Cryptosporidium parvum Subtilisin-Like Serine Protease (SUB1) Is Crucial for Parasite Egress from Host Cells.

Despite the severity and global burden of Cryptosporidium infection, treatments are less than optimal, and there is no effective vaccine. Egress from host cells is a key process for the completion of the life cycle of apicomplexan parasites. For Plasmodium species, subtilisin-like serine protease (SUB1) is a key mediator of egress. For Toxoplasma species, calcium-dependent protein kinases (CDPKs) are critical. In this study, we characterized Cryptosporidium SUB1 expression and evaluated its effect using an infection model. We found increased expression between 12 and 20 h after in vitro infection, prior to egress. We induced silencing of SUB1 (ΔSUB1) mRNA using SUB1 single-stranded antisense RNA coupled with human Argonaute 2. Silencing of SUB1 mRNA expression did not affect parasite viability, excystation, or invasion of target cells. However, knockdown led to a 95% decrease in the proportion of released merozoites in vitro (P < 0.0001). In contrast, silencing of CDPK5 had no effect on egress. Overall, our results indicate that SUB1 is a key mediator of Cryptosporidium egress and suggest that interruption of the life cycle at this stage may effectively inhibit the propagation of infection.

Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy.

Bumped kinase inhibitors (BKIs) of Cryptosporidium parvum calcium-dependent protein kinase 1 (CpCDPK1) are leading candidates for treatment of cryptosporidiosis-associated diarrhea. Potential cardiotoxicity related to anti-human ether-à-go-go potassium channel (hERG) activity of the first-generation anti-Cryptosporidium BKIs triggered further testing for efficacy. A luminescence assay adapted for high-throughput screening was used to measure inhibitory activities of BKIs against C. parvum in vitro. Furthermore, neonatal and interferon γ knockout mouse models of C. parvum infection identified BKIs with in vivo activity. Additional iterative experiments for optimum dosing and selecting BKIs with minimum levels of hERG activity and frequencies of other safety liabilities included those that investigated mammalian cell cytotoxicity, C. parvum proliferation inhibition in vitro, anti-human Src inhibition, hERG activity, in vivo pharmacokinetic data, and efficacy in other mouse models. Findings of this study suggest that fecal concentrations greater than parasite inhibitory concentrations correlate best with effective therapy in the mouse model of cryptosporidiosis, but a more refined model for efficacy is needed.

Preassembled Single-Stranded RNA-Argonaute Complexes: A Novel Method to Silence Genes in Cryptosporidium.

Cryptosporidiosis is a common cause of diarrhea morbidity and mortality worldwide. Research progress on this infection has been slowed by lack of methods to genetically manipulate Cryptosporidium parasites. Small interfering RNA (siRNA) is widely used to study gene function, but Cryptosporidium species lack the enzymes necessary to process siRNA. By preassembling complexes with the human enzyme Argonaute 2 (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA), we induced specific slicing in Cryptosporidium RNA targets. We demonstrated the reduction in expression of target genes at the mRNA and protein levels by transfecting live parasites with ssRNA-hAgo2 complexes. Furthermore we used this method to confirm the role of selected molecules during host cell invasion. This novel method provides a novel means of silencing Cryptosporidium genes to study their role in host-parasite interactions and as potential targets for chemotherapy.

A Novel Calcium-Dependent Kinase Inhibitor, Bumped Kinase Inhibitor 1517, Cures Cryptosporidiosis in Immunosuppressed Mice.

Cryptosporidium is recognized as one of the main causes of childhood diarrhea worldwide. However, the current treatment for cryptosporidiosis is suboptimal. Calcium flux is essential for entry in apicomplexan parasites. Calcium-dependent protein kinases (CDPKs) are distinct from protein kinases of mammals, and the CDPK1 of the apicomplexan Cryptosporidium lack side chains that typically block a hydrophobic pocket in protein kinases. We exploited this to develop bumped kinase inhibitors (BKIs) that selectively target CDPK1. We have shown that several BKIs of Cryptosporidium CDPK1 potently reduce enzymatic activity and decrease parasite numbers when tested in vitro. In the present work, we studied the anticryptosporidial activity of BKI-1517, a novel BKI. The half maximal effective concentration for Cryptosporidium parvum in HCT-8 cells was determined to be approximately 50 nM. Silencing experiments of CDPK1 suggest that BKI-1517 acts on CDPK1 as its primary target. In a mouse model of chronic infection, 5 of 6 SCID/beige mice (83.3%) were cured after treatment with a single daily dose of 120 mg/kg BKI-1517. No side effects were observed. These data support advancing BKI-1517 as a lead compound for drug development for cryptosporidiosis.

