Ac-HSP20 regulates autophagy and promotes the encystation of Acanthamoeba castellanii by inhibiting the PI3K/AKT/mTOR signaling pathway.

BACKGROUND: The encystation of Acanthamoeba castellanii has important ecological and medical significance. Blocking encystation is the key to preventing transmission and curing infections caused by A. castellanii. The formation of autophagosomes is one of the most important changes that occur during the encystation of Acanthamoeba. Our previous studies have shown that the heat shock protein 20 of A. castellanii (Ac-HSP20) is involved in its encystation. This study aimed to determine the role and mechanism of Ac-HSP20 in regulating autophagy involved in the encystation of A. castellanii. METHODS: Immunofluorescence assay, western blotting and transmission electron microscopy were used to analyze the dynamic changes in autophagy during the initiation and continuation of encystation. The knockdown of Ac-HSP20 was performed to clarify its regulation of encystation and autophagy and to elucidate the molecular mechanism by which Ac-HSP20 participates in autophagy to promote cyst maturation. RESULTS: The encystation rates and autophagosomes were significantly decreased by treatment with the autophagy inhibitor 3-MA. The autophagy marker LC3B and autophagic lysosomes increased with the induced duration of encystation and reached the maximum at 48 h. The encystation rate, LC3B expression and autophagosomes decreased when Ac-HSP20 was knocked down by siRNA transfection. In addition, the expression levels of Ac-HSP20 and LC3B increased and the expressions of p-AKT and p-mTOR decreased after 48 h of encystation without knockdown. However, the expressions of p-AKT and p-mTOR increased while the expression of LC3B decreased under the knockdown of Ac-HSP20. Furthermore, the protein expression of LC3B increased when the PI3K/AKT/mTOR signaling pathway was inhibited but decreased when the pathway was activated. CONCLUSIONS: The results demonstrated that autophagy is positively correlated with the encystation of A. castellanii, and Ac-HSP20 regulates autophagy to maintain the homeostasis of A. castellanii by inhibiting the PI3K /AKT /mTOR signaling pathway, thus promoting the maturation and stability of encystation.

Lysine methyltransferase 2 plays a key role in the encystation process in the parasite Giardia lamblia.

Histone post-translational modifications are extensively studied for their role in regulating gene transcription and cellular environmental adaptation. Research into these modifications has recently begun in the protozoan parasite Giardia lamblia, focusing on histone-modifying enzymes and specific post-translational changes. In the transformation from the trophozoite to the cyst form in the life cycle of this parasite, significant morphological and genetic alterations occur, culminating in the synthesis of cyst wall proteins responsible for forming the protective cyst wall. It has been previously demonstrated that histone deacetylation is required during encystation and that the enzyme lysine methyltransferase 1 is involved in the upregulation of encystation. Our study aims to extend the analysis to lysine methyltransferase 2 (GlKMT2) function. For this, two constructs were generated: one that downregulate the expression of GLKMT2 via antisense (glkmt2-as transgenic cells) and the other overexpressing GlKMT2 (glkmt2-ha transgenic cells). We found that the glktm2-as transgenic cells showed an arrest in progress at the late encystation stage. Consequently, the number of cysts produced was lower than that of the control cells. On the other hand, we found that the overexpression of GlKMT2 acts as a negative mutant of the enzyme. In this way, these glktm2-ha transgenic cells showed the same behavior during growth and encystation as glkmt2-as transgenic cells. This interplay between different enzymes acting during encystation reveals the complex process behind the differentiation of the parasite. Understanding how these enzymes play their role during the encystation of the parasite would allow the design of inhibitors to control the parasite.

A cell-cycle-dependent GARP-like transcriptional repressor regulates the initiation of differentiation in Giardia lamblia.

