Cryptosporidium life cycle small molecule probing implicates translational repression and an Apetala 2 transcription factor in macrogamont differentiation.

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2_3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation.

A Molecular Dynamics Simulation Study of the Effects of ßGln114 Mutation on the Dynamic Behavior of the Catalytic Site of the Tryptophan Synthase.

L-tryptophan (l-Trp), a vital amino acid for the survival of various organisms, is synthesized by the enzyme tryptophan synthase (TS) in organisms such as eubacteria, archaebacteria, protista, fungi, and plantae. TS, a pyridoxal 5'-phosphate (PLP)-dependent enzyme, comprises α and ß subunits that typically form an α2ß2 tetramer. The enzyme's activity is regulated by the conformational switching of its α and ß subunits between the open (T state) and closed (R state) conformations. Many microorganisms rely on TS for growth and replication, making the enzyme and the l-Trp biosynthetic pathway potential drug targets. For instance, Mycobacterium tuberculosis, Chlamydiae bacteria, Streptococcus pneumoniae, Francisella tularensis, Salmonella bacteria, and Cryptosporidium parasitic protozoa depend on l-Trp synthesis. Antibiotic-resistant salmonella strains have emerged, underscoring the need for novel drugs targeting the l-Trp biosynthetic pathway, especially for salmonella-related infections. A single amino acid mutation can significantly impact enzyme function, affecting stability, conformational dynamics, and active or allosteric sites. These changes influence interactions, catalytic activity, and protein-ligand/protein-protein interactions. This study focuses on the impact of mutating the ßGln114 residue on the catalytic and allosteric sites of TS. Extensive molecular dynamics simulations were conducted on E(PLP), E(AEX1), E(A-A), and E(C3) forms of TS using the WT, ßQ114A, and ßQ114N versions. The results show that both the ßQ114A and ßQ114N mutations increase protein backbone root mean square deviation fluctuations, destabilizing all TS forms. Conformational and hydrogen bond analyses suggest the significance of ßGln114 drifting away from cofactor/intermediates and forming hydrogen bonds with water molecules necessary for l-Trp biosynthesis. The ßQ114A mutation creates a gap between ßAla114 and cofactor/intermediates, hindering hydrogen bond formation due to short side chains and disrupting ß-sites. Conversely, the ßQ114N mutation positions ßAsn114 closer to cofactor/intermediates, forming hydrogen bonds with O3 of cofactors/intermediates and nearby water molecules, potentially disrupting the l-Trp biosynthetic mechanism.

A growth-based assay using fluorescent protein emission to screen for S-adenosylmethionine synthetase inhibitors.

The use of cell growth-based assays to identify inhibitory compounds is straightforward and inexpensive, but is also inherently insensitive and somewhat nonspecific. To overcome these limitations and develop a sensitive, specific cell-based assay, two different approaches were combined. To address the sensitivity limitation, different fluorescent proteins have been introduced into a bacterial expression system to serve as growth reporters. To overcome the lack of specificity, these protein reporters have been incorporated into a plasmid in which they are paired with different orthologs of an essential target enzyme, in this case l-methionine S-adenosyltransferase (MAT, AdoMet synthetase). Screening compounds that serve as specific inhibitors will reduce the growth of only a subset of strains, because these strains are identical, except for which target ortholog they carry. Screening several such strains in parallel not only reveals potential inhibitors but the strains also serve as specificity controls for one another. The present study makes use of an existing Escherichia coli strain that carries a deletion of metK, the gene for MAT. Transformation with these plasmids leads to a complemented strain that no longer requires externally supplied S-adenosylmethionine for growth, but its growth is now dependent on the activity of the introduced MAT ortholog. The resulting fluorescent strains provide a platform to screen chemical compound libraries and identify species-selective inhibitors of AdoMet synthetases. A pilot study of several chemical libraries using this platform identified new lead compounds that are ortholog-selective inhibitors of this enzyme family, some of which target the protozoal human pathogen Cryptosporidium parvum.

Research progress on phosphatidylinositol 4-kinase inhibitors.

