Toxoplasma gondii ROP18I inhibits host innate immunity through cGAS-STING signaling.

Toxoplasma gondii is an opportunistic protozoan, which widely infects humans and other warm-blooded animals. The type I interferon (IFN) such as IFN-α/ß is involved in cGAS-STING signaling to resist T. gondii infection. We found in RAW264.7 cells, that T. gondii virulence factor TgROP18I , inhibited IFN-ß production through interacting with interferon regulatory factor 3 (IRF3). Besides, TgROP18I interacted with p62 and Tumor Necrotic Factor Receptor Associated Factor 6 (TRAF6), which resulted in the inhibition of TRAF6-p62 interaction, and phosphorylation of p62. Furthermore, TgROP18I restricted the recruitment of ubiquitin, p62 and microtubule-associated protein light chain 3 (LC3) to the parasitophorous vacuole membrane (PVM) in IFN-γ-stimulated murine cell line L929 cells. In IFN-γ-stimulated human cells, TgROP18I restricted the decoration of PVM with ubiquitin, p62, and LC3, and bound with TRAF2, TRAF6, and p62, respectively. As a result, TgROP18I led to a successful parasitic replication in murine and human cells. Collectively, our study revealed the function of TgROP18I in suppressing host type I interferon responses in T. gondii infection for parasitic immune escape.

Strain-specific disruption of interferon-stimulated N-myc and STAT interactor (NMI) function by Toxoplasma gondii type I ROP18 in human cells.

Toxoplasma gondii rhoptry protein TgROP18 is a polymorphic virulence effector that targets immunity-related GTPases (IRGs) in rodents. Given that IRGs are uniquely diversified in rodents and not in other T. gondii intermediate hosts, the role of TgROP18 in manipulating non-rodent cells is unclear. Here we show that in human cells TgROP18I interacts with the interferon-gamma-inducible protein N-myc and STAT interactor (NMI) and that this is a property that is unique to the type I TgROP18 allele. Specifically, when expressed ectopically in mammalian cells only TgROP18I co-immunoprecipitates with NMI in IFN-γ-treated cells, while TgROP18II does not. In parasites expressing TgROP18I or TgROP18II, NMI only co-immunoprecipitates with TgROP18I and this is associated with allele-specific immunolocalization of NMI on the parasitophorous vacuolar membrane (PVM). We also found that TgROP18I reduces NMI association with IFN-γ-activated sequences (GAS) in the IRF1 gene promoter. Finally, we determined that polymorphisms in the C-terminal kinase domain of TgROP18I are required for allele-specific effects on NMI. Together, these data further define new host pathway targeted by TgROP18I and provide the first function driven by allelic differences in the highly polymorphic ROP18 locus.

Application of the Scorpion Neurotoxin AaIT against Insect Pests.

Androctonus australis Hector insect toxin (AaIT), an insect-selective toxin, was identified in the venom of the scorpion Androctonus australis. The exclusive and specific target of the toxin is the voltage-gated sodium channels of the insect, resulting in fast excitatory paralysis and even death. Because of its strict toxic selectivity and high bioactivity, AaIT has been widely used in experiments exploring pest bio-control. Recombinant expression of AaIT in a baculovirus or a fungus can increase their virulence to insect pests and diseases vectors. Likewise, transgenic plants expressing AaIT have notable anti-insect activity. AaIT is an efficient toxin and has great potential to be used in the development of commercial insecticides.

Analysis of the Differential Exosomal miRNAs of DC2.4 Dendritic Cells Induced by Toxoplasma gondii Infection.