Transcriptional regulation of differentiation is critical for parasitic pathogens to adapt to environmental changes and regulate transmission. In response to encystation stimuli, Giardia lamblia shifts the distribution of the cell cycle toward G2 and induces the expression of cyst wall proteins (CWPs) within 2 to 4 h, indicating that key regulatory steps occur within the first 4 h of encystation. However, the role of transcription factors (TFs) in encystation has primarily been investigated at later time points. How TFs initiate encystation and link it to the cell cycle remains enigmatic. Here, we systematically screened six putative early up-regulated TFs for nuclear localization, established their dynamic expression profiles, and determined their functional role in regulating encystation. We found a critical repressor, Golden2, ARR-B, Psr-1­like protein 1 (GARP)­like protein 4 (GLP4), that increases rapidly after 30 min of encystation stimuli and down-regulates encystation-specific markers, including CWPs and enzymes in the cyst N-acetylgalactosamine pathway. Depletion of GLP4 increases cyst production. Importantly, we observe that G2+M cells exhibit higher levels of CWP1, resulting from the activation of myeloblastosis domain protein 2 (MYB2), a TF previously linked to encystation in Giardia. GLP4 up-regulation occurs in G1+S cells, suggesting a role in repressing MYB2 and encystation-specific genes in the G1+S phase of the cell cycle. Furthermore, we demonstrate that depletion of GLP4 up-regulates MYB2 and promotes encystation while overexpression of GLP4 down-regulates MYB2 and represses encystation. Together, these results suggest that Giardia employs a dose-dependent transcriptional response that involves the cell-cycle­regulated repressor GLP4 to orchestrate MYB2 and entry into the encystation pathway.

A Detailed Gene Expression Map of Giardia Encystation.

Giardia intestinalis is an intestinal protozoan parasite that causes diarrheal infections worldwide. A key process to sustain its chain of transmission is the formation of infectious cysts in the encystation process. We combined deep RNAseq of a broad range of encystation timepoints to produce a high-resolution gene expression map of Giardia encystation. This detailed transcriptomic map of encystation confirmed a gradual change of gene expression along the time course of encystation, showing the most significant gene expression changes during late encystation. Few genes are differentially expressed early in encystation, but the major cyst wall proteins CWP-1 and -2 are highly up-regulated already after 3.5 h encystation. Several transcription factors are sequentially up-regulated throughout the process, but many up-regulated genes at 7, 10, and 14 h post-induction of encystation have binding sites in the upstream regions for the Myb2 transcription factor, suggesting that Myb2 is a master regulator of encystation. We observed major changes in gene expression of several meiotic-related genes from 10.5 h of encystation to the cyst stage, and at 17.5 h encystation, there are changes in many different metabolic pathways and protein synthesis. Late encystation, 21 h to cysts, show extensive gene expression changes, most of all in VSP and HCMP genes, which are involved in antigenic variation, and genes involved in chromatin modifications. This high-resolution gene expression map of Giardia encystation will be an important tool in further studies of this important differentiation process.

Signaling in temperature-induced resting cyst formation in the ciliated protozoan Colpoda cucullus.

The terrestrial ciliated protozoan Colpoda cucullus inhabits soil. When the habitat conditions become unfavorable, the vegetative cells of C. cucullus quickly transform into resting cysts. C. cucullus culture is established in our laboratory, and encystment is routinely induced by the addition of Ca2+ to overpopulated vegetative cells. However, an increase in Ca2+ concentration and overpopulation of vegetative cells do not always occur in natural. We investigated the effect of temperature and found that cyst formation was induced by a rapid increase of 5 °C within 2 min but not by a decrease. Moreover, an increase in intracellular Ca2+ concentrations is essential, but Ca2+ inflow does not necessarily occur during encystment. Ca2+ image analysis showed that Ca2+ is stored in vesicular structures and released into the cytoplasm within 60 s after temperature stimulation. Multiple signaling pathways are activated after the release of Ca2+ from vesicles, and cAMP is a candidate second messenger with a crucial role in the process of temperature-induced encystment. Further studies are needed to clarify the mechanism underlying the sensing of temperature and release of Ca2+ from vesicles.

Stage-Specific De Novo Synthesis of Very-Long-Chain Dihydroceramides Confers Dormancy to Entamoeba Parasites.

