Toxoplasma gondii chitinase-like protein TgCLP1 regulates the parasite cyst burden.

Toxoplasma, an important intracellular parasite of humans and animals, causes life-threatening toxoplasmosis in immunocompromised individuals. Although Toxoplasma secretory proteins during acute infection (tachyzoite, which divides rapidly and causes inflammation) have been extensively characterized, those involved in chronic infection (bradyzoite, which divides slowly and is surrounded by a cyst wall) remain uncertain. Regulation of the cyst wall is essential to the parasite life cycle, and polysaccharides, such as chitin, in the cyst wall are necessary to sustain latent infection. Toxoplasma secretory proteins during the bradyzoite stage may have important roles in regulating the cyst wall via polysaccharides. Here, we focused on characterizing the hypothetical T. gondii chitinase, chitinase-like protein 1 (TgCLP1). We found that the chitinase-like domain containing TgCLP1 is partially present in the bradyzoite microneme and confirmed, albeit partially, its previous identification in the tachyzoite microneme. Furthermore, although parasites lacking TgCLP1 could convert from tachyzoites to bradyzoites and make an intact cyst wall, they failed to convert from bradyzoites to tachyzoites, indicating that TgCLP1 is necessary for bradyzoite reactivation. Taken together, our findings deepen our understanding of the molecular basis of recrudescence and could contribute to the development of novel strategies for the control of toxoplasmosis.

Detection of Developmental Asexual Stage-Specific RNA Editing Events in Plasmodium falciparum 3D7 Malaria Parasite.

Transcriptional variation has been studied but post-transcriptional modification due to RNA editing has not been investigated in Plasmodium. We investigated developmental stage-specific RNA editing in selected genes in Plasmodium falciparum 3D7. We detected extensive amination- and deamination-type RNA editing at 8, 16, 24, 32, 40, and 46 h in tightly synchronized Plasmodium. Most of the editing events were observed in 8 and 16 h ring-stage parasites. Extensive A-to-G deamination-type editing was detected more during the 16 h ring stage (25%) than the 8 h ring stage (20%). Extensive U-to-C amination-type editing was detected more during the 16 h ring stage (31%) than the 8 h ring stage (22%). In 28S, rRNA editing converted the loop structure to the stem structure. The hemoglobin binding activity of PF3D7_0216900 was also altered due to RNA editing. Among the expressed 28S rRNA genes, PF3D7_0532000 and PF3D7_0726000 expression was higher. Increased amounts of the transcripts of these two genes were found, particularly PF3D7_0726000 in the ring stage and PF3D7_0532000 in the trophozoite and schizont stages. Adenosine deaminase (ADA) expression did not correlate with the editing level. This first experimental report of RNA editing will help to identify the editing machinery that might be useful for antimalarial drug discovery and malaria control.

Combination therapy with oral antiviral and anti-inflammatory drugs improves the efficacy of delayed treatment in a COVID-19 hamster model.

BACKGROUND: Pulmonary infection with SARS-CoV-2 stimulates host immune responses and can also result in the progression of dysregulated and critical inflammation. Throughout the pandemic, the management and treatment of COVID-19 has been continuously updated with a range of antiviral drugs and immunomodulators. Monotherapy with oral antivirals has proven to be effective in the treatment of COVID-19. However, treatment should be initiated in the early stages of infection to ensure beneficial therapeutic outcomes, and there is still room for further consideration on therapeutic strategies using antivirals. METHODS: We studied the therapeutic effects of monotherapy with the oral antiviral ensitrelvir or the anti-inflammatory corticosteroid methylprednisolone and combination therapy with ensitrelvir and methylprednisolone in a delayed dosing model of hamsters infected with SARS-CoV-2. FINDINGS: Combination therapy with ensitrelvir and methylprednisolone improved respiratory conditions and reduced the development of pneumonia in hamsters even when the treatment was started after 2 days post-infection. The combination therapy led to a differential histological and transcriptomic pattern in comparison to either of the monotherapies, with reduced lung damage and down-regulation of expression of genes involved in the inflammatory response. Furthermore, we found that the combination treatment is effective in case of infection with either the highly pathogenic delta or circulating omicron variants. INTERPRETATION: Our results demonstrate the advantage of combination therapy with antiviral and corticosteroid drugs in COVID-19 treatment from the perspective of lung pathology and host inflammatory responses. FUNDING: Funding bodies are described in the Acknowledgments section.