Multiplexed Recombinase Polymerase Amplification Assay To Detect Intestinal Protozoa.

This work describes a proof-of-concept multiplex recombinase polymerase amplification (RPA) assay with lateral flow readout that is capable of simultaneously detecting and differentiating DNA from any of the diarrhea-causing protozoa Giardia, Cryptosporidium, and Entamoeba. Together, these parasites contribute significantly to the global burden of diarrheal illness. Differential diagnosis of these parasites is traditionally accomplished via stool microscopy. However, microscopy is insensitive and can miss up to half of all cases. DNA-based diagnostics such as polymerase chain reaction (PCR) are far more sensitive; however, they rely on expensive thermal cycling equipment, limiting their availability to centralized reference laboratories. Isothermal DNA amplification platforms, such as the RPA platform used in this study, alleviate the need for thermal cycling equipment and have the potential to broaden access to more sensitive diagnostics. Until now, multiplex RPA assays have not been developed that are capable of simultaneously detecting and differentiating infections caused by different pathogens. We developed a multiplex RPA assay to detect the presence of DNA from Giardia, Cryptosporidium, and Entamoeba. The multiplex assay was characterized using synthetic DNA, where the limits-of-detection were calculated to be 403, 425, and 368 gene copies per reaction of the synthetic Giardia, Cryptosporidium, and Entamoeba targets, respectively (roughly 1.5 orders of magnitude higher than for the same targets in a singleplex RPA assay). The multiplex assay was also characterized using DNA extracted from live parasites spiked into stool samples where the limits-of-detection were calculated to be 444, 6, and 9 parasites per reaction for Giardia, Cryptosporidium, and Entamoeba parasites, respectively. This proof-of-concept assay may be reconfigured to detect a wide variety of targets by re-designing the primer and probe sequences.

A nanotherapy strategy significantly enhances anticryptosporidial activity of an inhibitor of bifunctional thymidylate synthase-dihydrofolate reductase from Cryptosporidium.

Cryptosporidiosis, a gastrointestinal disease caused by protozoans of the genus Cryptosporidium, is a common cause of diarrheal diseases and often fatal in immunocompromised individuals. Bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) from Cryptosporidium hominis (C. hominis) has been a molecular target for inhibitor design. C. hominis TS-DHFR inhibitors with nM potency at a biochemical level have been developed however drug delivery to achieve comparable antiparasitic activity in Cryptosporidium infected cell culture has been a major hurdle for designing effective therapies. Previous mechanistic and structural studies have identified compound 906 as a nM C. hominis TS-DHFR inhibitor in vitro, having µM antiparasitic activity in cell culture. In this work, proof of concept studies are presented using a nanotherapy approach to improve drug delivery and the antiparasitic activity of 906 in cell culture. We utilized PLGA nanoparticles that were loaded with 906 (NP-906) and conjugated with antibodies to the Cryptosporidium specific protein, CP2, on the nanoparticle surface in order to specifically target the parasite. Our results indicate that CP2 labeled NP-906 (CP2-NP-906) reduces the level of parasites by 200-fold in cell culture, while NP-906 resulted in 4.4-fold decrease. Moreover, the anticryptosporidial potency of 906 improved 15 to 78-fold confirming the utility of the antibody conjugated nanoparticles as an effective drug delivery strategy.

Nucleic acid test to diagnose cryptosporidiosis: lab assessment in animal and patient specimens.