Amoebiasis is a parasitic disease caused by Entamoeba histolytica infection and is a serious public health problem worldwide due to ill-prepared preventive measures as well as its high morbidity and mortality rates. Amoebiasis transmission is solely mediated by cysts. Cysts are produced by the differentiation of proliferative trophozoites in a process termed "encystation." Entamoeba encystation is a fundamental cell differentiation process and proceeds with substantial changes in cell metabolites, components, and morphology, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites that function as key factors for encystation. However, a comprehensive lipid analysis has not been reported, and the involved lipid metabolic pathways remain largely unknown. Here, we exploited the state-of-the-art untargeted lipidomics and characterized 339 molecules of 17 lipid subclasses. Of these, dihydroceramide (Cer-NDS) was found to be among the most induced lipid species during encystation. Notably, in encysting cells, amounts of Cer-NDS containing very long N-acyl chains (≥26 carbon) were more than 30-fold induced as the terminal product of a de novo metabolic pathway. We also identified three ceramide synthase genes responsible for producing the very-long-chain Cer-NDS molecules. These genes were upregulated during encystation. Furthermore, these ceramide species were shown to be indispensable for generating membrane impermeability, a prerequisite for becoming dormant cyst that shows resistance to environmental assault inside and outside the host for transmission. Hence, the lipid subclass of Cer-NDS plays a crucial role for Entamoeba cell differentiation and morphogenesis by alternating the membrane properties.IMPORTANCEEntamoeba is a protozoan parasite that thrives in its niche by alternating its two forms between a proliferative trophozoite and dormant cyst. Cysts are the only form able to transmit to a new host and are differentiated from trophozoites in a process termed "encystation." During Entamoeba encystation, cell metabolites, components, and morphology drastically change, which occur sequentially in an orchestrated manner. Lipids are plausibly among these metabolites. However, the involved lipid species and their metabolic pathways remain largely unknown. Here, we identified dihydroceramides (Cer-NDSs) containing very long N-acyl chains (C26 to C30) as a key metabolite for Entamoeba encystation by our state-of-the-art untargeted lipidomics. We also showed that these Cer-NDSs are critical to generate the membrane impermeability, a prerequisite for this parasite to show dormancy as a cyst that repels substances and prevents water loss. Hence, ceramide metabolism is essential for Entamoeba to maintain the parasitic lifestyle.

Ultra-structural analysis and morphological changes during the differentiation of trophozoite to cyst in Entamoeba invadens.

Entamoeba histolytica, a pathogenic parasite, is the causative organism of amoebiasis and uses human colon to complete its life cycle. It destroys intestinal tissue leading to invasive disease. Since it does not form cyst in culture medium, a reptilian parasite Entamoeba invadens serves as the model system to study encystation. Detailed investigation on the mechanism of cyst formation, information on ultra-structural changes and cyst wall formation during encystation are still lacking in E. invadens. Here, we used electron microscopy to study the ultrastructural changes during cyst formation and showed that the increase in heterochromatin patches and deformation of nuclear shape were early events in encystation. These changes peaked at ∼20 h post induction, and normal nuclear morphology was restored by 72 h. Two types of cellular structures were visible by 16 h. One was densely stained and consisted of the cytoplasmic mass with clearly visible nucleus. The other consisted of membranous shells with large vacuoles and scant cytoplasm. The former structure developed into the mature cyst while the latter structure was lost after 20 h, This study of ultra-structural changes during encystation in E. invadens opens up the possibilities for further investigation into the mechanisms involved in this novel process.

A myeloid leukemia factor homolog involved in encystation-induced protein metabolism in Giardia lamblia.

BACKGROUND: Giardia lamblia differentiates into resistant cysts as an established model for dormancy. Myeloid leukemia factor (MLF) proteins are important regulators of cell differentiation. Giardia possesses a MLF homolog which was up-regulated during encystation and localized to unknown cytosolic vesicles named MLF vesicles (MLFVs). METHODS: We used double staining for visualization of potential factors with role in protein metabolism pathway and a strategy that employed a deletion mutant, CDK2m3, to test the protein degradation pathway. We also explored whether autophagy or proteasomal degradation are regulators of Giardia encystation by treatment with MG132, rapamycin, or chloroquine. RESULTS: Double staining of MLF and ISCU or CWP1 revealed no overlap between their vesicles. The aberrant CDK2m3 colocalized with MLFVs and formed complexes with MLF. MG132 increased the number of CDK2m3-localized vesicles and its protein level. We further found that MLF colocalized and interacted with a FYVE protein and an ATG8-like (ATG8L) protein, which were up-regulated during encystation and their expression induced Giardia encystation. The addition of MG132, rapamycin, or chloroquine, increased their levels and the number of their vesicles, and inhibited the cyst formation. MLF and FYVE were detected in exosomes released from culture. CONCLUSIONS: The MLFVs are not mitosomes or encystation-specific vesicles, but are related with degradative pathway for CDK2m3. MLF, FYVE, and ATG8L play a positive role in encystation and function in protein clearance pathway, which is important for encystation and coordinated with Exosomes. GENERAL SIGNIFICANCE: MLF, FYVE, and ATG8L may be involved an encystation-induced protein metabolism during Giardia differentiation.