Bovine Piroplasma Populations in the Philippines Characterized Using Targeted Amplicon Deep Sequencing.

Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina's Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera Babesia (n = 58), Theileria (n = 17), Hepatozoon (n = 2), and Sarcocystis (n = 2). The ASV top hits were composed of 10 species: Babesia bovis, B. bigemina, Theileria orientalis, Babesia sp., Hepatozoon canis, Sarcocystis cruzi, T. annulata, T. equi, T. mutans, and Theileria sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.

Field-deployable multiplex detection method of SARS-CoV-2 and influenza virus using loop-mediated isothermal amplification and DNA chromatography.

A novel multiplex loop-mediated isothermal amplification (LAMP) method combined with DNA chromatography was developed for the simultaneous detection of three important respiratory disease-causing viruses: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, and influenza B virus. Amplification was performed at a constant temperature, and a positive result was confirmed by a visible colored band. An in-house drying protocol with trehalose was used to prepare the dried format multiplex LAMP test. Using this dried multiplex LAMP test, the analytical sensitivity was determined to be 100 copies for each viral target and 100-1000 copies for the simultaneous detection of mixed targets. The multiplex LAMP system was validated using clinical COVID-19 specimens and compared with the real-time qRT-PCR method as a reference test. The determined sensitivity of the multiplex LAMP system for SARS-CoV-2 was 71% (95% CI: 0.62-0.79) for cycle threshold (Ct) ≤ 35 samples and 61% (95% CI: 0.53-0.69) for Ct ≤40 samples. The specificity was 99% (95%CI: 0.92-1.00) for Ct ≤35 samples and 100% (95%CI: 0.92-1.00) for the Ct ≤40 samples. The developed simple, rapid, low-cost, and laboratory-free multiplex LAMP system for the two major important respiratory viral diseases, COVID-19 and influenza, is a promising field-deployable diagnosis tool for the possible future 'twindemic, ' especially in resource-limited settings.

COVID-19 Whole-Genome Resequencing with Redundant Tiling PCR and Subtract-Based Amplicon Normalization Successfully Characterized SARS-CoV-2 Variants in Clinical Specimens.

With an increasing number of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) sequences gathered worldwide, we recognize that deletion mutants and nucleotide substitutions that may affect whole-genome sequencing are accumulating. Here, we propose an additional strategy for tiling PCR for whole-genome resequencing, which can make the pipeline robust for mutations at the primer annealing site by a redundant amplicon scheme. We further demonstrated that subtracting overrepresented amplicons from the multiplex PCR products reduced the bias of the next-generation sequencing (NGS) library, resulting in decreasing required sequencing reads per sample. We applied this sequencing strategy to clinical specimens collected in Bangladesh. More than 80% out of the 304 samples were successfully sequenced. Less than 5% were ambiguous nucleotides, and several known variants were detected. With the additional strategies presented here, we believe that whole-genome resequencing of SARS-CoV-2 from clinical samples can be optimized.

Circular Whole-Transcriptome Amplification (cWTA) and mNGS Screening Enhanced by a Group Testing Algorithm (mEGA) Enable High-Throughput and Comprehensive Virus Identification.