Phosphatidylinositol 4-kinases (PI4Ks) could phosphorylate phosphatidylinositol (PI) to produce phosphatidylinositol 4-phosphate (PI4P) and maintain its metabolic balance and location. PI4P, the most abundant monophosphate inositol in eukaryotic cells, is a precursor of higher phosphoinositols and an essential substrate for the PLC/PKC and PI3K/Akt signaling pathways. PI4Ks regulate vesicle transport, signal transduction, cytokinesis, and cell unity, and are involved in various physiological and pathological processes, including infection and growth of parasites such as Plasmodium and Cryptosporidium, replication and survival of RNA viruses, and the development of tumors and nervous system diseases. The development of novel drugs targeting PI4Ks and PI4P has been the focus of the research and clinical application of drugs, especially in recent years. In particular, PI4K inhibitors have made great progress in the treatment of malaria and cryptosporidiosis. We describe the biological characteristics of PI4Ks; summarize the physiological functions and effector proteins of PI4P; and analyze the structural basis of selective PI4K inhibitors for the treatment of human diseases in this review. Herein, this review mainly summarizes the developments in the structure and enzyme activity of PI4K inhibitors.

Indole metabolites generated by microbiota inhibit Cryptosporidium growth.

Funkhouser-Jones et al. recently identified gut metabolites that affected Cryptosporidium growth. A key focus, indole, was shown to inhibit the parasite in vivo and in vitro by decreasing the host mitochondria function and the membrane potential of parasite mitosomes. These findings help clarify the role microflora and metabolites play in host resistance.

Photoswitchable Inhibitors to Optically Control Specific Kinase Activity.

Potent and selective small-molecule inhibitors are valuable tools to elucidate the functions of protein kinases within complex signaling networks. Incorporation of a photoswitchable moiety into the inhibitor scaffold offers the opportunity to steer inhibitor potency with temporal precision, while the challenge of selective inhibition can often be addressed by employing a chemical genetic approach, termed the analog-sensitive method. Here, we combine the perks of these two approaches and report photoswitchable azopyrazoles to target calcium-dependent protein kinase 1 (CDPK1) from Toxoplasma gondii, a kinase naturally susceptible to analog-sensitive kinase inhibitors due to its glycine gatekeeper residue. The most promising azopyrazoles display favorable photochemical properties, thermal stability, and a substantial difference in IC50 values between both photostationary states. Consequently, the CDPK1 kinase reaction can be controlled dynamically and reversibly by applying light of different wavelengths. Inhibition of CDPK1 by the azopyrazoles drastically relies on the nature of the gatekeeper residue as a successive increase in gatekeeper size causes a concurrent loss of inhibitory activity. Furthermore, two photoswitchable inhibitors exhibit activity against T. gondii and Cryptosporidium parvum infection in a cell culture model, making them a promising addition to the toolbox for dissecting the role of CDPK1 in the infectious cycle with high temporal control. Overall, this work merges the benefits of the analog-sensitive approach and photopharmacology without compromising inhibitory potency and thus holds great promise for application to other protein kinases in the future.

The relationship between serum cytokine profile and vitamin D in calves with neonatal diarrhea.

It is important to know the characteristics of the immunological response in newborn calf diarrhea, which is often caused by bacterial, viral and protozoal pathogens. Cytokinesare proteins that serve as chemical messengers to regulate theinnate and adaptive arms of theimmune response. Changes in circulatory cytokine levels provide valuable information for understanding the pathophysiological process and monitoring disease progression and inflammation. Vitamin D has important immunomodulatory effects, which include enhancing the innate immune system and inhibiting adaptative immune responses. The objective of this study was to evaluate the relationship between serum cytokine profile and vitamin D level in neonatal calves with diarrhea. The study population was comprised of 40 neonatal calves, 32 of which had diarrhea and 8 of which were healthy calves. The calves with diarrhea were allocated to four groups according to bacterial (Escherichia coli), viral (Rotavirus, Coronavirus) and protozoal (Cryptosporidium parvum) etiologies. Circulatory vitamin D metabolites (25-hydroxyvitamin D, 1,25-dihydroxyvitamin D) and cytokines (TNF-α, IFN-γ, IL-1ß, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, IL-13 and IL-17) in the calves were determined. There was no statistically significant difference among the groups in 25-hydroxyvitamin D levels. 1,25-dihydroxyvitamin D levels were higher in Coronavirus and E. coli groups compared to the controls. Serum levels of all cytokines except for IL-13, were higher in E. coli group than those of the control group. As a result, differences in serum cytokines and vitamin D levels according to etiological factors in calf diarrhea indicate that vitamin D may play a role in the immune response in the disease.

Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.

An immunoinformatics analysis: design of a multi-epitope vaccine against Cryptosporidium hominis by employing heat shock protein triggers the innate and adaptive immune responses.

Cryptosporidium hominis, an anthropologically transferred species in the Cryptosporidium genus, represents many clinical studies in several countries. Its growth in the recent decade is primarily owing to epidemiologic studies. This parasite has complicated life cycles that require differentiation through a variety of phases of development and passage across two or more hosts throughout their lifetimes. As they move from host to host and environment to environment, pathogenic organisms are continually exposed to unexpected changes in the circumstances under which they develop. Heat shock proteins (HSPs) are targets of the host immune response; they are involved in the progression of diseases and play a significant part in this process. It has been discovered that the immunodominant immunogenic antigens in parasite infections HSPs. In this study, we have generated a multi-epitope vaccine against Cryptosporidium hominis (C. hominis) by using heat shock proteins. The epitopes that were selected had a substantial binding affinity for the B- and T-cell reference set of alleles, a high antigenicity score, a nature that was not allergic, a high solubility, non-toxicity and good binders. The epitopes were incorporated into a chimeric vaccine by using appropriate linkers. In order to increase the immunogenicity of the connected epitopes and effectively activate both innate and adaptive immunity, an adjuvant was attached to the epitopes. We have also analyzed the physiochemical characteristics of the vaccine which were satisfactory and then lead to the development of a 3D model. In addition, the binding confirmation of the vaccine to the TLR-4 innate immune receptor was also determined using molecular docking and molecular dynamics (MD) simulation. The results of this simulation show that the vaccine has a strong binding affinity for TLR4, which indicates that the vaccine is highly effective. In general, the vaccine that has been described here has a good potential for inducing protective and targeted immunogenicity, however, this hypothesis is contingent upon more experimental testing.Communicated by Ramaswamy H. Sarma.

Cryptosporidium parvum maintains intracellular survival by activating the host cellular EGFR-PI3K/Akt signaling pathway.

Autophagy is a critical cellular mechanism in helping infected cells remove intracellular pathogens and is countered by pathogens maintaining intracellular survival by regulating autophagy through the manipulation of the host cellular signal transduction pathway. Cryptosporidium parvum is a zoonotic intracellular but extracytoplasmic protozoon that causes diarrhea in infants and young children worldwide. However, it is still unclear how Cryptosporidium adapts to intracellular survival. In the present study, we demonstrated that C. parvum could activate the EGFR-PI3K/Akt signaling pathway to promote intracellular survival in HCT-8 cells. The western blot results showed that C. parvum induced EGFR and Akt phosphorylation in HCT-8 cells. The EGFR inhibitor AG1478 decreased EGFR and Akt phosphorylation, and the PI3K inhibitor LY294002 impaired Akt phosphorylation induced by C. parvum in HCT-8 cells. Inhibition of EGFR or Akt decreased the number of intracellular parasites. Second, low-dose infection of C. parvum triggered complete autophagy and enhanced autophagic flux in HCT-8 cells. The expressions of mTOR and p62 were decreased, and the expressions of LC3 and Beclin1 were increased in C. parvum-infected HCT-8 cells. Transfection with siBeclin1 or siATG7 reduced LC3 accumulation, while lysosome inhibitor E64d+pepA increased LC3 accumulation induced by C. parvum in HCT-8 cells. Intracellular parasite proliferation was decreased when treated with autophagy inducer rapamycin, whereas autophagy inhibitor 3-MA, E64d+pep A, siBeclin1 or siATG7 increased intracellular parasites. Third, C. parvum inhibited autophagy killing to promote its own intracellular survival by activating EGFR-Akt signaling pathway. The EGFR inhibitor AG1478 enhanced autophagic flux, and Akt inhibitor IV increased LC3 accumulation and inhibited C. parvum proliferation in HCT-8 cells. Akt inhibitor IV-inhibited C. parvum proliferation was attenuated by E64d+pepA. In summary, C. parvum could maintain intracellular survival by inhibiting autophagy via EGFR-PI3K/Akt pathway. These results revealed a new mechanism for the interaction of C. parvum with host cells.

