Guan Zhu

Cryptosporidium parvum is a zoonotic protozoan pathogen that infects the small intestinal epithelia of humans and a wide range of other mammals [1]. It is also a frequent cause of waterborne and fo...

Current phenotypic screens for anti- Cryptosporidium compounds typically quantify intracellular parasite growth in host cell cultures after two days of infection. Here, we developed a rapid, host cell-free phenotypic assay that directly measures compound-induced loss of viability in excysted Cryptosporidium parvum sporozoites, the invasive stage that initiates infection. We first compared qRT-PCR, luminescence ATP, and resazurin fluorescence readouts for detecting viable sporozoites. The luminescence ATP assay provided the best balance of linear dynamic range, assay time, parasite input, and cost, and was therefore adapted for high-throughput screening. Screening 5,000 bioactive compounds at 40 μM identified 28 primary hits with ≥50% inhibition of sporozoite viability, including 14 with >60% inhibition. Secondary screening of these 14 compounds at 4 μM identified five hits retaining >50% inhibition: ZL0420, sulbactam, abexinostat, kojic acid, and SIB-1757. All five showed submicromolar activity against free sporozoites, with EC ₅₀ values of 0.073–0.311 μM. Four compounds, abexinostat, ZL0420, SIB-1757, and sulbactam, also inhibited intracellular parasite growth in vitro, with EC ₅₀ values of 0.316–11.87 μM and selectivity indices from >17 to >107. In an IFN-γ-knockout mouse model, these four compounds reduced oocyst shedding over the 35-day experiment by 53.7–79.0% based on area-under-the-curve analysis and improved body-weight trajectories and ileal histopathology. Biochemical assays further showed that abexinostat inhibited native parasite HDAC activity at low nanomolar concentrations, while sulbactam inhibited a β-lactamase-like activity in sporozoite lysates. These findings establish sporozoite viability as a rapid screening endpoint and identify anti- Cryptosporidium leads associated with targetable enzymatic activities in the invasive stage. Author summary: Cryptosporidium parvum is a major cause of diarrheal disease in humans and young animals, but treatment options remain limited. Most laboratory screens for new drugs against this parasite require infection of host cells and measurement of parasite growth after one or more days. We developed a faster approach that tests whether compounds can directly damage freshly excysted sporozoites — the parasite stage that first invades intestinal cells — or reduce their viability. This assay can be completed within a few hours and does not require host cells. Using this approach, we screened 5,000 bioactive compounds and identified several molecules that rapidly reduced sporozoite viability. Four of these compounds also inhibited parasite growth in cell culture and reduced infection severity in a mouse model, as measured by parasite shedding, body-weight changes, and intestinal pathology. We further showed that one compound inhibits parasite histone deacetylase activity, while another inhibits a β-lactamase-like activity present in sporozoites. Our study provides a rapid screening strategy and highlights vulnerable biological activities in the invasive stage of C. parvum .

Background: Cryptosporidium parvum is an obligate apicomplexan parasite and a major cause of diarrheal disease in humans and animals worldwide. Despite its public health importance, the molecular regulation of parasite metabolism, particularly at the post-translational level, remains poorly understood. This study aims to understand major pathways regulated by protein phosphorylation in sporozoites, the parasite invasive stage, by comparative analysis of the proteomic and phosphoproteomic profiles. Methods: The proteome and phosphoproteome of excysted sporozoites of C. parvum were determined by data-independent acquisition-based mass spectrometry. Highly abundant pathways at proteomic and phosphoproteomic levels were identified by functional enrichment. A relative phosphorylation index (RPI) was developed for comparative assessment of phosphorylation propensity across proteins. Results: Proteomics identified 2,272 proteins in the sporozoites, representing 58% of the predicted parasite proteome. Phosphoproteomics identified 8,994 phosphorylation sites across 2,141 proteins; after intensity filtering, 833 phosphoproteins were retained for downstream analysis, representing 36.7% of the detected proteome. The correlations between transcript and protein abundances are weak. Proteins involved in carbohydrate metabolism, particularly glycolysis, are highly abundant in the proteome and phosphoproteome. Proteins in carbohydrate metabolism, signaling, cytoskeletal organization, and host–parasite interaction are selectively phosphorylated. In carbohydrate metabolism, starch degradative enzymes exhibit greater phosphorylation propensity than glycolytic and fermentative enzymes. Conclusions: The C. parvum sporozoites translate more than half of the protein-coding genes, in which one-third of the proteome is phosphorylated in varied degrees. In the highly active energy metabolism, starch degradation enzymes show greater phosphorylation propensity than core glycolytic and fermentative enzymes. This study reveals the first phosphoproteome and the largest proteome for C. parvum , providing a resource for further investigations of the functional regulation by protein phosphorylation.