Metagenomic next-generation sequencing (mNGS) offers a hypothesis-free approach for pathogen detection, but its applicability in clinical diagnosis, in addition to other factors, remains limited due to complicated library construction. The present study describes a PCR-free isothermal workflow for mNGS targeting RNA, based on a multiple displacement amplification, termed circular whole-transcriptome amplification (cWTA), as the template is circularized before amplification. The cWTA approach was validated with clinical samples and nanopore sequencing. Reads homologous to dengue virus 2 and chikungunya virus were detected in clinical samples from Bangladesh and Brazil, respectively. In addition, the practicality of a high-throughput detection system that combines mNGS and a group testing algorithm termed mNGS screening enhanced by a group testing algorithm (mEGA) was established. This approach enabled significant library size reduction while permitting trackability between samples and diagnostic results. Serum samples of patients with undifferentiated febrile illnesses from Vietnam (n = 43) were also amplified with cWTA, divided into 11 pools, processed for library construction, and sequenced. Dengue virus 2, hepatitis B virus, and parvovirus B19 were successfully detected without prior knowledge of their existence. Collectively, cWTA with the nanopore platform opens the possibility of hypothesis-free on-site comprehensive pathogen diagnosis, while mEGA contributes to the scaling up of sample throughput. IMPORTANCE Given the breadth of pathogens that cause infections, a single approach that can detect a wide range of pathogens is ideal but is impractical due to the available tests being highly specific to a certain pathogen. Recent developments in sequencing technology have introduced mNGS as an alternative that provides detection of a wide-range of pathogens by detecting the presence of their nucleic acids in the sample. However, sequencing library preparation is still a bottleneck, as it is complicated, costly, and time-consuming. In our studies, alternative approaches to optimize library construction for mNGS were developed. This included isothermal nucleic acid amplification and expansion of sample throughput with a group testing algorithm. These methods can improve the utilization of mNGS as a diagnostic tool and can serve as a high-throughput screening system aiding infectious disease surveillance.

Single Cell Transcriptomes of In Vitro Bradyzoite Infected Cells Reveals Toxoplasma gondii Stage Dependent Host Cell Alterations.

Toxoplasma gondii bradyzoites establish chronic infections within their host cells. Recent studies have demonstrated that several parasite effector proteins are translocated to host cells during the bradyzoite stage of chronic infection. To understand the interaction between host cells and bradyzoites at the transcriptomic landscape level, we utilized single-cell RNA-sequencing (scRNA-Seq) to characterize the bradyzoite-induced host cell response. Distinct gene expression profiles were observed in infected host, cells with low parasite mapped reads, and mock (non-exposed) control cells. Gene set enrichment analysis showed that c-Myc and NF-κB signaling and energy metabolic pathways were upregulated by infection. Type I and II interferon response pathways were upregulated in cells with low parasite mapped reads compared to the non-exposed host control cells, and this upregulation effect was reversed in infected cells. Differences were observed in the host cells depending on the differentiation status of the parasites, as determined by BAG1 and SAG1 expression. NF-κB, inflammatory response pathways, and IFN-γ response pathways were downregulated in host cells containing T. gondiiBAG1+/SAG1-, whereas this downregulation effect was reversed in case of T. gondiiBAG1-/SAG1+. We also identified two distinct host cell subsets that contained T. gondiiBAG1+/SAG1-, one of which displayed distinct transcriptomes with upregulated c-Myc expression. Overall, these data clearly demonstrate that host cell transcriptional alteration by bradyzoite infection is different from that of tachyzoite infection, indicating fine-tuning of the host immune response.

TSS-seq of Toxoplasma gondii sporozoites revealed a novel motif in stage-specific promoters.

Toxoplasma gondii is one of the most common zoonotic protozoan parasites. It has three major infectious stages: rapidly multiplying tachyzoites (Tz), slowly replicating bradyzoites (Bz) and a resting/free-living stage, sporozoites (Sz). The regulatory mechanisms governing stage-specific gene expression are not fully understood. Few transcriptional start sites (TSS) are known for Sz. In this study, we obtained TSS of Sz using an oligo-capping method and RNA-seq analysis. We identified 1,043,503 TSS in the Sz transcriptome. These defined 38,973 TSS clusters, of which, 11,925 were expressed in Sz and 1535 TSS differentially expressed in Sz. Based on these data, we defined promoter regions and novel sporozoite stage-specific motifs using MEME. TGTANNTACA was distributed around -55 to -75 regions from each TSS. Interestingly, the same motif was reported in another apicomplexan, Plasmodium berghei, as a cis-element of female-specific gametocyte genes, implying the presence of common regulatory machinery. Further comparative analysis should better define the distribution and function of these elements in other members of this important parasitic phylum.