Toxoplasma gondii is an intracellular parasite that infects humans and other warm-blooded animals. Exosomes are endocytic-derived vesicles released by cells, representing an important mode of intercellular communication. In exosomes, specific molecules of proteins, lipids, and mRNAs or miRNAs have been detected, some of which are capable of transferring biologically active molecules to recipient cells. Dendritic cells (DCs) are the only antigen-presenting cells (APCs) that activate the initial immune response. In this study, high-throughput sequencing was used to analyze the exosomal miRNA profile of DC2.4 cells infected with Toxoplasma gondii for 28 h, compared with those of uninfected DC2.4 cells. Differential exosomal miRNAs (DEmiRs) from these two cell groups were analyzed. Through high-throughput sequencing, 3434 DEmiRs were obtained, and 12 stably enriched DEmiRNAs were verified by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) and selected for further analysis. The target genes of these 12 miRNAs were predicted with online analysis software and subjected to bioinformatics analyses including protein-protein interaction (PPI) network analysis, key driver analysis (KDA), gene ontology (GO) enrichment, and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis. These DEmiRs were found to be associated with a variety of biological processes and signaling pathways involved in host ubiquitin system, innate immunity, biosynthesis, and transferase activity and could be potential biomarkers for T. gondii infection.

Diagnostic Function of 3D Optical Coherence Tomography Images in Diagnosis of Vogt-Koyanagi-Harada Disease at Acute Uveitis Stage.

BACKGROUND This study analyzed the macular 3D-OCT images of Vogt-Koyanagi-Harada disease (VKH) in uveitis, explored the characteristics of 3D-OCT images of the macular region of VKH, and assessed which characteristics contribute most to VKH diagnosis. MATERIAL AND METHODS The 3D-OCT examination of 25 cases of VKH was performed on the macular area, and the image characteristics were analyzed. RESULTS Our study included a total of 50 eyes from 25 cases of VKH patients, 10 males and 15 females, aged 17 to 64 years, mean (39.44±11.60) years old. According to OCT B-scan images, 49 (98%) eyes had ERD, 49 (98%) eyes had nerve retinal edema, 36 (72%) eyes had endometrium-like structure (including cysts), 5 (10%) eyes had RPE folds, 35 (70%) eyes had changes in the internal septum, 49 (98%) eyes had RPE monolayer structure outside the ERD region. In ILM-RPE thickness, 49 (98%) eyes had retinal irregular thickening and 31 (62%) eyes had radial stripe changes. In ILM contour figure, 50 eyes (100%) showed exceptional uplift, 5 (10%) eyes had small focal uplift for PED on the RPE surface, and 48 (96%) eyes had wavy ups and downs. CONCLUSIONS In OCT B-scan imaging, the ERT, retinal edema of the retina, and the RPE monolayer structure outside the range are most likely to occur in VKH. The ILM-RPE thickness chart in 3D reconstruction showed irregular thickening of the retina. The ILM contour graph showed abnormal uplift, and RPE surface wavy ups and downs in VKH most likely to occur.

Juvenile hormone-activated phospholipase C pathway enhances transcriptional activation by the methoprene-tolerant protein.

Juvenile hormone (JH) is a key regulator of a wide diversity of developmental and physiological events in insects. Although the intracellular JH receptor methoprene-tolerant protein (MET) functions in the nucleus as a transcriptional activator for specific JH-regulated genes, some JH responses are mediated by signaling pathways that are initiated by proteins associated with plasma membrane. It is unknown whether the JH-regulated gene expression depends on the membrane-mediated signal transduction. In Aedes aegypti mosquitoes, we found that JH activated the phospholipase C (PLC) pathway and quickly increased the levels of inositol 1,4,5-trisphosphate, diacylglycerol, and intracellular calcium, leading to activation and autophosphorylation of calcium/calmodulin-dependent protein kinase II (CaMKII). When abdomens from newly emerged mosquitoes were cultured in vitro, the JH-activated gene expression was repressed substantially if specific inhibitors of PLC or CaMKII were added to the medium together with JH. In newly emerged female mosquitoes, RNAi-mediated depletion of PLC or CaMKII considerably reduced the expression of JH-responsive genes, including the Krüppel homolog 1 gene (AaKr-h1) and the early trypsin gene (AaET). JH-induced loading of MET to the promoters of AaKr-h1 and AaET was weakened drastically when either PLC or CaMKII was inactivated in the cultured tissues. Therefore, the results suggest that the membrane-initiated signaling pathway modifies the DNA-binding activity of MET via phosphorylation and thus facilitates the genomic responses to JH. In summary, this study reveals an interplay of genomic and nongenomic signaling mechanisms of JH.