The Acanthamoeba SBDS, a cytoskeleton-associated gene, is highly expressed during phagocytosis and encystation.

BACKGROUND/PURPOSE: Shwachman-Bodian-Diamond syndrome (SBDS) protein is widely present in eukaryotes from vertebrates to protozoa. However, there are several variants within species, and previous studies have shown evidence that they may have additional functions. There are two SBDS-related proteins in Acanthamoeba. One is an rRNA metabolism protein of the SBDS family (ACA1_142090), and the other is SBDS (ACA1_204560). Although there is a conserved SBDS domain in the Acanthamoeba SBDS (ACA1_204560; AcSBDS), its function has not been reported. The aims of this study were to characterize the expression of AcSBDS during phagocytosis and encystation. METHODS: AcSBDS-specific primer was designed to amplify the genomic AcSBDS of Acanthamoeba ATCC-30010. The AcSBDS expression was examined using reverse transcription polymerase chain reaction (RT-PCR) and immunostaining after phagocytosis and encystation treatment. RESULTS: In this study, we found that the mRNA expression level of AcSBDS increased rapidly and that alternative splice variants were detected during phagocytosis and encystation processes. The results of immunofluorescence staining showed that the AcSBDS proteins accumulated surrounding phagocytosed bacteria. CONCLUSION: Our results suggest that AcSBDS may not only have ribosomal maturation features but also have cytoskeleton-associated functions related to phagocytosis and encystation.

Identification and biochemical characterisation of Acanthamoeba castellanii cysteine protease 3.

BACKGROUND: Acanthamoeba spp. are free-living amoeba that are ubiquitously distributed in the environment. This study examines pathogenic Acanthamoeba cysteine proteases (AcCPs) belonging to the cathepsin L-family and explores the mechanism of AcCP3 interaction with host cells. METHODS: Six AcCP genes were amplified by polymerase chain reaction (PCR). Quantitative real-time PCR was used to analyse the relative mRNA expression of AcCPs during the encystation process and between pre- and post-reactivated trophozoites. To further verify the role of AcCP3 in these processes, AcCP3 recombinant proteins were expressed in Escherichia coli, and the hydrolytic activity of AcCP3 was determined. The influence of the AcCP3 on the hydrolytic activity of trophozoites and the toxicity of trophozoites to human corneal epithelial cells (HCECs) was examined by inhibiting AcCP3 expression using siRNA. Furthermore, the levels of p-Raf and p-Erk were examined in HCECs following coculture with AcCP3 gene knockdown trophozoites by Western blotting. RESULTS: During encystation, five out of six AcCPs exhibited decreased expression, and only AcCP6 was substantially up-regulated at the mRNA level, indicating that most AcCPs were not directly correlated to encystation. Furthermore, six AcCPs exhibited increased expression level following trophozoite reactivation with HEp-2 cells, particularly AcCP3, indicating that these AcCPs might be virulent factors. After refolding of recombinant AcCP3 protein, the 27 kDa mature protein from the 34 kDa pro-protein hydrolysed host haemoglobin, collagen and albumin and showed high activity in an acidic environment. After AcCP3 knockdown, the hydrolytic activity of trophozoite crude protein against gelatin was decreased, suggesting that these trophozoites had decreased toxicity. Compared with untreated trophozoites or negative control siRNA-treated trophozoites, AcCP3-knockdown trophozoites were less able to penetrate and damage monolayers of HCECs. Western blot analysis showed that the activation levels of the Ras/Raf/Erk/p53 signalling pathways in HCECs decreased after inhibiting the expression of trophozoite AcCP3. CONCLUSIONS: AcCP6 was correlated to encystation. Furthermore, AcCP3 was a virulent factor in trophozoites and participated in the activation of the Ras/Raf/Erk/p53 signalling pathways of host cells.

The dynamics of ultrastructural changes during Entamoeba invadens encystation.

