Metabolic plasticity, essentiality and therapeutic potential of ribose-5-phosphate synthesis in Toxoplasma gondii.

Ribose-5-phosphate (R5P) is a precursor for nucleic acid biogenesis; however, the importance and homeostasis of R5P in the intracellular parasite Toxoplasma gondii remain enigmatic. Here, we show that the cytoplasmic sedoheptulose-1,7-bisphosphatase (SBPase) is dispensable. Still, its co-deletion with transaldolase (TAL) impairs the double mutant's growth and increases 13C-glucose-derived flux into pentose sugars via the transketolase (TKT) enzyme. Deletion of the latter protein affects the parasite's fitness but is not lethal and is correlated with an increased carbon flux via the oxidative pentose phosphate pathway. Further, loss of TKT leads to a decline in 13C incorporation into glycolysis and the TCA cycle, resulting in a decrease in ATP levels and the inability of phosphoribosyl-pyrophosphate synthetase (PRPS) to convert R5P into 5'-phosphoribosyl-pyrophosphate and thereby contribute to the production of AMP and IMP. Likewise, PRPS is essential for the lytic cycle. Not least, we show that RuPE-mediated metabolic compensation is imperative for the survival of the ΔsbpaseΔtal strain. In conclusion, we demonstrate that multiple routes can flexibly supply R5P to enable parasite growth and identify catalysis by TKT and PRPS as critical enzymatic steps. Our work provides novel biological and therapeutic insights into the network design principles of intracellular parasitism in a clinically-relevant pathogen.

Toxoplasma gondii infection and testosterone alteration: A systematic review and meta-analyses.

BACKGROUND: Toxoplasma gondii (T. gondii) is a worldwide distributed protozoan parasite which has infected a wide range of warm-blooded animals and humans. The most common form of T. gondii infection is asymptomatic (latent); nevertheless, latent toxoplasmosis can induce various alterations of sex hormones, especially testosterone, in infected humans and animals. On the other hand, testosterone is involved in behavioral traits and reproductive functions in both sexes. Hence, the purpose of this systematic review is to summarize the available evidence regarding the association between T. gondii infection and testosterone alteration. METHODS: In the setting of a systematic review, an electronic search (any date to 10 January 2023) without language restrictions was performed using Science Direct, Web of Science, PubMed, Scopus, and Google Scholar. The PRISMA guidelines were followed. Following the initial search, a total of 12,306 titles and abstracts were screened initially; 12,281 were excluded due to the lack of eligibility criteria or duplication. Finally, 24 articles met the included criteria. A mean±standard deviation (SD) was calculated to assess the difference of testosterone between T. gondii positive and T. gondii negative humans. The possibility of publication bias was assessed using Egger's regression. P-value < 0.05 was considered statistically significant. RESULTS: This systematic review identified 24 articles (18 studies in humans and six studies in animals). Most human studies (13 out of 19) reported an increased level of testosterone following latent toxoplasmosis in males, while three studies reported decreased levels and two studies reported an insignificant change. Eleven articles (seven datasets in males and seven datasets in females) were eligible to be included in the data synthesis. Based on the random-effects model, the pooled mean± SD of testosterone in T. gondii positive than T. gondii negative was increased by 0.73 and 0.55 units in males and females, respectively. The Egger's regression did not detect a statistically significant publication bias in males and females (p = value = 0.95 and 0.71), respectively. Three studies in male animals (rats, mice, and spotted hyenas) and two studies in female animals (mice and spotted hyenas) reported a decline in testosterone in infected compared with non-infected animals. While, one study in female rats reported no significant changes of testosterone in infected than non-infected animals. Moreover, two studies in male rats reported an increased level of testosterone in infected than non-infected animals. CONCLUSIONS: This study provides new insights about the association between T. gondii infection and testosterone alteration and identifies relevant data gaps that can inform and encourage further studies. The consequence of increased testosterone levels following T. gondii infection could partly be associated with increased sexual behavior and sexual transmission of the parasite. On the other hand, declining testosterone levels following T. gondii infection may be associated with male reproductive impairments, which were observed in T. gondii-infected humans and animals. Furthermore, these findings suggest the great need for more epidemiological and experimental investigations in depth to understand the relationship between T. gondii infection and testosterone alteration alongside with future consequences of testosterone alteration.