Genome sequence of the Asian Tiger mosquito, Aedes albopictus, reveals insights into its biology, genetics, and evolution.

The Asian tiger mosquito, Aedes albopictus, is a highly successful invasive species that transmits a number of human viral diseases, including dengue and Chikungunya fevers. This species has a large genome with significant population-based size variation. The complete genome sequence was determined for the Foshan strain, an established laboratory colony derived from wild mosquitoes from southeastern China, a region within the historical range of the origin of the species. The genome comprises 1,967 Mb, the largest mosquito genome sequenced to date, and its size results principally from an abundance of repetitive DNA classes. In addition, expansions of the numbers of members in gene families involved in insecticide-resistance mechanisms, diapause, sex determination, immunity, and olfaction also contribute to the larger size. Portions of integrated flavivirus-like genomes support a shared evolutionary history of association of these viruses with their vector. The large genome repertory may contribute to the adaptability and success of Ae. albopictus as an invasive species.

Risk of drug resistance in Plasmodium falciparum malaria therapy-a systematic review and meta-analysis.

Plasmodium falciparum is responsible for the vast majority of the morbidity and mortality associated with malaria infection globally. Although a number of studies have reported the emergence of drug resistance in different therapies for P. falciparum infection, the degree of the drug resistance in different antimalarials is still unclear. This research investigated the risk of drug resistance in the therapies with different medications based on meta-analyses. Relevant original randomized control trials (RCTs) were searched in all available electronic databases. Pooled relative risks (RRs) with 95% confidence intervals (95% CIs) were used to evaluate the risk of drug resistance resulting from different treatments. Seventy-eight studies were included in the meta-analysis to compare drug resistance in the treatment of P. falciparum infections and yielded the following results: chloroquine (CQ) > sulfadoxine-pyrimethamine (SP) (RR = 3.67, p < 0.001 ), mefloquine (MQ) < SP (RR = 0.26, p < 0.001), artesunate + sulfadoxine-pyrimethamine (AS + SP) > artemether + lumefantrine (AL) (RR = 2.94, p < 0.001), dihydroartemisinin + piperaquine (DHA + PQ) < AL (RR = 0.7, p < 0.05), and non-artemisinin-based combination therapies (NACTs) > artemisinin-based combination therapies (ACTs) (RR = 1.93, p < 0.001); no significant difference was found in amodiaquine (AQ) vs. SP, AS + AQ vs. AS + SP, AS + AQ vs. AL, or AS + MQ vs. AL. These results presented a global view for the current status of antimalarial drug resistance and provided a guidance for choice of antimalarials for efficient treatment and prolonging the life span of the current effective antimalarial drugs.

Roles of Interferons in Pregnant Women with Dengue Infection: Protective or Dangerous Factors.

Dengue infection is a serious public health problem in tropical and subtropical areas. With the recent outbreaks of Zika disease and its reported correlation with microcephaly, the large number of pregnancies with dengue infection has become a serious concern. This review describes the epidemiological characteristics of pregnancy with dengue and the initial immune response to dengue infection, especially in IFNs production in this group of patients. Dengue is much more prevalent in pregnant women compared with other populations. The severity of dengue is correlated with the level of IFNs, while the serum IFN level must be sufficiently high to maintain the pregnancy and to inhibit virus replication.

[Secretion and Distribution of Rhoptry Protein 16 during Toxoplasma gondii Invasion into Host Cells].