SARS-CoV-2 Genome Sequences, Including One with an ORF7a Deletion, Obtained from Patients in Bangladesh.

Genomic sequences from a complete SARS-CoV-2 open reading frame (ORF) were obtained from 24 patients diagnosed in May 2020 in Dhaka, Bangladesh. All sequences belonged to clade 20A or 20B, and none were variants of concern. Interestingly, one sequence showed a 161-nucleotide deletion in ORF7a.

Toxoplasma gondii Matrix Antigen 1 Is a Secreted Immunomodulatory Effector.

Our studies on novel cyst wall proteins serendipitously led us to the discovery that cyst wall and vacuolar matrix protein MAG1, first identified a quarter of a century ago, functions as a secreted immunomodulatory effector. MAG1 is a dense granular protein that is found in the parasitophorous vacuolar matrix in tachyzoite vacuoles and the cyst wall and matrix in bradyzoite vacuoles. In the current study, we demonstrated that MAG1 is secreted beyond the parasitophorous vacuole into the host cytosol in both tachyzoites and bradyzoites. Secretion of MAG1 gradually decreases as the parasitophorous vacuole matures, but prominent MAG1 puncta are present inside host cells even at 4 and 6 days following infection. During acute murine infection, Δmag1 parasites displayed significantly reduced virulence and dissemination. In the chronic stage of infection, Δmag1 parasites generated almost no brain cysts. To identify the mechanism behind the attenuated pathology seen with Δmag1 parasites, various immune responses were screened in vitro using bone marrow-derived macrophages (BMDM). Infection of BMDM with Δmag1 parasites induced a significant increase in interleukin 1ß (IL-1ß) secretion, which is a hallmark of inflammasome activation. Heterologous complementation of MAG1 in BMDM cells prevented this Δmag1 parasite-induced IL-1ß release, indicating that secreted MAG1 in host cytosol dampens inflammasome activation. Furthermore, knocking out GRA15 (an inducer of IL-1ß release) in Δmag1 parasites completely inhibited all IL-1ß release by host cells following infection. These data suggest that MAG1 has a role as an immunomodulatory molecule and that by suppressing inflammasome activation, it would favor survival of the parasite and the establishment of latent infection.IMPORTANCEToxoplasma gondii is an Apicomplexan that infects a third of humans, causing encephalitis in AIDS patients and intellectual disabilities in congenitally infected patients. We determined that one of the cyst matrix proteins, MAG1, which had been thought to be an innate structural protein, can be secreted into the host cell and suppress the host immune reaction. This particular immune reaction is initiated by another parasite-secreted protein, GRA15. The intricate balance of inflammasome activation by GRA15 and suppression by MAG1 protects mice from acute death while enabling parasites to disseminate and establish chronic cysts. Our finding contributes to our understanding of how parasites persist in the host and how T. gondii modulates the host immune system.

Nullscript inhibits Cryptosporidium and Toxoplasma growth.

Cryptosporidium and Toxoplasma are parasites that have caused problems worldwide. Cryptosporidium causes severe watery diarrhoea and may be fatal in immunocompromised patients and in infants. Nitazoxanide is the only agent currently approved by the FDA, but its efficacy is limited. Toxoplasmosis is also a problem in the immunocompromised, as currently available treatment options have limited efficacy and patient tolerance can be poor. In the present investigation, we screened libraries of epigenetic compounds to identify those that inhibited C. parvum growth. Nullscript was identified as a compound with an inhibitory effect on C. parvum and T. gondii growth, and was less toxic to host cells. Nullscript was also able to significantly decrease oocyst excretion in C. parvum-infected SCID mice.