Gut Microbial Perturbation and Host Response Induce Redox Pathway Upregulation along the Gut-Liver Axis during Giardiasis in C57BL/6J Mouse Model.

Apicomplexan infections, such as giardiasis and cryptosporidiosis, negatively impact a considerable proportion of human and commercial livestock populations. Despite this, the molecular mechanisms of disease, particularly the effect on the body beyond the gastrointestinal tract, are still poorly understood. To highlight host-parasite-microbiome biochemical interactions, we utilised integrated metabolomics-16S rRNA genomics and metabolomics-proteomics approaches in a C57BL/6J mouse model of giardiasis and compared these to Cryptosporidium and uropathogenic Escherichia coli (UPEC) infections. Comprehensive samples (faeces, blood, liver, and luminal contents from duodenum, jejunum, ileum, caecum and colon) were collected 10 days post infection and subjected to proteome and metabolome analysis by liquid and gas chromatography-mass spectrometry, respectively. Microbial populations in faeces and luminal washes were examined using 16S rRNA metagenomics. Proteome-metabolome analyses indicated that 12 and 16 key pathways were significantly altered in the gut and liver, respectively, during giardiasis with respect to other infections. Energy pathways including glycolysis and supporting pathways of glyoxylate and dicarboxylate metabolism, and the redox pathway of glutathione metabolism, were upregulated in small intestinal luminal contents and the liver during giardiasis. Metabolomics-16S rRNA genetics integration indicated that populations of three bacterial families-Autopobiaceae (Up), Desulfovibrionaceae (Up), and Akkermanasiaceae (Down)-were most significantly affected across the gut during giardiasis, causing upregulated glycolysis and short-chained fatty acid (SCFA) metabolism. In particular, the perturbed Akkermanasiaceae population seemed to cause oxidative stress responses along the gut-liver axis. Overall, the systems biology approach applied in this study highlighted that the effects of host-parasite-microbiome biochemical interactions extended beyond the gut ecosystem to the gut-liver axis. These findings form the first steps in a comprehensive comparison to ascertain the major molecular and biochemical contributors of host-parasite interactions and contribute towards the development of biomarker discovery and precision health solutions for apicomplexan infections.

Exploration of aminoacyl-tRNA synthetases from eukaryotic parasites for drug development.

Parasitic diseases result in considerable human morbidity and mortality. The continuous emergence and spread of new drug-resistant parasite strains is an obstacle to controlling and eliminating many parasitic diseases. Aminoacyl-tRNA synthetases (aaRSs) are ubiquitous enzymes essential for protein synthesis. The design and development of diverse small molecule, drug-like inhibitors against parasite-encoded and expressed aaRSs have validated this enzyme family as druggable. In this work, we have compiled the progress to date towards establishing the druggability of aaRSs in terms of their biochemical characterization, validation as targets, inhibitor development, and structural interpretation from parasites responsible for malaria (Plasmodium), lymphatic filariasis (Brugia,Wuchereria bancrofti), giardiasis (Giardia), toxoplasmosis (Toxoplasma gondii), leishmaniasis (Leishmania), cryptosporidiosis (Cryptosporidium), and trypanosomiasis (Trypanosoma). This work thus provides a robust framework for the systematic dissection of aaRSs from these pathogens and will facilitate the cross-usage of potential inhibitors to jump-start anti-parasite drug development.

A multi-epitope vaccine candidate developed from unique immunogenic epitopes against Cryptosporidium hominis by utilizing an immunoinformatics-driven approach.