Entamoeba histolytica infection causes amoebiasis, which is a global public health problem. The major route of infection is oral ingestion of E. histolytica cysts, cysts being the sole form responsible for host-to-host transmission. Cysts are produced by cell differentiation from proliferative trophozoites in a process termed 'encystation'. Therefore, encystation is an important process from a medical as well as a biological perspective. Previous electron microscopy studies have shown the ultrastructure of precysts and mature cysts; however, the dynamics of ultrastructural changes during encystation were ambiguous. Here, we analysed a series of Entamoeba invadens encysting cells by transmission electron microscopy. Entamoeba invadens is a model for encystation and the cells were prepared by short interval time course sampling from in vitro encystation-inducing cultures. We related sampled cells to stage conversion, which was monitored in the overall population by flow cytometry. The present approach revealed the dynamics of ultrastructure changes during E. invadens encystation. Importantly, the results indicate a functional linkage of processes that are crucial in encystation, such as glycogen accumulation and cyst wall formation. Hence, this study provides a reference for studying sequential molecular events during Entamoeba encystation.

Transcriptomic Features of Echinococcus granulosus Protoscolex during the Encystation Process.

Cystic echinococcosis (CE) is a zoonotic infection caused by Echinococcus granulosus larvae. It seriously affects the development of animal husbandry and endangers human health. Due to a poor understanding of the cystic fluid formation pathway, there is currently a lack of innovative methods for the prevention and treatment of CE. In this study, the protoscoleces (PSCs) in the encystation process were analyzed by high-throughput RNA sequencing. A total of 32,401 transcripts and 14,903 cDNAs revealed numbers of new genes and transcripts, stage-specific genes, and differently expressed genes. Genes encoding proteins involved in signaling pathways, such as putative G-protein coupled receptor, tyrosine kinases, and serine/threonine protein kinase, were predominantly up-regulated during the encystation process. Antioxidant enzymes included cytochrome c oxidase, thioredoxin glutathione, and glutathione peroxidase were a high expression level. Intriguingly, KEGG enrichment suggested that differentially up-regulated genes involved in the vasopressin-regulated water reabsorption metabolic pathway may play important roles in the transport of proteins, carbohydrates, and other substances. These results provide valuable information on the mechanism of cystic fluid production during the encystation process, and provide a basis for further studies on the molecular mechanisms of growth and development of PSCs.

Characterization of Extracellular Vesicles from Entamoeba histolytica Identifies Roles in Intercellular Communication That Regulates Parasite Growth and Development.

Extracellular vesicles (EVs) secreted by eukaryotic and prokaryotic cells to transport lipids, proteins, and nucleic acids to the external environment have important roles in cell-cell communication through cargo transfer. We identified and characterized EVs from Entamoeba histolytica, a protozoan parasite and a human pathogen. Conditioned medium from amebic parasites contained particles consistent with the expected size and morphology of EVs. Mass spectrometry was used to characterize the EV proteome and showed that it was enriched in common exosome marker proteins, including proteins associated with vesicle formation, cell signaling, and metabolism, as well as cytoskeletal proteins. Additionally, the EVs were found to selectively package small RNAs (sRNA), which were protected within the vesicles against RNase treatment. Sequencing analysis of the sRNA contained in EVs revealed that the majority were 27 nucleotides (nt) in size and represented a subset of the cellular antisense small RNA population that has previously been characterized in Entamoeba RNA interference (RNAi) pathway proteins, including Argonaute, were also present in amebic EVs. Interestingly, we found that the amebic EVs impacted intercellular communication between parasites and altered encystation efficiency. EVs isolated from encysting parasites promoted encystation in other parasites, whereas EVs from metabolically active trophozoites impeded encystation. Overall, the data reveal that Entamoeba secrete EVs that are similar in size and shape to previously characterized exosomes from other organisms and that these EVs contain a defined protein and small RNA cargo and have roles in intercellular communication among parasites and influence growth kinetics.

The role of the Acanthamoeba castellanii Sir2-like protein in the growth and encystation of Acanthamoeba.