Diarrheal diseases cause more morbidity and mortality around the world than human immunodeficiency virus (HIV), malaria, or tuberculosis. Given that effective treatment of persistent diarrheal illness requires knowledge of the causative organism, diagnostic tests are of paramount importance. The protozoan parasites of the genus Cryptosporidium are increasingly recognized to be responsible for a significant portion of diarrhea morbidity. We present a novel nucleic acid test to detect the presence of Cryptosporidium species in DNA extracted from stool samples. The assay uses the isothermal amplification technique recombinase polymerase amplification (RPA) to amplify trace amounts of pathogen DNA extracted from stool to detectable levels in 30 min; products are then detected visually on simple lateral flow strips. The RPA-based Cryptosporidium assay (RPAC assay) was developed and optimized using DNA from human stool samples spiked with pathogen. It was then tested using DNA extracted from the stool of infected mice where it correctly identified the presence or absence of 27 out of 28 stool samples. It was finally tested using DNA extracted from the stool of infected patients where it correctly identified the presence or absence of 21 out of 21 stool samples. The assay was integrated into a foldable, paper and plastic device that enables DNA amplification with only the use of pipets, pipet tips, and a heater. The performance of the integrated assay is comparable to or better than polymerase chain reaction (PCR), without requiring the use of thermal cycling equipment. This platform can easily be adapted to detect DNA from multiple pathogens.

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase-dihydrofolate reductase.

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS-DHFR specific inhibitors.

A novel calcium-dependent protein kinase inhibitor as a lead compound for treating cryptosporidiosis.

Cryptosporidium parasites infect intestinal cells, causing cryptosporidiosis. Despite its high morbidity and association with stunting in the developing world, current therapies for cryptosporidiosis have limited efficacy. Calcium-dependent protein kinases (CDPKs) are essential enzymes in the biology of protozoan parasites. CDPK1 was cloned from the genome of Cryptosporidium parvum, and potent and specific inhibitors have been developed based on structural studies. In this study, we evaluated the anti-Cryptosporidium activity of a novel CDPK1 inhibitor, 1294, and demonstrated that 1294 significantly reduces parasite infection in vitro, with a half maximal effective concentration of 100 nM. Pharmacokinetic studies revealed that 1294 is well absorbed, with a half-life supporting daily administration. Oral therapy with 1294 eliminated Cryptosporidium parasites from 6 of 7 infected severe combined immunodeficiency-beige mice, and the parasites did not recur in these immunosuppressed mice. Mice treated with 1294 had less epithelial damage, corresponding to less apoptosis. Thus, 1294 is an important lead for the development of drugs for treatment of cryptosporidiosis.

A PCR Test Using the Mini-PCR Platform and Simplified Product Detection Methods Is Highly Sensitive and Specific to Detect Fasciola hepatica DNA Mixed in Human Stool, Snail Tissue, and Water DNA Specimens.

Fasciola hepatica has a complex lifecycle with multiple intermediate and definitive hosts and influenced by environmental factors. The disease causes significant morbidity in children and its prevalent worldwide. There is lack of data about distribution and burden of the disease in endemic regions, owing to poor efficacy of the different diagnostic methods used. A novel PCR-based test was developed by using a portable mini-PCR® platform to detect Fasciola sp. DNA and interpret the results via a fluorescence viewer and smartphone image analyzer application. Human stool, snail tissue, and water samples were used to extract DNA. Primers targeting the ITS-1 of the 18S rDNA gene of Fasciola sp. were used. The limit of detection of the mini-PCR test was 1 fg/µL for DNA samples diluted in water, 10 fg/µL for Fasciola/snail DNA scramble, and 100 fg/µL for Fasciola/stool DNA scramble. The product detection by agarose gel, direct visualization, and image analyses showed the same sensitivity. The Fh mini-PCR had a sensitivity and specificity equivalent to real-time PCR using the same specimens. Testing was also done on infected human stool and snail tissue successfully. These experiments demonstrated that Fh mini-PCR is as sensitive and specific as real time PCR but without the use of expensive equipment and laboratory facilities. Further testing of multiple specimens with natural infection will provide evidence for feasibility of deployment to resource constrained laboratories.

