falciparum in human erythrocytes to screen a library of 50 dihydrotriazines and 29 biguanides for their efficacy in vitro and compared their potency and therapeutic indices across different species and isolates. We identified nine analogs that inhibit the growth of all species, including the P. falciparum pyrimethamine-resistant strain HB3, with IC 50 values below 10 nM, and display excellent in vitro therapeutic indices. These compounds hold substantial promise as lead antifolates for further development as broad-spectrum antiparasitic drugs., Competing Interests: The authors declare no conflict of interest.

Coral reefs are a biodiversity hotspot, 1,2 and the association between coral and intracellular dinoflagellates is a model for endosymbiosis. 3,4 Recently, corals and related anthozoans have also been found to harbor another kind of endosymbiont, apicomplexans called corallicolids. 5 Apicomplexans are a diverse lineage of obligate intracellular parasites 6 that include human pathogens such as the malaria parasite, Plasmodium. 7 Global environmental sequencing shows corallicolids are tightly associated with tropical and subtropical reef environments, 5,8,9 where they infect diverse corals across a range of depths in many reef systems, and correlate with host mortality during bleaching events. 10 All of this points to corallicolids being ecologically significant to coral reefs, but it is also possible they are even more widely distributed because most environmental sampling is biased against parasites that maintain a tight association with their hosts throughout their life cycle. We tested the global distribution of corallicolids using a more direct approach, by specifically targeting potential anthozoan host animals from cold/temperate marine waters outside the coral reef context. We found that corallicolids are in fact common in such hosts, in some cases at high frequency, and that they infect the same tissue as parasites from topical coral reefs. Parasite phylogeny suggests corallicolids move between hosts and habitats relatively frequently, but that biogeography is more conserved. Overall, these results greatly expand the range of corallicolids beyond coral reefs, suggesting they are globally distributed parasites of marine anthozoans, which also illustrates significant blind spots that result from strategies commonly used to sample microbial biodiversity. • Coral parasites are found in cold-water ecosystems • They infect animals related to corals, such as anemones and corallimorphs • Biogeography is a major factor in parasite evolution • Current strategies for sampling microbial diversity overlook important parasites Corallicolids are coral parasites that were thought to be restricted to coral reef ecosystems. Trznadel et al. show that they are widely distributed, infecting related hosts, such as anemones, in cold marine environments. This shows that corallicolids are globally relevant parasites and raises questions about biases in microbial diversity surveys. [ABSTRACT FROM AUTHOR]

The growing emergence of resistance to current anti-theilerial agents necessitates the exploration of alternative approaches to drug discovery. This study evaluated the antiparasitic efficacy of 148 compounds derived from an epigenetic inhibitor library against the schizont stage of a Theileria annulata -infected cell line. Initial screening at a concentration of 10 µM identified 27 compounds exhibiting promising anti-theilerial activity. Further investigation, including determination of the 50% inhibitory concentration (IC 50 ) and host cell cytotoxicity assay, highlighted seven highly effective compounds (SAHA, BVT-948, Trichostatin A, Methylstat, Plumbagin, Ryuvidine, and TCE-5003) against T. annulata -infected cells. Analysis of the active compounds revealed their inhibitory action against various human targets, such as HDAC (SAHA and Trichostatin A), SET domain (Ryuvidine), PRMT (BVT-948 and TCE-5003), histone demethylase (Methylstat), and ROS/apoptosis inducer (Plumbagin). We identified gene orthologs of these targets in Theileria and conducted molecular docking studies, demonstrating effective binding of the compounds with their respective targets in the parasite, supported by in vitro data. Additionally, we performed in silico ADME/T predictions, which indicated potential mutagenic and hepatotoxic effects of Plumbagin, Methylstat, and TCE-5003, rendering them unsuitable for drug development. Conversely, SAHA, Trichostatin A, and BVT-948 showed promising characteristics and may represent potential candidates for future development as chemotherapeutic agents against tropical theileriosis. These findings provide valuable insights into the search for novel anti-theilerial drugs and offer a basis for further research in this area.IMPORTANCE Theileria annulata is a protozoan parasite responsible for tropical theileriosis, a devastating disease affecting cattle. Traditional chemotherapy has limitations, and the study explores the potential of epidrugs as an alternative treatment approach. Epidrugs are compounds that modify gene expression without altering the underlying DNA sequence, offering a novel way to combat parasitic infections. This research is pivotal as it addresses the urgent need for innovative therapies against T. annulata , contributing to the development of more effective and targeted treatments for infected livestock. Successful implementation of epidrugs could not only enhance the well-being of cattle but also have broader implications for the control of parasitic diseases, showcasing the paper's significance in advancing veterinary science and improving livestock health globally., Competing Interests: The authors declare no conflict of interest.

SUMMARYProtozoan parasite infection dramatically alters host metabolism, driven by immunological demand and parasite manipulation strategies. Immunometabolic checkpoints are often exploited by kinetoplastid and protozoan parasites to establish chronic infection, which can significantly impair host metabolic homeostasis. The recent growth of tools to analyze metabolism is expanding our understanding of these questions. Here, we review and contrast host metabolic alterations that occur in vivo during infection with Leishmania , trypanosomes, Toxoplasma , Plasmodium, and Cryptosporidium . Although genetically divergent, there are commonalities among these pathogens in terms of metabolic needs, induction of the type I immune responses required for clearance, and the potential for sustained host metabolic dysbiosis. Comparing these pathogens provides an opportunity to explore how transmission strategy, nutritional demand, and host cell and tissue tropism drive similarities and unique aspects in host response and infection outcome and to design new strategies to treat disease., Competing Interests: The authors declare no conflict of interest.