Objective: To examine the secretion and localization of Toxoplasma gondii Rhoptry protein 16 （ROP16） during invasion of different strains of T. gondii into host cells. Methods: The Tgrop16 gene was amplified by PCR on the cDNA of T. gondii RH strain, subcloned into the plasmid pET-32a（+）, and expressed in Escherichia coli BL21（DE3） under the induction of isopropyl ß-D-1-thiogalactopyranoside. New Zealand rabbit was immuned with the expressed recombinant protein TgROP16 to produce polyclonal anti-TgROP16 antibody. The specificity and sensitivity of the polyclonal antibody were examined by Western blotting and indirect ELISA, respectively. The transcriptional and protein levels of Tgrop16 in T. gondii RH strain and Pru strain were determined by real-time PCR and Western blotting, respectively. The secretion and distribution of TgROP16 in human foreskin fibroblasts (HFFs) during the invasion by T. gondii RH strain and Pru strain were examined by indirect immunofluorescence assay (IFA). Results: Western blotting showed a specific band at M(r) of ～100 000, indicating that the specific rabbit-derived anti-TgROP16 polyclonal antibody was capable of recognizing TgROP16. Indirect ELISA revealed a titer of 1:25 600 for the antibody. The relative expression level of Tgrop16 in Pru strain［（7.786±0.206）］ was 7 times than that in RH strain［（1.000±0.110）］（P<0.05） as detected by real-time PCR, and TgROP16 protein level was higher in RH strain than in Pru strain. IFA showed that TgROP16 was localized on the apical complex of the unrecruited tachyzoite of T. gondii before invasion and was secreted out of the recruited tachyzoite after invasion. Conclusion: The anti-TgROP16 polyclonal antibody has high specificity and sensitivity. The TgROP16 protein level is higher in the RH strain than in the Pru strain. For both strains, TgROP16 is localized on the apical complex of the unrecruited tachyzoite before invasion and secreted out of the recruited tachyzoite during invasion.

[Research Advances on Toxoplasma gondii Virulence Mediating Factors].

Toxoplasma gondii strains are grouped into three clonal lineages (Types I, II , and III) with different virulence. T. gondii rhoptry proteins ROP18, ROP16, and ROP5 are the major virulence factors, which are secreted into the host cell by T. gondii during invasion and mediate the strain virulence. This paper reviews the research progress of T. gondii virulence mediating factors.

[Observation of the Feline Intestinal Epithelial Cell Infected with Toxoplasma gondii].

Small intestine samples of neonatal cat were aseptically collected from the jejunum-ileum region and digested with collagenase XI/dispase I. Immunohistochemistry results showed that feline intestinal epithelial cells were successfully isolated and could be cultured. Cytokeratin was positive in the cytoplasm of feline intestinal epithelial cells. The cells were infected with the bradyzoites of Toxoplasma gondii Prugniaud strain, and the rupture of the cells was observed on the 72nd day post-infection. The sexual stage of T. gondii did not occur, however.

Association of periodontal disease with oral cancer: a meta-analysis.

The objective is to evaluate the association of periodontal disease with the risk of oral cancer. Literature retrieval, selection and assessment, data extraction, and meta-analyses were performed according to the RevMan 5.0 guidelines. In the meta-analysis, we utilized random-effect model to pool the odds ratio (OR) according to the test of heterogeneity. A total of five eligible studies included 1,191 oral cancer patients and 1,992 healthy control subjects were analyzed. By meta-analysis, we found a significant association of periodontal disease with oral cancer [OR = 3.53, 95 % CI (1.52-8.23); P = 0.003]. Patients with periodontal disease have increased susceptibility to oral cancer.

Phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for the GTPase Rac2.

We have discovered that the enzyme phospholipase D2 (PLD2) binds directly to the small GTPase Rac2, resulting in PLD2 functioning as a guanine nucleotide exchange factor (GEF), because it switches Rac2 from the GDP-bound to the GTP-bound states. This effect is large enough to be meaningful (∼72% decrease for GDP dissociation and 300% increase for GTP association, both with PLD2), it has a half-time of ∼7 min, is enhanced with increasing PLD2 concentrations, and compares favorably with other known GEFs, such as Vav-1. The PLD2-Rac2 protein-protein interaction is sufficient for the GEF function, because it can be demonstrated in vitro with just recombinant proteins without lipid substrates, and a catalytically inactive lipase (PLD2-K758R) has GEF activity. Apart from this function, exogenous phosphatidic acid by itself (300 pM) increases GTP binding and enhances PLD2-K758R-mediated GTP binding (by ∼34%) but not GDP dissociation. Regarding the PLD2-Rac2 protein-protein association, it involves, for PLD2, residues 263-266 within a Cdc42/Rac interactive binding region in the PH domain, as well as the PX domain, and it involves, for Rac2, residue N17 within its Switch-1 region. PLD2's GEF function is demonstrated in living cells, because silencing PLD2 results in reduced Rac2 activity, whereas PLD2-initiated Rac2 activation enhances cell adhesion, chemotaxis, and phagocytosis. There are several known GEFs, but we report that this GEF is harbored in a phospholipase. The benefit to the cell is that PLD2 brings spatially separated molecules together in a membrane environment, ready for fast intracellular signaling and cell function.