Human primary intestinal epithelial cells as an improved in vitro model for Cryptosporidium parvum infection.

The study of human intestinal pathogens has been limited by the lack of methods for the long-term culture of primary human intestinal epithelial cells (PECs). The development of infection models with PECs would allow a better understanding of host-parasite interactions. The objective of this study was to develop a novel method for prolonged in vitro cultivation of PECs that can be used to study Cryptosporidium infection. We isolated intact crypts from human intestines removed during weight loss surgery. The fragments of intestinal layers were cultivated with culture medium supplemented with growth factors and antiapoptotic molecules. After 7 days, the PECs formed self-regenerating cell clusters, forming villi that resemble intestinal epithelium. The PECs proliferated and remained viable for at least 60 days. The cells expressed markers for intestinal stem cells, epithelial cells, and mature enterocytes. The PECs were infected with Cryptosporidium. In contrast to older models in which parasite numbers decay, the burden of parasites increased for >120 h. In summary, we describe here a novel method for the cultivation of self-regenerating human epithelial cells from small intestinal crypts, which contain both intestinal stem cells and mature villus cells. We present data that suggest these cells support Cryptosporidium better than existing cell lines. PECs should provide an improved tool for studying host-parasite interactions involving Cryptosporidium and other intestinal pathogens.

MiniPCR as a portable equipment for the molecular diagnosis of american cutaneous leishmaniasis.

There is an urgent need to improve the diagnostic capacity of cutaneous leishmaniasis (CL) in rural health centers to improve the management of the disease in patients from remote regions where the infection is endemic. Microscopy of Giemsa-stained lesion smears is the standard-of-care diagnostic test in virtually all health centers, but its sensitivity is suboptimal (50-70%) and prone to false negative results. We evaluated the performance of a low-cost DNA extraction buffer (LAB) using a portable miniPCR™ equipment coupled with an inexpensive fluorescence viewer to detect Leishmania DNA with the naked eye or using a commercial photo app. Using ten-fold serial dilutions of Leishmania (V.) panamensis promastigotes the miniPCR-F test detected 10 parasites per µL, which was comparable to real-time PCR. Utilization of DNA from retrospective clinical samples preserved at -80 °C from Colombia (n = 28) or lesion exudate preserved in filter papers from Peru (n = 48) showed that the miniPCR-fluorescent test had a 100% sensitivity and > 90% specificity compared to real-time PCR. This study demonstrated the utility of LAB DNA extraction method for direct amplification of Leishmania using the miniPCR and reading of P51 results with the naked eye or via digital reading with a photo app. These preliminary results indicated that the miniPCR-F test workflow could be amenable to implementation in resource-limited health centers.

Benzoylbenzimidazole-based selective inhibitors targeting Cryptosporidium parvum and Toxoplasma gondii calcium-dependent protein kinase-1.

Calcium-dependent protein kinase-1 (CDPK1) from Cryptosporidium parvum (CpCDPK1) and Toxoplasma gondii (TgCDPK1) have become attractive targets for discovering selective inhibitors to combat infections caused by these protozoa. We used structure-based design to improve a series of benzoylbenzimidazole-based compounds in terms of solubility, selectivity, and potency against CpCDPK1 and TgCDPK1. The best inhibitors show inhibitory potencies below 50 nM and selectivity well above 200-fold over two human kinases with small gatekeeper residues.

Identification and functional characterization of the siRNA pathway in Taenia crassiceps by silencing Enolase A.

A gene silencing procedure on cysticerci of the taeniid cestode Taenia crassiceps is described. This is the first time this technique is reported in this species that is widely used as an animal model for human cysticercosis. Genome database searches were performed in order to find out if relevant genes involved in gene silencing and non-coding RNA processing, Argonaute and Dicer (AGO and Dcr) are present in T. crassiceps. We found three AGO and two Dcr orthologues that were designed TcAGO1, Tc2 and Tc3, as well as TcDcr1 and TcDcr2. In order to elucidate the evolutionary relationships of T. crassiceps TcAGO and TcDcr genes, separate phylogenetic analyses were carried out for each, including AGO and Dcr orthologues of other 20 platyhelminthes. Our findings showed a close phylogenetic relationship of TcAGO and TcDcr with those previously described for Echinococcus spp. Our RT-PCR studies demonstrated expression of all TcAGO and TcDcr orthologues. Our results show that the gene silencing machinery in T. crassiceps is functionally active by inducing silencing of TcEnoA (∼90%). These results clearly show that gene silencing using siRNAs can be used as a molecular methodology to study gene function in taeniid cestodes.

