Parasite Proliferation Research Articles

Toxoplasma gondii dense granule protein GRA24 drives MyD88-independent p38 MAPK activation, IL-12 production and induction of protective immunity.

The apicomplexan Toxoplasma gondii induces strong protective immunity dependent upon recognition by Toll-like receptors (TLR)11 and 12 operating in conjunction with MyD88 in the murine host. However, TLR11 and 12 proteins are not present in humans, inspiring us to investigate MyD88-independent pathways of resistance. Using bicistronic IL-12-YFP reporter mice on MyD88+/+ and MyD88-/- genetic backgrounds, we show that CD11c+MHCII+F4/80- dendritic cells, F4/80+ macrophages, and Ly6G+ neutrophils were the dominant cellular sources of IL-12 in both wild type and MyD88 deficient mice after parasite challenge. Parasite dense granule protein GRA24 induces p38 MAPK activation and subsequent IL-12 production in host macrophages. We show that Toxoplasma triggers an early and late p38 MAPK phosphorylation response in MyD88+/+ and MyD88-/- bone marrow-derived macrophages. Using the uracil auxotrophic Type I T. gondii strain cps1-1, we demonstrate that the late response does not require active parasite proliferation, but strictly depends upon GRA24. By i. p. inoculation with cps1-1 and cps1-1:Δgra24, we identified unique subsets of chemokines and cytokines that were up and downregulated by GRA24. Finally, we demonstrate that cps1-1 triggers a strong host-protective GRA24-dependent Th1 response in the absence of MyD88. Our data identify GRA24 as a major mediator of p38 MAPK activation, IL-12 induction and protective immunity that operates independently of the TLR/MyD88 cascade.

Expansion of Host Regulatory T Cells by Secreted Products of the Tapeworm Echinococcus multilocularis.

BackgroundAlveolar echinococcosis (AE), caused by the metacestode larval stage of the fox-tapeworm Echinococcus multilocularis, is a chronic zoonosis associated with significant modulation of the host immune response. A role of regulatory T-cells (Treg) in generating an immunosuppressive environment around the metacestode during chronic disease has been reported, but the molecular mechanisms of Treg induction by E. multilocularis, particularly parasite immunoregulatory factors involved, remain elusive so far.Methodology/Principal FindingsWe herein demonstrate that excretory/secretory (E/S) products of the E. multilocularis metacestode promote the formation of Foxp3+ Treg from CD4+ T-cells in vitro in a TGF-β-dependent manner, given that this effect was abrogated by treatment with antibody to mammalian TGF-β. We also show that host T-cells secrete elevated levels of the immunosuppressive cytokine IL-10 in response to metacestode E/S products. Within the E/S fraction of the metacestode we identified an E. multilocularis activin A homolog (EmACT) that displays significant similarities to mammalian Transforming Growth Factor-β (TGF-β/activin subfamily members. EmACT obtained from heterologous expression failed to directly induce Treg expansion from naïve T cells but required addition of recombinant host TGF-β to promote CD4+ Foxp3+ Treg conversion in vitro. Furthermore, like in the case of metacestode E/S products, EmACT-treated CD4+ T-cells secreted higher levels of IL-10. These observations suggest a contribution of EmACT to in vitro expansion of Foxp3+ Treg by the E. multilocularis metacestode. Using infection experiments we show that intraperitoneally injected metacestode tissue expands host Foxp3+ Treg, confirming the expansion of this cell type in vivo during parasite establishment.Conclusion/SignificanceIn conclusion, we herein demonstrate that E. multilocularis larvae secrete factors that induce the secretion of IL-10 by T-cells and contribute to the expansion of TGF-b-driven Foxp3+ Treg, a cell type that has been reported crucial for generating a tolerogenic environment to support parasite establishment and proliferation. Among the E/S factors of the parasite we identified a factor with structural and functional homologies to mammalian activin A which might play an important role in these activities.

Nanobiotechnological modules as molecular target tracker for the treatment and prevention of malaria: options and opportunity.

Malaria is one of the major infectious diseases that remains a constant challenge to human being mainly due to the emergence of drug-resistant strains of parasite and also the availability of drugs, which are non-specific for their pharmacodynamic activity and known to be associated with multiple side effects. The disease has acquired endemic proportions in tropical countries where the hygienic conditions are not satisfactory while the environmental conditions favor the proliferation of parasite and its transmission, particularly through the female anopheles. It is obvious that to square up the problems, there is a need for designing and development of more effective drugs, which can combat the drug-resistant strains of the parasite. Molecular biology of the parasite and its homing into host cellular tropics provide multiple drug targets that could judiciously be considered for engineering and designing of new generation antimalarial drugs and also drug delivery systems. Though the recent reports document that against malaria parasite the vaccine could be developed, nevertheless, due to smart mutational change overs by the parasite, it is able to bypass the immune surveillance. The developed vaccine therefore failed to assure absolute protection against the malarial infection. In the conventional mode of treatment antimalarial drugs, the dose and dosage regimen that is followed at large crops up the contraindicative manifestations, and hence compromising the effective treatment. The emerging trends and new updates in contemporary biological sciences, material sciences, and drug delivery domain have enabled us with the availability of a multitude of mode and modules which could plunge upon the nanotechnology in particular to treat this challenging infection. The nanotechnology-based option may be tuned or customized as per the requirements to mark and target i.e. the infected RBCs, for targeted drug delivery. Graphical abstract