An immunoinformatics-based strategy is being investigated to identify prospective multi-subunit vaccine candidates against Cryptosporidium hominis (C. hominis). We used a systematic technique based on protein structure to create a competent multi-subunit vaccine candidate against C. hominis, with the likelihood of antigenicity, allergenicity, and transmembrane helices as the screening criteria. Using the suitable linkers, the best-screened epitopes such as B-cell epitopes (BCL), Helper T-lymphocytes (HTL), and cytotoxic T-lymphocytes (CTL) were linked together to intensify and develop the presentation and processing of the antigenic molecules. The greatest 3 D model of the component protein was created with the help of modeling software called Raptorax. The validation of the modeled protein was accomplished via the use of PROCHECK. Furthermore, using the ClusPro web server, the projected modeled structure was docked with known receptor TLR-4 to determine their interactions. A molecular dynamics (MD) simulation was used to investigate the stability of the multi-subunit vaccine bound with TLR-4 based on the docking score. Aside from that, the codon optimization and in silico expression demonstrate the possibility of high expression and simple purification of the vaccine product resulting from codon optimization. The overall findings indicated that the multi-subunit vaccine might be a viable vaccination candidate against C. hominis.Communicated by Ramaswamy H. Sarma.

Association between asymptomatic infections and linear growth in 18-24-month-old Malawian children.

Inadequate diet and frequent symptomatic infections are considered major causes of growth stunting in low-income countries, but interventions targeting these risk factors have achieved limited success. Asymptomatic infections can restrict growth, but little is known about their role in global stunting prevalence. We investigated factors related to length-for-age Z-score (LAZ) at 24 months by constructing an interconnected network of various infections, biomarkers of inflammation (as assessed by alpha-1-acid glycoprotein [AGP]), and growth (insulin-like growth factor 1 [IGF-1] and collagen X biomarker [CXM]) at 18 months, as well as other children, maternal, and household level factors. Among 604 children, there was a continuous decline in mean LAZ and increased mean length deficit from birth to 24 months. At 18 months of age, the percentage of asymptomatic children who carried each pathogen was: 84.5% enterovirus, 15.5% parechovirus, 7.7% norovirus, 4.6% rhinovirus, 0.6% rotavirus, 69.6% Campylobacter, 53.8% Giardia lamblia, 11.9% malaria parasites, 10.2% Shigella, and 2.7% Cryptosporidium. The mean plasma IGF-1 concentration was 12.5 ng/ml and 68% of the children had systemic inflammation (plasma AGP concentration >1 g/L). Shigella infection was associated with lower LAZ at 24 months through both direct and indirect pathways, whereas enterovirus, norovirus, Campylobacter, Cryptosporidium, and malaria infections were associated with lower LAZ at 24 months indirectly, predominantly through increased systemic inflammation and reduced plasma IGF-1 and CXM concentration at 18 months.

[miRNA Expression Profile in Ileocecal Adenocarcinoma Cells Infected with Cryptosporidium]. / Cryptosporidium ile Enfekte Edilmis Ileoçekal Adenokarsinom Hücrelerindeki miRNA Ekspresyon Profili.