Imidazole derivatives as antiparasitic agents and use of molecular modeling to investigate the structure-activity relationship.

Toxoplasmosis is a common parasitic disease caused by Toxoplasma gondii. Limitations of available treatments motivate the search for better therapies for toxoplasmosis. In this study, we synthesized a series of new imidazole derivatives: bis-imidazoles (compounds 1-8), phenyl-substituted 1H-imidazoles (compounds 9-19), and thiopene-imidazoles (compounds 20-26). All these compounds were assessed for in vitro potential to restrict the growth of T. gondii. To explore the structure-activity relationships, molecular analyses and bioactivity prediction studies were performed using a standard molecular model. The in vitro results, in combination with the predictive model, revealed that the imidazole derivatives have excellent selectivity activity against T. gondii versus the host cells. Of the 26 compounds screened, five imidazole derivatives (compounds 10, 11, 18, 20, and 21) shared a specific structural moiety and exhibited significantly high selectivity (> 1176 to > 27,666) towards the parasite versus the host cells. These imidazole derivatives are potential candidates for further studies. We show evidence that supports the antiparasitic action of the imidazole derivatives. The findings are promising in that they reinforce the prospects of imidazole derivatives as alternative and effective antiparasitic therapy as well as providing evidence for a probable biological mechanism.

MAG2, a Toxoplasma gondii Bradyzoite Stage-Specific Cyst Matrix Protein.

Toxoplasma gondii causes a chronic infection that affects a significant portion of the world's population, and this latent infection is the source of reactivation of toxoplasmosis. An attribute of the slowly growing bradyzoite stage of the parasite is the formation of a cyst within infected cells, allowing the parasite to escape the host's immune response. In this study, a new bradyzoite cyst matrix antigen (MAG) was identified through a hybridoma library screen. This cyst matrix antigen, matrix antigen 2 (MAG2), contains 14 tandem repeats consisting of acidic, basic, and proline residues. Immunoblotting revealed that MAG2 migrates at a level higher than its predicted molecular weight, and computational analysis showed that the structure of MAG2 is highly disordered. Cell fractionation studies indicated that MAG2 was associated with both insoluble and soluble cyst matrix material, suggesting that it interacts with the intracyst network (ICN). Examination of the kinetics of MAG2 within the cyst matrix using fluorescence recovery after photobleaching (FRAP) demonstrated that MAG2 does not readily diffuse within the cyst matrix. Kinetic studies of MAG1 demonstrated that this protein has different diffusion kinetics in tachyzoite and bradyzoite vacuoles and that its mobility is not altered in the absence of MAG2. In addition, deletion of MAG2 does not influence growth, cystogenesis, or cyst morphology.IMPORTANCE This report expands on the list of characterized Toxoplasma gondii cyst matrix proteins. Using fluorescence recovery after photobleaching (FRAP), we have shown that matrix proteins within the cyst matrix are not mainly in a mobile state, providing further evidence of how proteins behave within the cyst matrix. Understanding the proteins expressed during the bradyzoite stage of the parasite reveals how the parasite functions during chronic infection.

Validation of a new serum granulocyte-macrophage colony-stimulating factor autoantibody testing kit.