Seromolecular study on the prevalence and risk factors of Toxoplasma gondii infection in pregnant women referred to a gynecology hospital in Urmia, northwest part of Iran in 2022.

Toxoplasmosis is a frequent infection among the human population. The infection can cause devastating complications for the fetus during pregnancy. The present study aimed to determine the serological and molecular prevalence of the infection and molecular characterization of Toxoplasma gondii isolates among pregnant women referred to Kowsar Hospital, Urmia, Iran. In a cross-sectional study, 340 blood samples were collected from pregnant women referred to Kowsar Hospital, Urmia, Iran from May to July 2022. Anti-T. gondii IgG and IgM seropositivity were determined by enzyme-linked immunosorbent assay. PCR was carried out by targeting the GRA6 gene of the parasite on all patients' buffy coats. Anti-T. gondii IgG and IgM antibodies were positive in two (0.6%) women, and 101 (29.7%) women had anti-T. gondii IgG and 70.3% were seronegative. PCR was positive in two IgM-positive women, and both isolates belonged to T. gondii carrying the GRA6 allele of lineage I. The risk of infection was significantly higher in women who had constant contact with cats and soil, and who were residents of rural areas. The two IgM-positive women were asymptomatic regarding acute toxoplasmosis. According to the results of the present study, the prevalence of toxoplasmosis in pregnant women in Urmia is similar to its prevalence in other areas in northwestern Iran, and despite the low prevalence of acute infection, it should not be ignored.

Inhibition of Foxp3 expression in the placenta of mice infected intraperitoneally by toxoplasma gondii tachyzoites: insights into the PPARγ/miR-7b-5p/Sp1 signaling pathway.

BACKGROUND: Toxoplasma gondii, an obligate intracellular parasitic protozoa, infects approximately 30% of the global population. Contracting T. gondii at the primary infection of the mother can result in neonatal microcephaly, chorioretinitis, hydrocephalus, or mortality. Our previous study indicated that pregnant mice infected with T. gondii displayed a decrease in both the number and the suppressive ability of regulatory T cells, accompanied by the reduced Forkhead box P3 (Foxp3). Numerous studies have proved that microRNAs (miRNAs) are implicated in T. gondii infection, but there is meager evidence on the relationship between alterations of miRNAs and downregulation of Foxp3 induced by T. gondii. METHODS: Quantitative reverse transcription polymerase chain reaction was utilized to detect the transcriptions of miRNAs and Foxp3. Protein blotting and immunofluorescence were used to detect the expressions of Foxp3 and related transcription factors. The structure of mouse placenta was observed by hematoxylin and eosin (HE) staining. To examine the activity of miR-7b promoter and whether miR-7b-5p targets Sp1 to suppress Foxp3 expression, we constructed recombinant plasmids containing the full-length/truncated/mutant miR-7b promoter sequence or wildtype/mutant of Sp1 3' untranslated region (3' UTR) to detect the fluorescence activity in EL4 cells. RESULTS: In T. gondii-infected mice, miR-7b transcription was significantly elevated, while Foxp3 expression was decreased in the placenta. In vitro, miR-7b mimics downregulated Foxp3 expression, whereas its inhibitors significantly upregulated Foxp3 expression. miR-7b promoter activity was elevated upon the stimulation of T. gondii antigens, which was mitigated by co-transfection of mutant miR-7b promoter lacking peroxisome proliferator-activated receptor γ (PPARγ) target sites. Additionally, miR-7b mimics diminished Sp1 expression, while miR-7b inhibitors elevated its expression. miR-7b mimics deceased the fluorescence activity of Sp1 3' untranslated region (3' UTR), but it failed to impact the fluorescence activity upon the co-transfection of mutant Sp1 3' UTR lacking miR-7b target site. CONCLUSIONS: T. gondii infection and antigens promote miR-7b transcription but inhibit Foxp3 protein and gene levels. T. gondii antigens promote miR-7b promoter activity by a PPARγ-dependent mechanism. miR-7b directly binds to Sp1 3' UTR to repress Sp1 expression. Understanding the regulatory functions by which T. gondii-induced miR-7b suppresses Foxp3 expression can provide new perspectives for the possible therapeutic avenue of T. gondii-induced adverse pregnancy outcomes.