The parasite Cryptosporidium is a leading cause of life-threatening diarrhoeal disease, and effective treatment is not available. Clofazimine, an antimicrobial used for treatment of leprosy and tuberculosis, was found to have potent anti- Cryptosporidium activity but it failed in a human trial. This was attributed to poor bioavailability. Here we observed differential clofazimine susceptibility among C. parvum parasite isolates, which we exploit to identify a single genomic locus encoding the type II NADH dehydrogenase (NDH2) in an unbiased genetic cross. Targeted genetic ablation of ndh2 resulted in high-level clofazimine resistance and biochemical studies demonstrated NDH2-mediated electron transfer to clofazimine. Through genomic analyses, we uncovered heterogeneity at the ndh2 locus for C. parvum and C. hominis , and widespread carriage of a conserved attenuated allele across multiple continents. This heterogeneity allows parasites genomically linked through frequent sexual recombination to adjust to changing NDH2 requirements and predisposes Cryptosporidium to evade clofazimine treatment.

The oocyst of Cryptosporidium is a highly resilient transmission stage that protects sporozoites in the environment and mediates infection through a specialized opening known as the suture. While recent studies have identified Cryptosporidium oocyst wall proteins (COWPs) as structural components of the oocyst wall, the functional roles of individual COWPs and the molecular basis of suture biology remain poorly understood. Here, we performed a detailed characterization of two COWP family members, COWP4 and COWP6, combining immunolocalization, ultrastructural analysis, protein interaction assays, and genetic manipulation. We show that COWP4 is strictly localized to the oocyst suture, whereas COWP6 is distributed throughout the oocyst wall inner layer and enriched at the suture. COWP6 is additionally present in sporozoites and is secreted during parasite motility and host cell invasion, where it exhibits high-affinity binding to host cells. Structural analyses indicate that both proteins are cysteine-rich and likely form disulfide-stabilized architectures consistent with roles in wall assembly. Functional analyses reveal that COWP4 is essential for proper suture formation, excystation, and parasite infectivity, establishing the suture as a genetically defined gatekeeper for parasite transmission. In contrast, COWP6 functions as a multifunctional protein linking oocyst wall architecture to host interaction. We further demonstrate that COWP4 and COWP6 interact, suggesting coordinated assembly within the oocyst wall. Together, these findings provide a functional dissection of the Cryptosporidium oocyst suture and reveal distinct, non-redundant roles for individual COWPs. This work advances our understanding of oocyst wall biology and identifies COWP4 as an essential determinant of parasite transmission that may be targeted to disrupt the infectious cycle. Author summary: Cryptosporidium is a major cause of diarrheal disease worldwide and is transmitted through environmentally resistant oocysts that protect infectious stages of the parasite. A defining feature of the oocyst is a specialized seam-like structure, called the suture, through which parasites exit to initiate infection. Although oocyst wall proteins (COWPs) have been identified, how individual proteins contribute to the structure and function of the oocyst wall, particularly the suture, has remained unclear. In this study, we investigated two oocyst wall proteins, COWP4 and COWP6, and uncovered distinct roles for each. We found that COWP4 is specifically localized to the suture and is essential for its proper formation, enabling parasites to exit the oocyst and establish infection. In contrast, COWP6 is distributed more broadly in the oocyst wall and also functions beyond it, being secreted during parasite movement and interacting with host cells. These findings reveal that the oocyst wall is composed of specialized proteins with non-redundant functions and identify the suture as a critical control point for parasite transmission. Understanding these mechanisms may help guide the development of new strategies to block infection by disrupting oocyst integrity or parasite release. Highlights: COWP4 is a suture-specific protein essential for oocyst excystation and infectivity Genetic disruption of COWP4 produces non-infectious oocysts with impaired viability The oocyst suture is a genetically defined gatekeeper for parasite transmission COWP6 localizes to the wall inner layer and is enriched at the suture COWP6 is secreted by sporozoites and binds host cells with nanomolar affinity · COWP4 and COWP6 directly interact, linking wall architecture to host interaction