[Preparation and purification of polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and its application in immunofluorescence localization].

OBJECTIVE: To prepare and purify polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and apply it to immunofluorescence localization. METHODS: The constructed recombinant plasmid pET32a-ROP2 was transformed into E. coli BL21 (DE3) and the protein was expressed under the condition of 0.5 mmol/L IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body, respectively. The washed inclusion bodies were dissolved in urea. New Zealand White rabbits were immunized with 200 microg purified recombinant ROP2 mixing with the same volume of Freund's adjuvant for 3 times at interval of 14 days and 19 days, respectively. Rabbit serum was collected at 10 days after the last immunization. Polyclonal antibody in rabbit serum was purified with HiTrap Protein G HP affinity purification column. Indirect ELISA and Western blotting were used to detect antibody titer and specificity of polyclonal antibody against the recombinant ROP2. The polyclonal antibody was used to the localization of ROP2 on the parasitophorous vacuole membrane in human foreskin fibroblasts infected by Toxoplasma tachyzoites by the immunofluorescence method. RESULTS: The recombinant ROP2 protein was obtained and specific rabbit-derived polyclonal antibody was prepared. Indirect ELISA confirmed that the rabbit-derived polyclonal antibody titer reached 1:102400, and the recombinant ROP2 protein was recognized by specific polyclonal antibody. Immunofluorescence localization test showed that the ROP2 protein was located on the parasitophorous vacuole membrane. CONCLUSION: The rabbit-derived polyclonal antibody against ROP2 is prepared, and used in immunofluorescence localization of ROP2 on parasitophorous vacuole membrane.

Toxoplasma gondii infection positively associated with schizophrenia: Evidences from UK Biobank cohort and case-controlled studies.

Toxoplasma gondii (T. gondii) is an opportunistic pathogen affecting about 1/3 of world population. While often asymptomatic in immunocompetent individuals, it can lead to severe toxoplasmosis in immunocompromised patients. Recent research has unveiled a potential link between T. gondii infection and neuropsychiatric diseases. We implemented both a cohort study and a case control study to further identify this association. In the cohort study, we analyzed data from the UK Biobank database, which included 8814 individuals tested for T. gondii SAG1 antibodies and free of neuropsychiatric disorders at baseline. Among them, 22.52% (n = 1985) tested positive for SAG1 antibody. Over an average follow-up period of 12.26 years, Cox proportional hazards models and logistic regression analysis revealed a significant association between the SAG1 seropositivity at baseline and the incidence of schizophrenia (HR: 5.89; 95% CI: 1.69-20.53). In our case-control study, 239 patients diagnosed with schizophrenia and 455 healthy individuals were involved. Using the modified agglutination test (MAT) to detect T. gondii antibodies, logistic regression analysis showed a higher prevalence of T. gondii infection among schizophrenia patients (10.04%) compared to healthy controls (3.74%). T. gondii infection emerged as a significant risk factor for schizophrenia (OR: 3.33; 95% CI: 1.68-6.61). However, our investigations did not reveal a robust association between T. gondii infection and other neuropsychiatric conditions, including Alzheimer's disease, dementia, anxiety, depression, neurodegenerative disorders, and peripheral neurological disorders such as neurological and plexus disorders.

Protein disulfide isomerase PDI8 is indispensable for parasite growth and associated with secretory protein processing in Toxoplasma gondii.