Toxoplasma gondii is an apicomplexan parasite that is the cause of toxoplasmosis, a potentially lethal disease for immunocompromised individuals. During in vivo infection, the parasites encounter various growth environments, such as hypoxia. Therefore, the metabolic enzymes in the parasites must adapt to such changes to fulfill their nutritional requirements. Toxoplasma can de novo biosynthesize some nutrients, such as heme. The parasites heavily rely on their own heme production for intracellular survival. Notably, the antepenultimate step within this pathway is facilitated by coproporphyrinogen III oxidase (CPOX), which employs oxygen to convert coproporphyrinogen III to protoporphyrinogen IX through oxidative decarboxylation. Conversely, some bacteria can accomplish this conversion independently of oxygen through coproporphyrinogen dehydrogenase (CPDH). Genome analysis found a CPDH ortholog in Toxoplasma . The mutant Toxoplasma lacking CPOX displays significantly reduced growth, implying that T. gondii CPDH (TgCPDH) potentially functions as an alternative enzyme to perform the same reaction as CPOX under low-oxygen conditions. In this study, we demonstrated that TgCPDH exhibits CPDH activity by complementing it in a heme synthesis-deficient Salmonella mutant. Additionally, we observed an increase in TgCPDH expression in Toxoplasma when it grew under hypoxic conditions. However, deleting TgCPDH in both wild-type and heme-deficient parasites did not alter their intracellular growth under both ambient and low-oxygen conditions. This research marks the first report of a CPDH-like protein in eukaryotic cells. Although TgCPDH responds to hypoxic conditions and possesses enzymatic activity, our findings revealed that it does not directly affect acute Toxoplasma infections in vitro and in vivo ., Importance: Toxoplasma gondii is a ubiquitous parasite capable of infecting a wide range of warm-blooded hosts, including humans. During its life cycle, these parasites must adapt to varying environmental conditions, including situations with low-oxygen levels, such as intestine and spleen tissues. Our research, in conjunction with studies conducted by other laboratories, has revealed that Toxoplasma primarily relies on its own heme production during acute infections. Intriguingly, in addition to this classical heme biosynthetic pathway, the parasites encode a putative oxygen-independent coproporphyrinogen dehydrogenase (CPDH), suggesting its potential contribution to heme production under varying oxygen conditions, a feature typically observed in simpler organisms like bacteria. Notably, so far, CPDH has only been identified in some bacteria for heme biosynthesis. Our study discovered that Toxoplasma harbors a functional enzyme displaying CPDH activity, which alters its expression in the parasites when they face fluctuating oxygen levels in their surroundings., Competing Interests: The authors declare that that they have no conflicts of interest concerning the contents of this article.

The zoonotic Cryptosporidium parvum is a global contributor to infantile diarrheal diseases and opportunistic infections in immunocompromised or weakened individuals. Like other apicomplexans, it possesses several specialized secretory organelles, including micronemes, rhoptry, and dense granules. However, the understanding of cryptosporidial micronemal composition and secretory pathway remains limited. Here, we report a new micronemal protein in C. parvum , namely, thrombospondin (TSP)-repeat domain-containing protein-4 (CpTSP4), providing insights into these ambiguities. Immunostaining and enzyme-linked assays show that CpTSP4 is prestored in the micronemes of unexcysted sporozoites but secreted during sporozoite excystation, gliding, and invasion. In excysted sporozoites, CpTSP4 is also distributed on the two central microtubules unique to Cryptosporidium . The secretion and microtubular distribution could be completely blocked by the selective kinesin-5 inhibitors SB-743921 and SB-715992, resulting in the accumulation of CpTSP4 in micronemes. These support the kinesin-dependent microtubular trafficking of CpTSP4 for secretion. We also localize γ-tubulin, consistent with kinesin-dependent anterograde trafficking. Additionally, recombinant CpTSP4 displays nanomolar binding affinity to the host cell surface, for which heparin acts as one of the host ligands. A novel heparin-binding motif is identified and validated biochemically for its contribution to the adhesive property of CpTSP4 by peptide competition assays and site-directed mutagenesis. These findings shed light on the mechanisms of intracellular trafficking and secretion of a cryptosporidial micronemal protein and the interaction of a TSP-family protein with host cells.IMPORTANCE Cryptosporidium parvum is a globally distributed apicomplexan parasite infecting humans and/or animals. Like other apicomplexans, it possesses specialized secretory organelles in the zoites, in which micronemes discharge molecules to facilitate the movement and invasion of zoites. Although past and recent studies have identified several proteins in cryptosporidial micronemes, our understanding of the composition, secretory pathways, and domain-ligand interactions of micronemal proteins remains limited. This study identifies a new micronemal protein, namely, CpTSP4, that is discharged during excystation, gliding, and invasion of C. parvum sporozoites. The CpTSP4 secretion depends on the intracellular trafficking on the two Cryptosporidium -unique microtubes that could be blocked by kinesin-5/Eg5 inhibitors. Additionally, a novel heparin-binding motif is identified and biochemically validated, which contributes to the nanomolar binding affinity of CpTSP4 to host cells. These findings indicate that kinesin-dependent microtubular trafficking is critical to CpTSP4 secretion, and heparin/heparan sulfate is one of the ligands for this micronemal protein., Competing Interests: The authors declare no conflict of interest.