Coccidiosis caused by Eimeria spp. remains a major constraint to poultry production worldwide, and the discovery of new anticoccidial agents is hindered by the lack of scalable in vitro screening systems. Here, we developed a phenotypic high-throughput screening (HTS) platform based on a transgenic E. tenella line coexpressing enhanced yellow fluorescent protein (EYFP) and nanoluciferase (Nluc), enabling sensitive and quantitative assessment of parasite growth in vitro. Using Madin-Darby bovine kidney (MDBK) cells as host cells, parasite replication was reliably monitored within a 45 h infection window via a luminescence readout. The assay exhibited strong linearity with the parasite inoculum and was validated using known anticoccidials, monensin and salinomycin, whose expected dose–response profiles were obtained. The platform was applied to screen 1,770 compounds, identifying 28 primary hits (> 50% inhibition). After the samples were filtered for their cytotoxicity and drug-like properties, three compounds (alendronate, pemetrexed, and torsemide) were selected for further evaluation. These compounds exhibited micromolar in vitro activity (EC ₅₀ ~ 8–10 μM) with favorable selectivity indices, particularly for pemetrexed and torsemide (> 100). In a chicken infection model, compared with the vehicle control, all three compounds significantly reduced oocyst shedding. Using the anticoccidial index (ACI), a composite metric incorporating oocyst shedding, mortality, weight gain, and lesion severity, torsemide was most effective (ACI = 163.3), followed by pemetrexed (ACI = 153.5). Alendronate showed only marginal activity (ACI = 110.8). Overall, this study establishes a robust, scalable in vitro HTS platform for anticoccidial drug discovery and highlights several approved drugs as potential leads for further optimization.

Cryptosporidium parvum is an obligate intracellular apicomplexan parasite and a major cause of diarrheal disease in humans and animals worldwide. Despite its public health importance, the molecular regulation of parasite metabolism, particularly at the post-translational level, remains poorly understood. Here, we present a comprehensive proteomic analysis and the first phosphoproteomic profile of excysted C. parvum sporozoites, the invasive stage responsible for host cell entry. Using data-independent acquisition–based mass spectrometry, we identified 2,272 proteins, representing approximately 58% of the predicted parasite proteome, and 8,994 phosphorylation sites across 833 phosphoproteins. Comparative analyses revealed weak correlations between transcript and protein abundance, underscoring extensive post- transcriptional regulation in sporozoites. Functional enrichment analyses showed that proteins involved in carbohydrate metabolism, particularly glycolysis, are highly abundant in the sporozoite proteome. In contrast, phosphoproteomic data revealed that many core glycolytic enzymes exhibit relatively low phosphorylation propensity, suggesting limited reliance on phosphorylation-based regulation for basal energy metabolism at this stage. To integrate proteomic and phosphoproteomic measurements acquired independently, we developed a relative phosphorylation index (RPI) that enables comparative assessment of phosphorylation propensity across proteins. Application of this metric highlighted selective phosphorylation of proteins associated with signaling, cytoskeletal organization, and host–parasite interaction, while key metabolic entry-point enzymes, such as hexokinase, showed high abundance but minimal detectable phosphorylation, in contrast to their mammalian counterparts. Together, these findings provide the most comprehensive molecular resource to date for C. parvum sporozoites and reveal a phosphorylation landscape that emphasizes regulatory and structural processes over core metabolic flux. This work establishes a foundation for understanding parasite-specific regulatory strategies and may inform the identification of novel therapeutic targets against cryptosporidiosis. Author Summary: Cryptosporidium parvum is a microscopic parasite that causes diarrheal disease in humans and animals worldwide, yet we still know surprisingly little about how it regulates its biology at the molecular level. In this study, we set out to better understand how the parasite prepares for infection by examining the proteins present in sporozoites, the life stage that actively invades host cells. We generated the most comprehensive protein catalog to date for C. parvum sporozoites and, for the first time, mapped thousands of chemical modifications known as protein phosphorylation. These modifications often act as molecular switches that control how proteins behave. We found that proteins involved in basic energy production are extremely abundant but show relatively little phosphorylation, while proteins linked to cell structure, movement, and interaction with the host are more frequently modified. This suggests that the parasite prioritizes regulation of invasion-related processes over fine-tuning its core metabolism at this stage. By providing a detailed molecular resource and a new way to compare protein phosphorylation patterns, our work lays the groundwork for future studies of parasite biology and may help guide the search for new strategies to combat cryptosporidiosis.