BACKGROUND: The encystation of Acanthamoeba leads to the development of resilient cysts from vegetative trophozoites. This process is essential for the survival of parasites under unfavorable conditions. Previous studies have reported that, during the encystation of A. castellanii, the expression levels of encystation-related factors are upregulated. However, the regulatory mechanisms for their expression during the encystation process remains unknown. Proteins in the sirtuin family, which consists of nicotinamide adenine dinucleotide-dependent deacetylases, are known to play an important role in various cellular functions. In the present study, we identified the Acanthamoeba silent-information regulator 2-like protein (AcSir2) and examined its role in the growth and encystation of Acanthamoeba. METHODS: We obtained the full-length sequence for AcSir2 using reverse-transcription polymerase chain reaction. In Acanthamoeba transfectants that constitutively overexpress AcSir2 protein, SIRT deacetylase activity was measured, and the intracellular localization of AcSir2 and the effects on the growth and encystation of trophozoites were examined. In addition, the sirtuin inhibitor salermide was used to determine whether these effects were caused by AcSir2 overexpression RESULTS: AcSir2 was classified as a class-IV sirtuin. AcSir2 exhibited functional SIRT deacetylase activity, localized mainly in the nucleus, and its transcription was upregulated during encystation. In trophozoites, AcSir2 overexpression led to greater cell growth, and this growth was inhibited by treatment with salermide, a sirtuin inhibitor. When AcSir2 was overexpressed in the cysts, the encystation rate was significantly higher; this was also reversed with salermide treatment. In AcSir2-overexpressing encysting cells, the transcription of cellulose synthase was highly upregulated compared with that of control cells, and this upregulation was abolished with salermide treatment. Transmission electron microscope-based ultrastructural analysis of salermide-treated encysting cells showed that the structure of the exocyst wall and intercyst space was impaired and that the endocyst wall had not formed. CONCLUSIONS: These results indicate that AcSir2 is a SIRT deacetylase that plays an essential role as a regulator of a variety of cellular processes and that the regulation of AcSir2 expression is important for the growth and encystation of A. castellanii.

Molecular survey for cyst-forming coccidia (Toxoplasma gondii, Neospora caninum, Sarcocystis spp.) in Mediterranean periurban micromammals.

Rodents and other micromammals constitute important reservoirs of infectious diseases; their role in the life cycle of apicomplexan parasites such as Toxoplasma gondii, Neospora caninum, and Sarcocystis spp. still needs clarification. In the present study, we analyzed by PCR and Sanger sequencing methods the presence of specific parasite DNA within brain and heart tissues of 313 individuals of five synanthropic small mammal species (Apodemus sylvaticus, Mus spretus, M. musculus, Rattus rattus, and Crocidura russula) collected in Barcelona metropolitan area (NE Spain). In addition, PCR-RFLP and microsatellites were also used as tools for genotypic characterization of T. gondii and N. caninum, respectively. Specific DNA of T. gondii, N. caninum, and Sarcocystis spp. was detected in 0.3% (n = 1), 1.3% (n = 4), and 3.8% (n = 12) of the animals, respectively. No mixed infections were observed. Crocidura russula stood out as the main host for Sarcocystis spp. Toxoplasma gondii-specific DNA detected in a house rat was genetically characterized by PCR-RFLP, presenting type II and III alleles (SAG1 [II], SAG3 [II], GRA6 [II], c22-8 [III], Apico [III]). Also, unsuccessful DNA sequencing and microsatellite typing were attempted in N. caninum-positive samples, which suggested a lack of PCR specificity and open avenues to speculate the host competence of rodents for N. caninum. Likewise, Sarcocystis spp. identity was studied by alignment and phylogenetic analyses of cox1 and 28S rRNA sequences from the 14 positive samples. It resulted in at least three unknown organisms closely similar (95.7-100% cox1-sequence homology) to Sarcocystis pantherophisi from the Eastern rat snake (Pantherophis alleghaniensis) (KU891603), suggesting together with 28S rRNA sequences analyses, three Sarcocystis sp. with a life cycle conformed by rodents as intermediate host (IH) and snakes as definitive hosts (DH) infecting the periurban micromammals surveyed. Prevalence figures found in this first survey carried out in Spain agree with other international studies focused on periurban areas. Further surveys should be conducted in farms and their surroundings in order to unravel the role of wild micromammals in the epidemiology of such protozoan parasites affecting our livestock, and therefore human population.

Antiamoebic activity of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one library against Acanthamoeba castellanii.