The α subunit of AMP-activated protein kinase is critical for the metabolic success and tachyzoite proliferation of Toxoplasma gondii.

Toxoplasma gondii is a zoonotic parasite infecting humans and nearly all warm-blooded animals. Successful parasitism in diverse hosts at various developmental stages requires the parasites to fine tune their metabolism according to environmental cues and the parasite's needs. By manipulating the ß and γ subunits, we have previously shown that AMP-activated protein kinase (AMPK) has critical roles in regulating the metabolic and developmental programmes. However, the biological functions of the α catalytic subunit have not been established. T. gondii encodes a canonical AMPKα, as well as a KIN kinase whose kinase domain has high sequence similarities to those of classic AMPKα proteins. Here, we found that TgKIN is dispensable for tachyzoite growth, whereas TgAMPKα is essential. Depletion of TgAMPKα expression resulted in decreased ATP levels and reduced metabolic flux in glycolysis and the tricarboxylic acid cycle, confirming that TgAMPK is involved in metabolic regulation and energy homeostasis in the parasite. Sequential truncations at the C-terminus found an α-helix that is key for the function of TgAMPKα. The amino acid sequences of this α-helix are not conserved among various AMPKα proteins, likely because it is involved in interactions with TgAMPKß, which only have limited sequence similarities to AMPKß in other eukaryotes. The essential role of the less conserved C-terminus of TgAMPKα provides opportunities for parasite specific drug designs targeting TgAMPKα.

[Use of LDBIO Diagnostics® TOXOPLASMA ICT IGG-IGM in association with Siemens Atellica® IM in toxoplasmosis serological screening]. / Place du test rapide LDBIO Diagnostics® TOXOPLASMA ICT IGG-IGM en complément de la sérologie Siemens Atellica® IM dans la stratégie diagnostique de la toxoplasmose.

According to French recommendations for serological screening of toxoplasmosis, some profiles must be confirmed by additional methods, extending the time taken to produce results. Thus, the Laborizon Bretagne technical platform in Nantes studied the place of the LDBIO Diagnostics® TOXOPLASMA ICT IGG-IGM (ICT) test in addition to Siemens Atellica® serology. IgG-/IgM+ and equivocal or weak positive IgG/IgM- (IgGEq/IgM-) profiles on Atellica® will be confirmed by ICT, Alinity® Abbott and Platelia® Biorad. Among the 66 IgGEq/IgM- profiles, the concordance is perfect between ICT and complementary techniques: 21 weak positives were confirmed positive, 8 equivocal were considered negative and 37 were confirmed positive. Concerning the 76 IgG-/IgM+ profiles, 68 are negative and 7 are positive by complementary techniques and ICT. One discordance was observed. The Atellica®/ICT combination allows excellent discrimination of IgG-/IgM+ and IgGEq/IgM serological profiles with consistent diagnostic orientation in 99.3% of cases. Only 1 sample was found to be discordant but required monitoring at 15 days. The observed performances are compatible with routine use. This test simplifies the analytical process, improves the time to obtain results, while guaranteeing an excellent level of quality.

Systematic Review on the Relationship between Toxoplasmosis and Mental Disorders.

BACKGROUND: Toxoplasmosis is a worldwide parasitic zoonosis caused by the protozoan Toxoplasma gondii. In cases of vertical infection, and in immunosuppressed people by the human immunodeficiency virus (HIV) serious clinical conditions may appear, while immunocompetent people do not present symptoms. However, T. gondii infection has been linked to several mental disorders for decades. OBJECTIVE: To substantiate the possible relationship between T. gondii and mental disorders and suggest control and prevention strategies. MATERIAL AND METHODS: A systematic review has been carried out to analyze the relationship between T. gondii exposure (presence of IgG) and the onset of mental disorders in minors and adults. The etiopathogenic mechanisms described by the authors have also been included and the systems of surveillance, prevention and control of infection have been evaluated. RESULTS: Several processes linked to the presence of cysts and the reactivation of the parasite in certain situations produce an immune and inflammatory response. Also, direct and indirect actions on different neurotransmitters. These mechanisms, together with other environmental and genetic factors, would predispose to different psychiatric pathologies. CONCLUSIONS: Due to the limits of the study, no conclusions can be drawn in childhood and adolescence. However, the results of this systematic review show a possible association of schizophrenia, bipolar disorder and compulsive disorder with T. gondii infection in adults. There is a need to improve control, integrated surveillance and extend prevention measures to the entire population.