Very recently, a modest but significant efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) inhalation therapy for the treatment of mild to moderate autoimmune pulmonary alveolar proteinosis (aPAP) has been reported. As the ability to measure the level of GM-CSF autoantibody (GMAb) in the serum is required to decide the indication for this therapy, we developed a high-performance GMAb testing kit for clinical use. As the kit succeeded in reducing nonspecific IgG binding to the ELISA plate, the predictive performance shown in the training study to discriminate aPAP patients from healthy subjects was perfect, providing a cut-off value of 1.65 U·mL-1 in 78 patients with aPAP and 90 healthy subjects in an operator-blinded manner using logistic regression analysis. As in the validation study, serum samples from another 213 patients with aPAP were also blinded and evaluated in an operator-blinded manner against external 207 samples from patients with other types of PAP and patients exhibiting various ground-glass opacities on chest high-resolution computed tomography that require discrimination from PAP. The logistic regression analysis of these validation data sets revealed values of 97.6% and 100% for specificity and sensitivity, respectively. Thus, this new GMAb testing kit is reliable for the diagnosis of aPAP and differential diagnosis of other lung diseases.

The Toxoplasma gondii Cyst Wall Interactome.

A characteristic of the latent cyst stage of Toxoplasma gondii is a thick cyst wall that forms underneath the membrane of the bradyzoite vacuole. Previously, our laboratory group published a proteomic analysis of purified in vitro cyst wall fragments that identified an inventory of cyst wall components. To further refine our understanding of the composition of the cyst wall, several cyst wall proteins were tagged with a promiscuous biotin ligase (BirA*), and their interacting partners were screened by streptavidin affinity purification. Within the cyst wall pulldowns, previously described cyst wall proteins, dense granule proteins, and uncharacterized hypothetical proteins were identified. Several of the newly identified hypothetical proteins were validated to be novel components of the cyst wall and tagged with BirA* to expand the model of the cyst wall interactome. Community detection of the cyst wall interactome model revealed three distinct clusters: a dense granule, a cyst matrix, and a cyst wall cluster. Characterization of several of the identified cyst wall proteins using genetic strategies revealed that MCP3 affects in vivo cyst sizes. This study provides a model of the potential protein interactions within the cyst wall and the groundwork to understand cyst wall formation.IMPORTANCE A model of the cyst wall interactome was constructed using proteins identified through BioID. The proteins within this cyst wall interactome model encompass several proteins identified in a prior characterization of the cyst wall proteome. This model provides a more comprehensive understanding of the composition of the cyst wall and may lead to insights on how the cyst wall is formed.

Enrichment and Proteomic Characterization of the Cyst Wall from In Vitro Toxoplasma gondii Cysts.

The tissue cyst of Toxoplasma gondii, found in latent infection, serves a critical role in both transmission and reactivation of this organism. Within infected cells, slowly replicating parasites (bradyzoites) are surrounded by a cyst matrix, cyst wall, and cyst membrane. The cyst wall is clearly delineated by ultrastructural analysis; however, the composition and function of this layer in host-parasite interactions are not fully understood. In order to understand the composition of the cyst wall, a proteomic analysis of purified cyst wall fragments, that were enriched with Percoll gradients and subsequently immunoprecipitated with CST1 antibody, was performed. Known cyst wall proteins, such as CST1, BPK1, MCP4, MAG1, GRA2, GRA3, and GRA5, were identified in this preparation by liquid chromatography-tandem mass spectrometry (LC-MS/MS). In addition, dense granule proteins (GRAs) not previously shown to associate with the cyst wall, as well as uncharacterized hypothetical proteins, were identified in this cyst wall preparation. Several of these hypothetical cyst wall (CST) proteins were epitope tagged, and immunofluorescence assays confirmed their localization as novel cyst matrix and cyst wall proteins. Expression of two of these newly identified cyst wall proteins was eliminated by gene knockout (CST2-KO and CST3-KO). CST2-KO parasites were highly attenuated in virulence and did not establish detectable cyst burdens. This targeted proteomic approach allowed the identification of new components of the cyst wall that probably have roles in the parasite/host interface.IMPORTANCEToxoplasma gondii is a highly prevalent parasite worldwide that presents life-threatening risks to immunocompromised and pregnant individuals. Whereas the life stage responsible for acute infection can be treated, the life stage responsible for chronic infection is refractory to currently available therapeutics. Little is known about the protein composition of the cyst wall, an amorphous structure formed by parasites that is suspected to facilitate persistence within muscle and nervous tissue during chronic (latent) infection. By implementing a refined approach to selectively purify cyst wall fragments, we identified several known and novel cyst wall proteins from our sample preparations. We confirmed the localizations of several proteins from this data set and identified one that is involved in parasite virulence. These data will propel further studies on cyst wall structure and function, leading to therapeutic strategies that can eliminate the chronic infection stage.