Protein disulfide isomerase, containing thioredoxin (Trx) domains, serves as a vital enzyme responsible for oxidative protein folding (the formation, reduction, and isomerization of disulfide bonds in newly synthesized proteins) in the endoplasmic reticulum (ER). However, the role of ER-localized PDI proteins in parasite growth and their interaction with secretory proteins remain poorly understood. In this study, we identified two ER-localized PDI proteins, TgPDI8 and TgPDI6, in Toxoplasma gondii. Conditional knockdown of TgPDI8 resulted in a significant reduction in intracellular proliferation and invasion abilities, leading to a complete block in plaque formation on human foreskin fibroblast monolayers, whereas parasites lacking TgPDI6 did not exhibit any apparent fitness defects. The complementation of TgPDI8 with mutant variants highlighted the critical role of the CXXC active site cysteines within its Trx domains for its enzymatic activity. By utilizing TurboID-based proximity labeling, we uncovered a close association between PDI proteins and canonical secretory proteins. Furthermore, parasites lacking TgPDI8 showed a significant reduction in the expression of secretory proteins, especially those from micronemes and dense granules. In summary, our study elucidates the roles of TgPDI8 and sets the stage for future drug discovery studies. IMPORTANCE: Apicomplexans, a phylum of intracellular parasites, encompass various zoonotic pathogens, including Plasmodium, Cryptosporidium, Toxoplasma, and Babesia, causing a significant economic burden on human populations. These parasites exhibit hypersensitivity to disruptions in endoplasmic reticulum (ER) redox homeostasis, necessitating the presence of ER-localized thioredoxin (Trx) superfamily proteins, particularly protein disulfide isomerase (PDI), for proper oxidative folding. However, the functional characteristics of ER-localized PDI proteins in Toxoplasma gondii remain largely unexplored. In this study, we identified two ER-localized proteins, namely, TgPDI8 and TgPDI6, and demonstrated the indispensable role of TgPDI8 in parasite survival. Through a comprehensive multi-omics analysis, we elucidated the crucial role of TgPDI8 in the processing of secretory proteins in T. gondii. Additionally, we introduced a novel ER-anchored TurboID method to label and identify canonical secretory proteins in T. gondii. This research opens up new avenues for understanding oxidative folding and the secretory pathway in apicomplexan parasites, laying the groundwork for future advancements in antiparasitic drug development.

Population-based cohort study of Toxoplasma gondii P22 antibody positivity correlation with anxiety.

BACKGROUND: Accumulating evidence suggests that latent infection with Toxoplasma gondii (T. gondii) is associated with a variety of neuropsychiatric and behavioral conditions. This research aims to explore the potential correlation between T. gondii antibody positivity and neuropsychiatric disorders through a comprehensive prospective cohort study. METHODS: The cohort study utilized the UK Biobank database to recruit 8814 individuals with no prior diagnosis of neuropsychiatric disorders. Cox proportional hazards models were employed to investigate the associations between T. gondii P22 antibody seropositivity (P22+) and the development of various types of neuropsychiatric disorders. RESULTS: Of the population, 14.65 % tested positive for T. gondii P22 antibody. The presence of T. gondii P22 antibody showed a slight inverse association with epilepsy (HR: 0.28; 95 % CI: 0.10-0.77), while it was positively associated with an increased risk of developing anxiety disorders (HR: 1.38; 95 % CI: 1.04-1.83). LIMITATIONS: The study sample consisted mostly of white British individuals aged 40 to 69 years old. Although we adjusted for potential confounders, there may be other unmeasured and residual confounding factors that could have influenced our reported associations. CONCLUSIONS: The findings suggested an increased risk of anxiety and potential evidence of epilepsy associated with T. gondii P22+. However, our analysis did not reveal an increased risk of several other neuropsychiatric conditions including Alzheimer's disease, dementia, substance abuse disorders, depression, and neurodegenerative disorders, associated with P22 antibody seropositivity.

Intestinal microbiota imbalance resulted by anti-Toxoplasma gondii immune responses aggravate gut and brain injury.