Effect of concurrent infection of Helicobacter pylori with Toxoplasma gondii infection on gastric pathology.

BACKGROUND: Toxoplasma gondii (T. gondii) and Helicobacter pylori (H. pylori) are among the most prevalent foodborne parasitic and bacterial infections worldwide. However, the concurrent impact of coinfection on gastric pathology has yet to be studied in depth. The effect of coinfection generally either adds a synergetic or antagonistic impact; we aimed in the current work to assess the impact of T. gondii coinfection on the progression of H. pylori-associated gastric pathology and reporting H. pylori virulent strains. The study was conducted on 82 patients complaining of persistent gastrointestinal symptoms with failed treatment response and prone to endoscopy. They were subjected to stool examination to detect H. pylori antigen, serological screening for latent toxoplasmosis, endoscopy, histopathological examination, and molecular detection of H. pylori virulence strains in gastric biopsies. Out of the 82 patients, 62 patients were positive for H. pylori antigen in stool and 55 patients confirmed positivity by histopathology; out of them, 37 patients had isolated Vac As1 variants, 11 patients had combined Vac As1 and Cag A variants, and 7 patients had combined Vac As1, Cag A and VacAs2 variants. Patients with the combined two or three variances showed significantly deteriorated histopathological features than patients with a single Vac As1 variant (P < 0.05). Latent toxoplasmosis was positive among 35/82 patients. Combined H. pylori and Toxoplasma gondii infection had significantly marked inflammation than patients with isolated infection (P < 0.05). CONCLUSION: Screening for toxoplasmosis among H. pylori-infected patients is recommended as it is considered a potential risk factor for gastric inflammation severity. H. pylori gastric inflammation may be heightened by Toxoplasma coinfection.

Toxoplasma gondii modulates the host cell cycle, chromosome segregation, and cytokinesis irrespective of cell type or species origin.

BACKGROUND: Toxoplasma gondii is an apicomplexan intracellular obligate parasite and the etiological agent of toxoplasmosis in humans, domestic animals and wildlife, causing miscarriages and negatively impacting offspring. During its intracellular development, it relies on nutrients from the host cell, controlling several pathways and the cytoskeleton. T. gondii has been proven to control the host cell cycle, mitosis and cytokinesis, depending on the time of infection and the origin of the host cell. However, no data from parallel infection studies have been collected. Given that T. gondii can infect virtually any nucleated cell, including those of humans and animals, understanding the mechanism by which it infects or develops inside the host cell is essential for disease prevention. Therefore, we aimed here to reveal whether this modulation is dependent on a specific cell type or host cell species. METHODS: We used only primary cells from humans and bovines at a maximum of four passages to ensure that all cells were counted with appropriate cell cycle checkpoint control. The cell cycle progression was analysed using fluorescence-activated cell sorting (FACS)-based DNA quantification, and its regulation was followed by the quantification of cyclin B1 (mitosis checkpoint protein). The results demonstrated that all studied host cells except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Additionally, we used an immunofluorescence assay to track mitosis and cytokinesis in uninfected and T. gondii-infected cells. RESULTS: The results demonstrated that all studied host cell except bovine colonic epithelial cells (BCEC) were arrested in the S-phase, and none of them were affected in cyclin B1 expression. Our findings showed that the analysed cells developed chromosome segregation problems and failed to complete cytokinesis. Also, the number of centrosomes per mitotic pole was increased after infection in all cell types. Therefore, our data suggest that T. gondii modulates the host cell cycle, chromosome segregation and cytokinesis during infection or development regardless of the host cell origin or type.

Deciphering the epidemiological dynamics: Toxoplasma gondii seroprevalence in mainland China's food animals, 2010-2023.