Cryptosporidium spp. is an opportunistic protozoan transmitted by fecal-oral route via oocysts. The agent may cause severe infection especially in individuals with suppressed immune system, due to its intracellular location and ability to cause auto-infection. MicroRNAs (miRNAs) are non-translated endogenous RNA molecules with an average of 22 nucleotides in length that regulate the expression of genes involved in important biological functions such as proliferation, differentiation, apoptosis and immune response. Recent studies have focused on the role of miRNAs in pathogenesis of infectious diseases and their potential to be used as biomarkers. The aim of this study was to determine the miRNA profile of human ileocecal adenocarcinoma (HCT-8) cells at 24 hours of infection with Cryptosporidium spp. In the study, the HCT-8 cell line was infected with Cryptosporidium spp. that were isolated from infected human stool samples and RNA was isolated from the cells 24 hours after infection. After this process, cDNA synthesis was performed and the expression of 95 human miRNA profiles were investigated by polymerase chain reaction (PCR) method. Fold changes of expression were determined by comparison with Cryptosporidium spp. uninfected cell lines. Sequence information of miRNAs and their target genes were performed via TargetScanHuman7.1 and miRDB websites, while gene ontology (GO) pathways of target genes were analyzed with the mirPath v.3 program. It was detected that the expression of 10 miRNAs were upregulated and 11 of them were downregulated compared with the control group. It was observed that, this 21 differentially expressed miRNAs were mainly associated with apoptosis, mitotic cell cycle, and immune response. Hsa-miR-612, hsa-miR-6763-5p, hsa-miR-188-5p, hsa-miR-664b-3p, hsa-miR-210-3p, hsa-let-7e-5p hsa-let-7b-3p, hsa-miR-4787-3p, hsa-miR-548ab, hsa-miR-3714 and hsamiR-4803 were found to be associated with apoptosis; and hsa-miR-612, hsa-miR-664b-3p, hsa-miR210-3p, hsa-let-7e-5p, hsa-let-7b-3p, hsa-miR-548ab, and hsa-miR4803 were found to be associated with mitotic cell cycle. The balance of proliferation and apoptosis is very significant in the development of infection and cancer. It is thought that determination of the effect of miRNAs on proliferation-apoptosis balance could provide information related to the etiopathogenesis and prognosis of infections, and on the role of microorganisms in carcinogenesis. In this study, 12 differentially expressed miRNAs were found to be associated with immune response. This may emphasize the role of miRNAs in the prevention and treatment of infections. It was concluded that, miRNAs could be used in the diagnosis, treatment and prevention of infections with the determination of miRNA's role in the infection mechanism as a result of the increasing number of studies.

Characterization of Dense Granule Metalloproteinase INS-16 in Cryptosporidium parvum.

The protozoan pathogen Cryptosporidium parvum infects intestinal epithelial cells and causes diarrhea in humans and young animals. Among the more than 20 genes encoding insulinase-like metalloproteinases (INS), two are paralogs with high sequence identity. In this study, one of them, INS-16 encoded by the cgd3_4270 gene, was expressed and characterized in a comparative study of its sibling, INS-15 encoded by the cgd3_4260 gene. A full-length INS-16 protein and its active domain I were expressed in Escherichia coli, and antibodies against the domain I and an INS-16-specific peptide were produced in rabbits. In the analysis of the crude extract of oocysts, a ~60 kDa fragment of INS-16 rather than the full protein was recognized by polyclonal antibodies against the specific peptide, indicating that INS-16 undergoes proteolytic cleavage before maturation. The expression of the ins-16 gene peaked at the invasion phase of in vitro C. parvum culture, with the documented expression of the protein in both sporozoites and merozoites. Localization studies with antibodies showed significant differences in the distribution of the native INS-15 and INS-16 proteins in sporozoites and merozoites. INS-16 was identified as a dense granule protein in sporozoites and macrogamonts but was mostly expressed at the apical end of merozoites. We screened 48 candidate INS-16 inhibitors from the molecular docking of INS-16. Among them, two inhibited the growth of C. parvum in vitro (EC50 = 1.058 µM and 2.089 µM). The results of this study suggest that INS-16 may have important roles in the development of C. parvum and could be a valid target for the development of effective treatments.

DNA topoisomerases in the unicellular protozoan parasites: Unwinding the mystery.

DNA topoisomerases are a group of enzymes present ubiquitously in all organisms from unicellular protozoan parasites to humans. These enzymes control the topological problems caused by DNA double helix in the cell during nucleic acid metabolism. Certain types of topoisomerases present in unicellular parasites are quite different from human topoisomerases (hTop) concerning structure, expression, and function. Many protozoan parasites causing fatal diseases have DNA topoisomerases, which play vital roles in their survival. Given the fact that the structures of the protozoan parasite topoisomerases are different from humans, DNA topoisomerase acts as an essential target for potent drug development for parasitic diseases. Moreover, various studies revealed the therapeutic potential of these drugs targeting the parasitic topoisomerases. Therefore, the characterization of parasitic topoisomerases is pivotal for the development of future potential drug targets. Considering the importance of this ubiquitous enzyme as a potential drug target, we describe in detail all the reported protozoan topoisomerases in an organized manner including Leishmania, Trypanosoma, Plasmodium, Giardia, Entamoeba, Babesia, Theileria, Crithidia, Cryptosporidium, Toxoplasma, etc. This review highlights the unique attributes associated with the structure and function of different types of DNA topoisomerases from the unicellular protozoan parasites. So, it would be beneficial for researchers to obtain awareness about the currently characterized topoisomerases and the ones that need better characterization, understand the structure-function relationship of parasitic topoisomerases, to develop the potent anti-parasitic drugs, and also provides a future platform for therapeutic development.