Isothermal recombinase polymerase amplification-lateral flow detection of SARS-CoV-2, the etiological agent of COVID-19.

The rapid detection of novel pathogens including SARS-CoV-2 necessitates the development of easy-to-use diagnostic tests that can be readily adapted and utilized in both clinical laboratories and field settings. Delay in diagnosis has facilitated the rapid spread of this novel virus throughout the world resulting in global mortality that will surpass 2.5 million people. Development of point-of-care diagnostic assays that can be performed in rural or decentralized health care centers to expand testing capacity is needed. We developed a qualitative test based on recombinase-polymerase-amplification coupled with lateral flow reading (RPA-LF) for rapid detection of SARS-CoV-2. The RPA-LF detected SARS-CoV-2 with a limit of detection of 35.4 viral cDNA nucleocapsid (N) gene copies/µL. Additionally, the RPA-LF was able to detect 0.25-2.5 copies/µL of SARS-CoV-2 N gene containing plasmid. We evaluated 37 nasopharyngeal samples using CDC's N3, N1 and N2 RT-real-time PCR assays for SARS-CoV-2 as reference test. We found a 100 % concordance between RPA-LF and RT-qPCR reference test as determined by 18/18 positive and 19/19 negative samples. All positive samples had Ct values between 19-37 by RT-qPCR. The RPA-LF primers and probe did not cross react with other relevant betacoronaviruses such as SARS and MERS. This is the first isothermal amplification test paired with lateral flow developed for qualitative detection of COVID-19 allowing rapid viral detection and with prospective applicability in resource limited and decentralized laboratories.

Direct RT-PCR amplification of SARS-CoV-2 from clinical samples using a concentrated viral lysis-amplification buffer prepared with IGEPAL-630.

The pandemic of 2019 caused by the novel coronavirus (SARS-CoV-2) is still rapidly spreading worldwide. Nucleic acid amplification serves as the gold standard method for confirmation of COVID-19 infection. However, challenges faced for diagnostic laboratories from undeveloped countries includes shortage of kits and supplies to purify viral RNA. Therefore, it is urgent to validate alternative nucleic acid isolation methods for SARS-CoV-2. Our results demonstrate that a concentrated viral lysis amplification buffer (vLAB) prepared with the nonionic detergent IGEPAL enables qualitative detection of SARS-CoV-2 by direct Reverse Transcriptase-Polymerase Chain Reaction (dRT-PCR). Furthermore, vLAB was effective in inactivating SARS-CoV-2. Since this method is inexpensive and no RNA purification equipment or additional cDNA synthesis is required, this dRT-PCR with vLAB should be considered as an alternative method for qualitative detection of SARS-CoV-2.

Diagnostic Efficacy of Recombinase-Polymerase-Amplification Coupled with Lateral Flow Strip Reading in Patients with Cutaneous Leishmaniasis from the Amazonas Rainforest of Perú.