Background: Toxoplasma gondii (T. gondii) is a significant protozoan pathogen among food animals. Despite the threat to public health by T. gondii infections, there's limited understanding of its seroprevalence and trends in food animals across mainland China. This study aimed to estimate the seroprevalence of T. gondii infections among swine, sheep, goats, chickens, and cattle in mainland China from 2010 to 2023. Methods: We searched cross-sectional studies published between 2010 and 2023 that reported the prevalence of T. gondii in food animals from databases including PubMed, Embase, Web of Science, China Biology Medicine Disc (CBM), China National Knowledge Infrastructure (CNKI), Wanfang data, and the China Science and Technology Journal Database (CQVIP). We performed subgroup analyses to explore the impact of different factors on the seroprevalence of T. gondii. Pooled estimates of T. gondii seroprevalence were calculated with a random-effects model. Results: An analysis of 184 studies involving 211985 animals revealed a T. gondii overall seroprevalence of 15.3% (95% CI: 13.1-17.8). Although the seroprevalence of food animals across mainland China was relatively stable from 2010 to 2023, notable variations were observed across different animal types and regions (P < 0.01), along with changes in geographical distribution. Sample type, detection method, animal age, and history of abortion were identified as key risk factors for T. gondii seroprevalence. Conclusion: The study conducted a meta-analysis on the seroprevalence of T. gondii in mainland China's Food Animals from 2010 to 2023, and identified key risk factors. These findings advance our understanding of T. gondii infection dynamics, offering critical insights for developing control strategies and guiding public health policies.

Phosphoproteomic analysis reveals changes in A-Raf-related protein phosphorylation in response to Toxoplasma gondii infection in porcine macrophages.

BACKGROUND: Toxoplasma gondii is an obligate intracellular protozoan parasite that causes severe threats to humans and livestock. Macrophages are the cell type preferentially infected by T. gondii in vivo. Protein phosphorylation is an important posttranslational modification involved in diverse cellular functions. A rapidly accelerated fibrosarcoma kinase (A-Raf) is a member of the Raf family of serine/threonine protein kinases that is necessary for MAPK activation. Our previous research found that knockout of A-Raf could reduce T. gondii-induced apoptosis in porcine alveolar macrophages (3D4/21 cells). However, limited information is available on protein phosphorylation variations and the role of A-Raf in macrophages infected with T. gondii. METHODS: We used immobilized metal affinity chromatography (IMAC) in combination with liquid chromatography tandem mass spectrometry (LC-MS/MS) to profile changes in phosphorylation in T. gondii-infected 3D4/21 and 3D4/21-ΔAraf cells. RESULTS: A total of 1647 differentially expressed phosphorylated proteins (DEPPs) with 3876 differentially phosphorylated sites (DPSs) were identified in T. gondii-infected 3D4/21 cells (p3T group) when compared with uninfected 3D4/21 cells (pho3 group), and 959 DEPPs with 1540 DPSs were identified in the p3T group compared with infected 3D4/21-ΔAraf cells (p3KT group). Venn analysis revealed 552 DPSs corresponding to 406 DEPPs with the same phosphorylated sites when comparing p3T/pho3 versus p3T/p3KT, which were identified as DPSs and DEPPs that were directly or indirectly related to A-Raf. CONCLUSIONS: Our results revealed distinct responses of macrophages to T. gondii infection and the potential roles of A-Raf in fighting infection via phosphorylation of crucial proteins.

Trx4, a novel thioredoxin protein, is important for Toxoplasma gondii fitness.