BACKGROUND: Toxoplasma gondii infection affects a significant portion of the global population, leading to severe toxoplasmosis and, in immunocompromised patients, even death. During T. gondii infection, disruption of gut microbiota further exacerbates the damage to intestinal and brain barriers. Therefore, identifying imbalanced probiotics during infection and restoring their equilibrium can regulate the balance of gut microbiota metabolites, thereby alleviating tissue damage. METHODS: Vimentin gene knockout (vim-/-) mice were employed as an immunocompromised model to evaluate the influence of host immune responses on gut microbiota balance during T. gondii infection. Behavioral experiments were performed to assess changes in cognitive levels and depressive tendencies between chronically infected vim-/- and wild-type (WT) mice. Fecal samples were subjected to 16S ribosomal RNA (rRNA) sequencing, and serum metabolites were analyzed to identify potential gut probiotics and their metabolites for the treatment of T. gondii infection. RESULTS: Compared to the immunocompetent WT sv129 mice, the immunocompromised mice exhibited lower levels of neuronal apoptosis and fewer neurobehavioral abnormalities during chronic infection. 16S rRNA sequencing revealed a significant decrease in the abundance of probiotics, including several species of Lactobacillus, in WT mice. Restoring this balance through the administration of Lactobacillus murinus and Lactobacillus gasseri significantly suppressed the T. gondii burden in the intestine, liver, and brain. Moreover, transplantation of these two Lactobacillus spp. significantly improved intestinal barrier damage and alleviated inflammation and neuronal apoptosis in the central nervous system. Metabolite detection studies revealed that the levels of various Lactobacillus-related metabolites, including indole-3-lactic acid (ILA) in serum, decreased significantly after T. gondii infection. We confirmed that L. gasseri secreted much more ILA than L. murinus. Notably, ILA can activate the aromatic hydrocarbon receptor signaling pathway in intestinal epithelial cells, promoting the activation of CD8+ T cells and the secretion of interferon-gamma. CONCLUSION: Our study revealed that host immune responses against T. gondii infection severely disrupted the balance of gut microbiota, resulting in intestinal and brain damage. Lactobacillus spp. play a crucial role in immune regulation, and the metabolite ILA is a promising therapeutic compound for efficient and safe treatment of T. gondii infection.

Toxoplasma gondii bradyzoite-specific BAG1 is nonessential for cyst formation due to compensation by other heat-shock proteins.

BACKGROUND: Toxoplasma gondii is an opportunistic pathogenic protozoan that infects all warm-blooded animals, including humans, and causes zoonotic toxoplasmosis. The bradyzoite antigen 1 (BAG1), known as heat-shock protein (HSP)30, is a specific antigen expressed during the early stage of T. gondii tachyzoite-bradyzoite conversion. METHODS: A bag1 gene knockout strain based on the T. gondii type II ME49 was constructed and designated as ME49Δbag1. The invasion, proliferation, and cyst formation efficiency in the cell model and survival in the mouse model were compared between the ME49 and ME49Δbag1 strains after infection. Quantitative polymerase chain reaction (qPCR) was used to detect the transcriptional level of important genes, and western-blot was used to detect protein levels. RESULTS: ME49Δbag1 displayed significantly inhibited cyst formation, although it was not completely blocked. During early differentiation induced by alkaline and starvation conditions in vitro, the proliferation of ME49Δbag1 was significantly accelerated relative to the ME49 strain. Meanwhile, the transcription of the HSP family and bradyzoite formation deficient 1 (bfd1) were significantly enhanced. The observed upregulation suggests a compensatory mechanism to counterbalance the impaired stress responses of T. gondii following bag1 knockout. On the other hand, the elevated transcription levels of several HSP family members, including HSP20, HSP21, HSP40, HSP60, HSP70, and HSP90, along with BFD1, implied the involvement of alternative regulatory factors in bradyzoite differentiation aside from BAG1. CONCLUSIONS: The data suggested that when bag1 was absent, the stress response of T. gondii was partially compensated by increased levels of other HSPs, resulting in the formation of fewer cysts. This highlighted a complex regulatory network beyond BAG1 influencing the parasite's transformation into bradyzoites, emphasizing the vital compensatory function of HSPs in the T. gondii life cycle adaptation.