Acanthamoeba castellanii is a free-living amoeba which can cause a blinding keratitis and fatal granulomatous amoebic encephalitis. The treatment of Acanthamoeba infections is challenging due to formation of cyst. Quinazolinones are medicinally important scaffold against parasitic diseases. A library of nineteen new 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives was synthesized to evaluate their antiamoebic activity against Acanthamoeba castellanii. One-pot synthesis of 3-aryl-6,7-dimethoxyquinazolin-4(3H)-ones (1-19) was achieved by reaction of 2-amino-4,5-dimethoxybenzoic acid, trimethoxymethane, and different substituted anilines. These compounds were purified and characterized by standard chromatographic and spectroscopic techniques. Antiacanthamoebic activity of these compounds was determined by amoebicidal, encystation, excystation and host cell cytopathogenicity in vitro assays at concentrations of 50 and 100 µg/mL. The IC50 was found to be between 100 and 50 µg/mL for all the compounds except compound 5 which did not exhibit amoebicidal effects at these concentrations. Furthermore, lactate dehydrogenase assay was also performed to evaluate the in vitro cytotoxicity of these compounds against human keratinocyte (HaCaT) cells. The results revealed that eighteen out of nineteen derivatives of quinazolinones significantly decreased the viability of A. castellanii. Furthermore, eighteen out of nineteen tested compounds inhibited the encystation and excystation, as well as significantly reduced the A. castellanii-mediated cytopathogenicity against human cells. Interestingly, while tested against human normal cell line HaCaT keratinocytes, all compounds did not exhibit any overt cytotoxicity. Furthermore, a detailed structure-activity relationship is also studied to optimize the most potent hit from these synthetic compounds. This report presents several potential lead compounds belonging to 3-aryl-6,7-dimethoxyquinazolin-4(3H)-one derivatives for drug discovery against infections caused by Acanthamoeba castellanii.

Temporal expression of Toxoplasma stage-specific genes in brain tissue: coincidence with parasitological and histopathological findings in mice models.

In the intermediate hosts, tachyzoites of T. gondii predominate in the acute stage while bradyzoites persist inside tissue cysts with the potential for reactivation. The two stages exhibit different metabolic and antigenic characters. The present study aimed to investigate temporal expression of Toxoplasma SAG1 and BAG1 genes in the brain tissue and the coincident parasitological and histopathological findings in mice models of toxoplasmosis. The study included group A: mice infected with RH strain and sacrificed 7 days post-infection (p.i.); group B: mice infected with RH strain and treated with sulfamethoxazole-trimethoprim (30 mg/kg/day and 150 mg/kg/day respectively) 24 h p.i. until sacrificed at days 5, 10, or 20 post-treatment; group C: mice infected with ME-49 strain and sacrificed at days 7, 27, 47, or 67 p.i; and group D: mice infected with ME-49 strain and received dexamethasone daily starting at day 68 p.i. and scarified at days 6 or 10 post-treatment. All mice were inspected daily for abnormal physical signs. Peritoneal exudate and brain homogenate were examined for detection of Toxoplasma stages. Brain sections were examined histopathologically. SAG1 and BAG1 gene expression was evaluated using reverse transcription real-time polymerase chain reaction and the ΔΔCt method. Results revealed that marked BAG1 upregulation is consistent with detection of Toxoplasma cysts and degenerative changes while predominance of tachyzoites and inflammatory infiltrate is compatible with SAG1 upregulation. The study sheds light on the potential for using stage-specific gene expression pattern as markers for evaluation of toxoplasmosis disease progression in clinical settings.

Pathological findings in genital organs of bulls naturally infected with Besnoitia besnoiti.

Bulls chronically affected by bovine besnoitiosis can suffer from sterility. There is limited information about the distribution of Besnoitia cysts and their associated lesions within the male genital organs. This work describes the gross and histological abnormalities in the genital organs of 6 bulls chronically infected with Besnoitia besnoiti, including both clinically (n = 4) and subclinically (n = 2) affected cases. Parasitic cysts were observed in the genital organs of all the clinically affected bulls. The tissue cysts were most commonly found within the pampiniform plexus (4/4), where they were often seen within venous vascular walls and associated with vasculitis, followed by epididymis (3/4), tunica albuginea (2/4), and penis (1/4). In decreasing order of their frequency, observed abnormalities included seminiferous tubule degeneration, testicular fibrosis, testicular necrosis, lack of/or diminished numbers of spermatozoa, testicular atrophy, and Leydig cell hyperplasia. Only one of the subclinically infected bulls had few Besnoitia cysts within the pampinoform plexus, which was associated to small areas of necrosis and mineralization in the ipsilateral testicle. Results indicate that Besnoitia cysts and genital abnormalities are frequent in bulls chronically affected by bovine besnoitiosis, while they are mild and scarce in subclinically affected ones. Moreover, present data show that Besnotia-associated testicular lesions can occur without the presence of cysts within the testicular parenchyma. B. besnoiti cysts seem to have a tropism for the vascular structures of the spermatic chord, which may cause testicular abnormalities via vascular damage, reduced blood flow, and/or impaired thermoregulation and subsequently lead to the observed testicular lesions.