BACKGROUND: To successfully replicate within the host cell, Toxoplasma gondii employs several mechanisms to overcome the host cell defenses and mitigate the harmful effects of the free radicals resulting from its own metabolic processes using effectors such as thioredoxin proteins. In this study, we characterize the location and functions of a newly identified thioredoxin in T. gondii, which was named Trx4. METHODS: We characterized the functional role of Trx4 in T. gondii Type I RH and Type II Pru strains by gene knockout and studied its subcellular localization by endogenous protein HA tagging using CRISPR-Cas9 gene editing. The enzyme-catalyzed proximity labeling technique, the TurboID system, was employed to identify the proteins in proximity to Trx4. RESULTS: Trx4 was identified as a dense granule protein of T. gondii predominantly expressed in the parasitophorous vacuole (PV) and was partially co-localized with GRA1 and GRA5. Functional analysis showed that deletion of trx4 markedly influenced the parasite lytic cycle, resulting in impaired host cell invasion capacity in both RH and Pru strains. Mutation of Trx domains in Trx4 in RH strain revealed that two Trx domains were important for the parasite invasion. By utilizing the TurboID system to biotinylate proteins in proximity to Trx4, we identified a substantial number of proteins, some of which are novel, and others are previously characterized, predominantly distributed in the dense granules. In addition, we uncovered three novel proteins co-localized with Trx4. Intriguingly, deletion of trx4 did not affect the localization of these three proteins. Finally, a virulence assay demonstrated that knockout of trx4 resulted in a significant attenuation of virulence and a significant reduction in brain cyst loads in mice. CONCLUSIONS: Trx4 plays an important role in T. gondii invasion and virulence in Type I RH strain and Type II Pru strain. Combining the TurboID system with CRISPR-Cas9 technique revealed many PV-localized proximity proteins associated with Trx4. These findings suggest a versatile role of Trx4 in mediating the processes that occur in this distinctive intracellular membrane-bound vacuolar compartment.

Toxoplasma gondii infection-induced host cellular DNA damage is strain-dependent and leads to the activation of the ATM-dependent homologous recombination pathway.

Toxoplasma gondii is a globally occurring apicomplexan parasite that infects humans and animals. Globally, different typical and atypical haplotypes of T. gondii induce varying pathologies in hosts. As an obligate intracellular protozoon, T. gondii was shown to interfere with host cell cycle progression, leading to mitotic spindle alteration, chromosome segregation errors and cytokinesis failure which all may reflect chromosomal instability. Referring to strain-dependent virulence, we here studied the potential of different T. gondii strains (RH, Me49 and NED) to drive DNA damage in primary endothelial host cells. Utilizing microscopic analyses, comet assays and γ-H2AX quantification, we demonstrated a strain-dependent induction of binucleated host cells, DNA damage and DNA double strand breaks, respectively, in T. gondii-infected cells with the RH strain driving the most prominent effects. Interestingly, only the NED strain significantly triggered micronuclei formation in T. gondii-infected cells. Focusing on the RH strain, we furthermore demonstrated that T. gondii-infected primary host cells showed a DNA damage response by activating the ATM-dependent homologous recombination (HR) pathway. In contrast, key molecules of the nonhomologous DNA end joining (NHEJ) pathway were either not affected or downregulated in RH-infected host cells, suggesting that this pathway is not activated by infection. In conclusion, current finding suggests that T. gondii infection affects the host cell genome integrity in a strain-dependent manner by causing DNA damage and chromosomal instability.

Detection of Toxoplasma gondii in artisanal salted meat products sold in street markets of the Ilhéus-Itabuna microregion.

This study aimed to detect Toxoplasma gondii in artisanal salted meat products sold in street markets in the Ilhéus-Itabuna microregion and to assess the salt concentration used in their preparation and its influence on the parasite's viability. A total of 125 samples of various artisanal meat products sold in street markets located in the Ilhéus-Itabuna microregion were collected during 2021. Serological analysis using indirect hemagglutination (HAI) and molecular analysis (PCR) were performed on these samples to detect the presence of the parasite. Möhr's method was utilized to determine the sodium chloride concentration in the samples. Of all samples, 21 were subjected to a bioassay in albino mice to verify the viability of possible tissue cysts. Among the 125 meat products, 10 (8%) tested positive in the serological analysis including four cured pork sausages, five beef sun-dried meats, and one mixed fresh sausage (pork and chicken). None of 125 samples tested positive in the molecular analysis. On bioassay, all mice tested negative for the presence of the parasite. The NaCl concentration in the positive samples ranged from 2.9% to 8%. The results demonstrated that the salt concentration in the collected samples was sufficient to inactivate the parasite T. gondii.

Potent hydroxamate-derived compounds arrest endodyogeny of Toxoplasma gondii tachyzoites.