Inhibitory action of phenothiazinium dyes against Neospora caninum

Neospora caninum is an Apicomplexan parasite related to important losses in livestock, causing abortions and decreased fertility in affected cows. Several chemotherapeutic strategies have been developed for disease control; however, no commercial treatment is available. Among the candidate drugs against neosporosis, phenothiazinium dyes, offer a low cost-efficient approach to parasite control. We report the anti-parasitic effects of the phenothiaziums Methylene Blue (MB), New Methylene Blue (NMB), 1,9–Dimethyl Methylene Blue (DMMB) and Toluidine Blue O (TBO) on N. caninum, using in vitro and in vivo models. The dyes inhibited parasite proliferation at nanomolar concentrations (0.019–1.83 μM) and a synergistic effect was achieved when Methylene Blue was combined with New Methylene Blue (Combination Index = 0.84). Moreover, the phenothiazinium dyes improved parasite clearance when combined with Pyrimethamine (Pyr). Combination of Methylene Blue + 1,9–Dimethyl Methylene Blue demonstrated superior efficacy compared to Pyrimethamine based counterparts in an in vivo model of infection. We also observed that Methylene Blue, New Methylene Blue and 1,9–Dimethyl Methylene Blue increased by 5000% the reactive oxygen species (ROS) levels in N. caninum tachyzoites. Phenothiazinium dyes represent an accessible group of candidates with the potential to compound future formulations for neosporosis control.

Productivity and bioactivity of enokipodins A-D of Flammulina rossica and Flammulina velutipes.

Enokipodins are antimicrobial sesquiterpenes produced by Flammulina velutipes in a mycelial culture medium. To date, enokipodin production has not been reported in other members of the genus Flammulina. Hence, in this study, the production of enokipodins A, B, C, and D by F. velutipes and F. rossica was investigated. Some strains of F. rossica were confirmed to produce at least one of the four enokipodins in the culture medium. However, some strains of F. velutipes did not produce any of the enokipodins. In an antibacterial assay using liquid medium, enokipodin B showed the strongest growth inhibitory activity against Bacillus subtilis among the four types of enokipodins. Enokipodin B inhibited the spore germination of some plant pathogenic fungi. Enokipodins B and D exerted moderate anti-proliferative activity against some cancer cell lines, and enokipodins A and C inhibited the proliferation of the malarial parasite, Plasmodium falciparum.

VEGF Production Is Regulated by the AKT/ERK1/2 Signaling Pathway and Controls the Proliferation of Toxoplasma gondii in ARPE-19 Cells.

The retina is the primary site of Toxoplasma gondii infection in the eye, and choroidal neovascularization in ocular toxoplasmosis is one of the most important causes of visual impairment. Vascular endothelial growth factor (VEGF) is one of the key regulators of blood vessel development, however, little is known about the mechanisms of T. gondii-induced VEGF production in ocular toxoplasmosis. Here, we investigate the effect of T. gondii on VEGF production regulation in human retinal pigment epithelium ARPE-19 cells and attempted to unveil the underlying mechanism of this event by focusing on the interaction between parasite and the selected host intracellular signaling pathways. T. gondii infection increased the expression of VEGF mRNA and protein in ARPE-19 cells in parasite burden- and infection time-dependent manner. The proportional increase of VEGF upstream regulators, HIF-1α and HO-1, was also observed. T. gondii induced the activation of host p-AKT, p-ERK1/2, and p-p38 MAPK in ARPE-19 cells in a parasite-burden dependent manner. However, VEGF expression decreased after the pre-treatment with PI3K inhibitors (LY294002 and GDC-0941), ERK1/2 inhibitor (PD098059), and p38 MAPK inhibitor (SB203580), but not JNK inhibitor (SP600125), in a dose-dependent manner. The anti-VEGF agent bevacizumab or VEGF siRNA transfection prominently inhibited the activation of p-AKT and p-ERK1/2, but not p-p38 MAPK and JNK1/2 in T. gondii-infected ARPE-19 cells. Bevacizumab treatment or VEGF siRNA transfection significantly inhibited the proliferation of T. gondii tachyzoites in the host cell, dose-dependently, but not invasion of parasites. VEGF-receptor 2 (VEGF-R2) antagonist, SU5416, attenuated VEGF production and tachyzoite proliferation in T. gondii-infected ARPE-19 cells in a dose-dependent manner. Collectively, T. gondii prominently induces VEGF production in ARPE-19 cells, and VEGF and AKT/ERK1/2 signaling pathways mutually regulate each other in T. gondii-infected ARPE-19 cells, but not p38 MAPK and JNK1/2 signaling pathways. VEGF and VEGF-R2 control the parasite proliferation in T. gondii-infected ARPE-19 cells. From this study, we revealed the putative mechanisms for VEGF induction as well as the existence of positive feedback between VEGF and PI3K/MAPK signaling pathways in T. gondii-infected retinal pigment epithelium.