Cutaneous leishmaniasis (CL) is highly prevalent in rural and sylvatic regions of Latin America, with an estimated 55,000 annual cases. Diagnosis in resource-limited areas still relies on microscopy of dermal scrapings, while more sensitive methods like PCR are not attainable due to costs and lack of adequate health infrastructure. Isothermal amplification of Leishmania DNA can be performed without sophisticated equipment and training and may become a point of care (POC) test for health care centers with scarce resources. We evaluated the efficacy of recombinase-polymerase-amplification (RPA-LF) to diagnose CL in 226 patients attending a clinic in Puerto Maldonado within the Peruvian Amazon basin. Conventional PCR targeting kinetoplast DNA (kDNA-PCR) was used as the gold standard. Eight of 226 patients were considered true negatives (microscopy, kDNA-PCR, and RPA-LF negative), while RPA-LF resulted positive in 186 of 204 kDNA-PCR positive patients, yielding 91.2% (confidence interval [CI] = 86.5-94.4%) sensitivity and 93% (CI 88.6-95.8%) positive predictive value. There were 14% (32/226) discrepant samples alternating positive and negative results in similar proportions between both tests. Quantitative PCR used to resolve the discrepancies suggested that they occurred in samples with scarce parasite numbers as determined by high cycle threshold (Ct) values (≥32; cutoff 35.5). Microscopy had the lowest sensitivity of all methods (45.4%). Nested real-time PCR performed in 71 samples determined that Leishmania (Viannia) braziliensis was highly prevalent (69/71), and Leishmania (Viannia) lainsoni was present in only two isolates. Results indicated that RPA-LF has POC potential for CL endemic areas, yet further simplification and optimization coupled with field validation will be necessary to confirm its broad applicability.

A Novel Method to Silence Genes in Cryptosporidium.

The parasites of the genus Cryptosporidium are important causes of diarrheal diseases, specifically cryptosporidiosis, worldwide. A major bottleneck for developing drugs and vaccines against cryptosporidiosis is the lack of methods to study gene function in this parasite. Silencing of genes by RNA interference (RNAi) is a powerful method to investigate gene function that has been widely used in the identification of targets for several pathogens. Unfortunately, as Cryptosporidium does not possess the enzymes of the RNAi pathway, its genes cannot be silenced by standard siRNA technology. To circumvent that problem, we have developed a novel strategy to knock down Cryptosporidium genes by reconstituting the effector arm of the siRNA pathway. We have induced silencing of several genes in Cryptosporidium by transfecting parasites with hybrid complexes formed between recombinant human Argonaute (hAgo2) and Cryptosporidium single-stranded RNA (ssRNA). This novel methodology provides an effective strategy to study the role of selected genes in host-parasite interactions, and also can be used to identify potential targets for chemotherapy. The standardized methodology based on this strategy is described in this chapter.

Novel drug targets for treatment of cryptosporidiosis.

Introduction Cryptosporidium species are protozoan parasites that are important causes of diarrheal disease including waterborne outbreaks, childhood diarrhea in resource-poor countries, and diarrhea in compromised hosts worldwide. Recent studies highlight the importance of cryptosporidiosis in childhood diarrhea, malnutrition, and death in resource-poor countries. Despite this, only a single drug, nitazoxanide, has demonstrated efficacy in human cryptosporidiosis and its efficacy is limited in malnourished children and patients with HIV. Areas covered In this review, we highlight work on potential targets for chemotherapy and review progress on drug development. A number of new targets have been identified for chemotherapy and progress has been made at developing drugs for these targets. Targets include parasite kinases, nucleic acid synthesis and processing, proteases, and lipid metabolism. Other groups have performed high-throughput screening to identify potential drugs. Several compounds have advanced to large animal studies. Expert opinion Development of drugs for cryptosporidiosis has been plagued by a lack of success. Barriers have included poor correlations between in vitro activity and clinical success as well as frequent unanticipated adverse effects. Without a clear pathway forward, it is wise to maintain a diverse development pipeline. Drug developers should also realize that success will likely require a sustained, methodical effort.

Cryptosporidium parvum cyclic GMP-dependent protein kinase (PKG): An essential mediator of merozoite egress.

Cryptosporidiosis is an obligate intracellular pathogen causing diarrhea. Merozoite egress is essential for infection to spread between host cells. However, the mechanisms of egress have yet to be defined. We hypothesized that Cyclic GMP-Dependent Protein Kinase G (PKG) may be involved in Cryptosporidium egress. In this study, Cryptosporidium parvum PKG was silenced by using antisense RNA sequences. PKG-silencing significantly inhibited egress of merozoites from infected HCT-8 cells into the supernatant and led to retention of intracellular forms within the host cells. This data identifies PKG as a key mediator of merozoite egress, a key step in the parasite lifecycle.