Toxoplasmosis is a zoonosis that is a worldwide health problem, commonly affecting fetal development and immunodeficient patients. Treatment is carried out with a combination of pyrimethamine and sulfadiazine, which can cause cytopenia and intolerance and does not lead to a parasitological cure of the infection. Lysine deacetylases (KDACs), which remove an acetyl group from lysine residues in histone and non-histone proteins are found in the Toxoplasma gondii genome. Previous work showed the hydroxamate-type KDAC inhibitors Tubastatin A (TST) and Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) were effective against T. gondii. In the present study, the effects of three hydroxamates (KV-24, KV-30, KV-46), which were originally designed to inhibit human KDAC6, showed different effects against T. gondii. These compounds contain a heterocyclic cap group and a benzyl linker bearing the hydroxamic acid group in para-position. All compounds showed selective activity against T. gondii proliferation, inhibiting tachyzoite proliferation with IC50 values in a nanomolar range after 48h treatment. Microscopy analyses showed that after treatment, tachyzoites presented mislocalization of the apicoplast, disorganization of the inner membrane complex, and arrest in the completion of new daughter cells. The number of dividing cells with incomplete endodyogeny increased significantly after treatment, indicating the compounds can interfere in the late steps of cell division. The results obtained in this work that these new hydroxamates should be considered for future in vivo tests and the development of new compounds for treating toxoplasmosis.

Prevalence and factors associated with Leishmania spp. and Toxoplasma gondii infections in apparently healthy horses in Eastern Spain.

Leishmaniasis and toxoplasmosis are two of the most common parasitic zoonoses. Leishmaniasis is endemic to 98 countries around the world, whereas toxoplasmosis is widely distributed throughout the world, causing significant health expenditure. Horses can play a relevant role in the transmission of the disease, being a silent reservoir, as clinical signs are not common. Serum samples from 166 horses living in eastern Spain (Mediterranean basin) were analysed to determine the presence of antibodies against Leishmania spp. and T. gondii by ELISA (Enzyme-linked Immunosorbent Assay.) The risk factors evaluated were the geographical area and the relative humidity and average temperature, and epidemiological factors such as sex, reproductive status, age, breed, morphotype, living with other domestic animals, use and access to the outdoors. Seroprevalence of Leishmania spp. and T. gondii infection was found 28.92%, and 16.27% respectively, whereas co-infection of the two parasites was found only in two males. Leishmania seroprevalence was high in castrated males and several mesodolichomorphic equine breeds used for teaching, as well as in outdoor animals. The most elevated seroprevalence was found in winter with higher levels of rainfall, whereas high seroprevalence of T. gondii was found in crossbreeding animals and those used for breeding. High seroprevalence of Leishmania spp. and T. gondii was found in horses of the Mediterranean basin. These data suggest that horses can act as a silent reservoir and that this species has high potential for transmission to humans, outdoor animals and in geographical areas with high average rainfall.

Global prevalence and risk factors associated with Toxoplasma gondii infection in wild birds: A systematic review and meta-analysis.

A systematic review and meta-analysis were performed to identify the global prevalence and factors associated with Toxoplasma gondii infection in wild birds. Six bibliographic databases (Chinese National Knowledge Infrastructure, VIP Chinese Journal Database, Wanfang Data, PubMed, Web of science and ScienceDirect) were searched from inception to February 2023. The search yielded 1220 records of which 659 articles underwent full-text evaluation, which identified 49 eligible articles and 16,030 wild bird samples that were included in the meta-analysis. The estimated pooled global prevalence of T. gondii infection in wild birds was 16.6%. Out of the variables tested, publication year after 2020 and climate type were significantly associated with T. gondii infection (P<0.01). Our data indicate that the prevalence of T. gondii in wild birds can be influenced by epidemiological variables. Further research is needed to identify the biological, environmental, anthropogenic, and geographical risk factors which impact the ecology and prevalence of T. gondii in wild birds.

Cryogenic electron tomography reveals novel structures in the apical complex of Plasmodium falciparum.