The Long Non-Coding RNA Nostrill Regulates Transcription of Irf7 Through Interaction With NF-κB p65 to Enhance Intestinal Epithelial Defense Against Cryptosporidium parvum.

The cells of the intestinal epithelium establish the frontline for host defense against pathogens in the gastrointestinal tract and play a vital role in the initiation of the immune response. Increasing evidence supports the role of long non-coding RNAs (lncRNAs) as critical regulators of diverse cellular processes, however, their role in antimicrobial host defense is incompletely understood. In this study, we provide evidence that the lncRNA Nostrill is upregulated in the intestinal epithelium following infection by Cryptosporidium parvum, a globally prevalent apicomplexan parasite that causes significant diarrheal disease and an important opportunistic pathogen in the immunocompromised and AIDS patients. Induction of Nostrill in infected intestinal epithelial cells was triggered by NF-κB signaling and was observed to enhance epithelial defense by decreasing parasitic infection burden. Nostrill participates in the transcriptional regulation of C. parvum-induced Irf7 expression through interactions with NF-κB p65, and induction of Nostrill promotes epigenetic histone modifications and occupancy of RNA polymerase II at the Irf7 promoter. Our data suggest that the induction of Nostrill promotes antiparasitic defense against C. parvum and enhances intestinal epithelial antimicrobial defense through contributions to transcriptional regulation of immune-related genes, such as Irf7.

Construction of a recombinant food-grade Lactococcus lactis expressing P23 protein of Cryptosporidium parvum.

Cryptosporidium parvum infects enterocytes in diverse vertebrates, including humans, and causes diarrheal illness. However, no effective drugs are available for this protozoan infection. The P23 protein of C. parvum is a protective antigen, considered a potential candidate for developing an effective vaccine against cryptosporidiosis. In this study, the complementary DNA (cDNA) of the p23 gene was subcloned to Escherichia coli DH5α, with one nucleotide difference. The constructed plasmid pNZ8149-P23 was transferred by electroporation to Lactococcus lactis NZ3900, and the recombinant L. lactis NZ3900/pNZ8149-P23 strain was screened in Elliker-medium by adding bromocresolpurple indicator. A 23-kDa protein was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) after nisin induction in LM17 broth medium, suggesting that P23 protein was in the form of glycosylation. Simultaneously, an optimal induction time of 9 h was determined, and the density of OD600 = 2.7 was tested. Through western blot and indirect immunofluorescence (IIF) analysis, the immunocompetence of expressed P23 antigen was identified, and its location of release to the cell interior of recombinant L. lactis was manifested. The first report of a food-grade genetically engineered L. lactis strain expressing a P23 antigen of C. parvum is herein presented. This result provides a novel and safe utilization method of P23 against C. parvum infection.

QSAR and deep learning model for virtual screening of potential inhibitors against Inosine 5' Monophosphate dehydrogenase (IMPDH) of Cryptosporidium parvum.

Cryptosporidium parvum (Cp) causes a gastro-intestinal disease called Cryptosporidiosis. C. parvum Inosine 5' monophosphate dehydrogenase (CpIMPDH) is responsible for the production of guanine nucleotides. In the present study, 37 known urea-based congeneric compounds were used to build a 2D and 3D QSAR model against CpIMPDH. The built models were validated based on OECD principles. A deep learning model was adopted from a framework called Deep Purpose. The model was trained with 288 known active compounds and validated using a test set. From the training set of the 3D QSAR, a pharmacophore model was built and the best pharmacophore hypotheses were scored and sorted using a phase-hypo score. A phytochemical database was screened using both the pharmacophore model and a deep learning model. The screened compounds were considered for glide XP docking, followed by quantum polarized ligand docking. Finally, the best compound among them was considered for molecular dynamics simulation study.