Nanoparticles show potential to retard bradyzoites in vitro formation of Toxoplasma gondii.

Toxoplasmosis is a common parasitic disease caused by Toxoplasma gondii (Nicolle et Manceaux, 1908), an obligate parasite capable of infecting a range of cell types in almost all warm-blooded animals. Upon infecting an intermediate host, the parasites differentiate into tachyzoites which rapidly infect host tissues. Usually, the invading parasites are cleared by the immune system and administered drugs, but some tachyzoites differentiate into bradyzoites forming tissue cysts. These tissue cysts could serve as a source for re-infection and exacerbations. Currently, treatment for toxoplasmosis is limited and, moreover, there are no drugs for treating the cystic stage thus rendering toxoplasmosis a global burden. Recently, we demonstrated that inorganic nanoparticles showed promising activity against the tachyzoite stage T. gondii. In the present study, we evaluated nanoparticles for effect on bradyzoite formation in vitro. Data revealed that the nanoparticles limited bradyzoite burden in vitro. Further, the nanoparticles decreased the bradyzoite-specific BAG-1 promoter activity relative to the untreated control under a bradyzoite-inducing culture condition, even though this reduction in BAG-1 promoter activity waned with increasing concentrations of nanoparticles. In contrast, a parallel experiment under normal cell culture conditions showed that the nanoparticle treatment mildly increased the BAG-1 promoter activity relative to the untreated control. Taken together, the findings are evidence that nanoparticles not only possess anti-tachyzoite potential but they also have anti-bradyzoite potential in vitro.

Exploring Amino Acid-Capped Nanoparticles for Selective Anti-Parasitic Action and Improved Host Biocompatibility.

Toxoplasma gondii causes toxoplasmosis, a common infection against which better drugs are needed. Recently, we showed that inorganic nanoparticles have anti-Toxoplasma activity. Here, we sought to enhance the anti-parasitic efficacy and host biocompatibility of these nanoparticles by modifying their surface with amino acids. The amino acids used were selected based on the nutritional requirements of Toxoplasma gondii. Amino acid-capped nanoparticles (amino-NPs) were synthesized, purified, and then screened for anti-Toxoplasma activity in in vitro infection models. The amino-NPs showed enhanced anti-parasitic selectivity as well as improved host biocompatibility. Oxidative stress, modulation of host HIF-1α, and activation of the kynurenine pathway contributed to the anti-parasitic action of the amino-NPs. Our findings provide additional support for the potential use of nanoparticles as innovative anti-parasitic agents. Findings glean additional perspective that highlight prospects of nanoparticles not only as innovative source of anti-parasitic agents but also provide evidence for probable biological mechanism.

Screening of chemical compound libraries identified new anti-Toxoplasma gondii agents.

Toxoplasma gondii is the etiological agent of toxoplasmosis, a common parasitic disease that affects nearly one-third of the human population. The primary infection can be asymptomatic in healthy individuals but may prove fatal in immunocompromised individuals. Available treatment options for toxoplasmosis patients are limited, underscoring the urgent need to identify and develop new therapies. Non-biased screening of libraries of chemical compounds including the repurposing of well-characterized compounds is emerging as viable approach to achieving this goal. In the present investigation, we screened libraries of natural product and FDA-approved compounds to identify those that inhibited T. gondii growth. We identified 32 new compounds that potently inhibit T. gondii growth. Our findings are new and promising, and further strengthen the prospects of drug repurposing as well as the screening of a wide range of chemical compounds as a viable source of alternative anti-parasitic therapeutic agents.