Metformin-coated silver nanoparticles exhibit anti-acanthamoebic activities against both trophozoite and cyst stages.

Acanthamoeba castellanii is an opportunistic protozoan responsible for serious human infections including Acanthamoeba keratitis and granulomatous amoebic encephalitis. Despite advances in antimicrobial therapy and supportive care, infections due to Acanthamoeba are a major public concern. Current methods of treatment are not fully effective against both the trophozoite and cyst forms of A. castellanii and are often associated with severe adverse effects, host cell cytotoxicity and recurrence of infection. Therefore, there is an urgent need to develop new therapeutic approaches for the treatment and management of Acanthamoebic infections. Repurposing of clinically approved drugs is a viable avenue for exploration and is particularly useful for neglected and rare diseases where there is limited interest by pharmaceutical companies. Nanotechnology-based drug delivery systems offer promising approaches in the biomedical field, particularly in diagnosis and drug delivery. Herein, we conjugated an antihyperglycemic drug, metformin with silver nanoparticles and assessed its anti-acanthamoebic properties. Characterization by ultraviolet-visible spectrophotometry and atomic force microscopy showed successful formation of metformin-coated silver nanoparticles. Amoebicidal and amoebistatic assays revealed that metformin-coated silver nanoparticles reduced the viability and inhibited the growth of A. castellanii significantly more than metformin and silver nanoparticles alone at both 5 and 10 µM after 24 h incubation. Metformin-coated silver nanoparticles also blocked encystation and inhibited the excystation in Acanthamoeba after 72 h incubation. Overall, the conjugation of metformin with silver nanoparticles was found to enhance its antiamoebic effects against A. castellanii. Furthermore, the pretreatment of A. castellanii with metformin and metformin-coated silver nanoparticles for 2 h also reduced the amoebae-mediated host cell cytotoxicity after 24 h incubation from 73% to 10% at 10 µM, indicating that the drug-conjugated silver nanoparticles confer protection to human cells. These findings suggest that metformin-coated silver nanoparticles hold promise in the improved treatment and management of Acanthamoeba infections.

Toxoplasma gondii metacaspase 2 is an important factor that influences bradyzoite formation in the Pru strain.

Toxoplasma gondii is an important zoonotic protozoan of the phylum Apicomplexa that can infect nearly all warm-blooded animals. The parasite can exist as the interconvertible tachyzoite or bradyzoite forms, leading to acute or latent infection, respectively. No drug has been reported to penetrate the cyst wall and reduce bradyzoite survival and proliferation till now. The transcriptional level of metacaspases 2 (TgMCA2) in T. gondii is significantly upregulated during the formation of bradyzoites in the Pru strain, indicating that it may play an important role in the formation of bradyzoites. To further explore the function of TgMCA2, we constructed a TgMCA2 gene-knockout variant of the Pru strain (Δmca2). Comparative analysis revealed that the proliferative capacity of Pru Δmca2 increased, while the invasion and egressing properties were not affected by the knockout. Further data shows that the tachyzoites of Δmca2 failed to induce differentiation and form bradyzoites in vitro, and the transcriptional levels of some of the bradyzoite-specific genes (such as BAG1, LDH2, and SAG4A) in Δmca2 were significantly lower compared with that in the Pru strain at the bradyzoite stage. In vivo, no cysts were detected in Δmca2-infected mice. Further determination of parasite burden in Δmca2- and Pru-infected mice brain tissue at the genetic level showed that the gene load was significantly lower than that in Pru. In summary, we confirmed that TgMCA2 contributes to the formation of bradyzoites, and could provide an important foundation for the development of attenuated vaccines for the prevention of T. gondii infection.