Intracellular infectious agents, like the malaria parasite, Plasmodium falciparum, face the daunting challenge of how to invade a host cell. This problem may be even harder when the host cell in question is the enucleated red blood cell, which lacks the host machinery co-opted by many pathogens for internalization. Evolution has provided P. falciparum and related single-celled parasites within the phylum Apicomplexa with a collection of organelles at their apical end that mediate invasion. This apical complex includes at least two sets of secretory organelles, micronemes and rhoptries, and several structural features like apical rings and a putative pore through which proteins may be introduced into the host cell during invasion. We perform cryogenic electron tomography (cryo-ET) equipped with Volta Phase Plate on isolated and vitrified merozoites to visualize the apical machinery. Through tomographic reconstruction of cellular compartments, we see new details of known structures like the rhoptry tip interacting directly with a rosette resembling the recently described rhoptry secretory apparatus (RSA), or with an apical vesicle docked beneath the RSA. Subtomogram averaging reveals that the apical rings have a fixed number of repeating units, each of which is similar in overall size and shape to the units in the apical rings of tachyzoites of Toxoplasma gondii. Comparison of these polar rings in Plasmodium and Toxoplasma parasites also reveals them to have a structurally conserved assembly pattern. These results provide new insight into the essential and structurally conserved features of this remarkable machinery used by apicomplexan parasites to invade their respective host cells. IMPORTANCE: Malaria is an infectious disease caused by parasites of the genus Plasmodium and is a leading cause of morbidity and mortality globally. Upon infection, Plasmodium parasites invade and replicate in red blood cells, where they are largely protected from the immune system. To enter host cells, the parasites employ a specialized apparatus at their anterior end. In this study, advanced imaging techniques like cryogenic electron tomography (cryo-ET) and Volta Phase Plate enable unprecedented visualization of whole Plasmodium falciparum merozoites, revealing previously unknown structural details of their invasion machinery. Key findings include new insights into the structural conservation of apical rings shared between Plasmodium and its apicomplexan cousin, Toxoplasma. These discoveries shed light on the essential and conserved elements of the invasion machinery used by these pathogens. Moreover, the research provides a foundation for understanding the molecular mechanisms underlying parasite-host interactions, potentially informing strategies for combating diseases caused by apicomplexan parasites.

Multicenter evaluation of the Toxoplasma gondii Real-TM (Sacace) kit performance for the molecular diagnosis of toxoplasmosis.

The molecular detection of Toxoplasma gondii DNA is a key tool for the diagnosis of disseminated and congenital toxoplasmosis. This multicentric study from the Molecular Biology Pole of the French National Reference Center for toxoplasmosis aimed to evaluate Toxoplasma gondii Real-TM PCR kit (Sacace). The study compared the analytical and clinical performances of this PCR assay with the reference PCRs used in proficient laboratories. PCR efficiencies varied from 90% to 112%; linearity zone extended over four log units (R2 > 0.99) and limit of detection varied from 0.01 to ≤1 Tg/mL depending on the center. Determined on 173 cryopreserved DNAs from a large range of clinical specimens, clinical sensitivity was 100% [106/106; 95 confidence interval (CI): 96.5%-100%] and specificity was 100% (67/67; 95 CI: 94.6%-100%). The study revealed two potential limitations of the Sacace PCR assay: the first was the inconsistency of the internal control (IC) when added to the PCR mixture. This point was not found under routine conditions when the IC was added during the extraction step. The second is a lack of practicality, as the mixture is distributed over several vials, requiring numerous pipetting operations. Overall, this study provides useful information for the molecular diagnosis of toxoplasmosis; the analytical and clinical performances of the Sacace PCR kit were satisfactory, the kit having sensitivity and specificity similar to those of expert center methods and being able to detect low parasite loads, at levels where multiplicative analysis gives inconsistently positive results. Finally, the study recommends multiplicative analysis in particular for amniotic fluids, aqueous humor, and other single specimens.

Iron Stress Affects the Growth and Differentiation of Toxoplasma gondii.

Iron is an indispensable nutrient for the survival of Toxoplasma gondii; however, excessive amounts can lead to toxicity. The parasite must overcome the host's "nutritional immunity" barrier and compete with the host for iron. Since T. gondii can infect most nucleated cells, it encounters increased iron stress during parasitism. This study assessed the impact of iron stress, encompassing both iron depletion and iron accumulation, on the growth of T. gondii. Iron accumulation disrupted the redox balance of T. gondii while enhancing the parasite's ability to adhere in high-iron environments. Conversely, iron depletion promoted the differentiation of tachyzoites into bradyzoites. Proteomic analysis further revealed proteins affected by iron depletion and identified the involvement of phosphotyrosyl phosphatase activator proteins in bradyzoite formation.