Accounting for red blood cell accessibility reveals distinct invasion strategies in Plasmodium falciparum strains.

The growth of the malaria parasite Plasmodium falciparum in human blood causes all the symptoms of malaria. To proliferate, non-motile parasites must have access to susceptible red blood cells, which they invade using pairs of parasite ligands and host receptors that define invasion pathways. Parasites can switch invasion pathways, and while this flexibility is thought to facilitate immune evasion, it may also reflect the heterogeneity of red blood cell surfaces within and between hosts. Host genetic background affects red blood cell structure, for example, and red blood cells also undergo dramatic changes in morphology and receptor density as they age. The in vivo consequences of both the accessibility of susceptible cells, and their heterogeneous susceptibility, remain unclear. Here, we measured invasion of laboratory strains of P. falciparum relying on distinct invasion pathways into red blood cells of different ages. We estimated invasion efficiency while accounting for red blood cell accessibility to parasites. This approach revealed different tradeoffs made by parasite strains between the fraction of cells they can invade and their invasion rate into them, and we distinguish “specialist” strains from “generalist” strains in this context. We developed a mathematical model to show that generalist strains would lead to higher peak parasitemias in vivo compared to specialist strains with similar overall proliferation rates. Thus, the ecology of red blood cells may play a key role in determining the rate of P. falciparum parasite proliferation and malaria virulence.

Th1-Th2 Cross-Regulation Controls Early Leishmania Infection in the Skin by Modulating the Size of the Permissive Monocytic Host Cell Reservoir

The impact of T helper (Th) 1 versus Th2 immunity onintracellular infections is attributed to classical versus alternative activation of macrophages leading to resistance or susceptibility. However, observations in multiple infectious settings demonstrate deficiencies in mediators of Th1-Th2 immunity, which have paradoxical or no impact. We report that prior to influencing activation, Th1/Th2 immunity first controls the size of the permissive host cell reservoir. During early Leishmania infection of the skin, IFN-γ- or STAT6-mediated changes in phagocyte activation were counteracted by changes in IFN-γ-mediated recruitment of permissive CCR2+ monocytes. Monocytes were required for early parasite expansion andacquired an alternatively activated phenotype despite the Th1 dermal environment required fortheir recruitment. Surprisingly, STAT6 did not enhance intracellular parasite proliferation, but rather modulated the size and permissiveness of the monocytic host cell reservoir via regulation of IFN-γ and IL-10. These observations expand our understanding of the Th1-Th2 paradigm during infection.

A Novel wx2 Gene of Toxoplasma gondii Inhibits the Parasitic Invasion and Proliferation in vitro and Attenuates Virulence in vivo via Immune Response Modulation.

Toxoplasma gondii (T. gondii) is an obligate intracellular apicomplexan protozoan that can parasitize most warm-blooded animals and cause severe diseases in immunocompromised individuals or fetal abnormalities in pregnant woman. The treatment of toxoplamosis has been limited by effective drugs. Our previous work indicated that the novel gene wx2 of T. gondii may serve as a vaccine antigen candidate. To further investigate the molecular functions of wx2 in highly virulent T. gondii (RH strain), a wx2 gene deletion mutant RH strain (KO-wx2) was established using CRISPR-Cas9. The phynotype of KO-wx2 was analyzed by plaque, invasion, and replication assays in vitro as well as in vivo virulence assays. The results indicated that the targeted deletion of the wx2 gene significantly inhibited in vitro parasite growth and replication in the host cells as well as attenuated parasite virulence in the mouse model. Notably, the percentage of pro-inflammatory factors of interferon gamma (IFN-γ) and interlukin-17A (IL-17A) and anti-inflammatory factor of interlukin-10 (IL-10) in the lymph nodes were upregulated in mice infected with the KO-wx2 strain. Our data suggested that the wx2 gene plays an important role in the process of the parasite’s life cycle and virulence in mice. In addition, it also plays an important role in the host’s immunity reaction, mainly via Th1 and Th17 cellular immunity, not Th2.

Screening of compound libraries for inhibitors of Toxoplasma growth and invasion.

Toxoplasma gondii can infect virtually all warm-blooded animals, including humans. It can differentiate between rapidly replicating tachyzoites that cause acute infection and slowly growing bradyzoites in tissue cysts. Treatment options for toxoplasmosis are challenging because current therapies cannot eradicate the latent T. gondii infection that is mainly caused by the bradyzoite forms. Accordingly, recurrence of infection is a problem for immunocompromised patients and congenitally infected patients. Protein kinases have been widely studied in eukaryotic cells, and while little is known about signaling in Toxoplasma infection, it is likely that protein kinases play a key role in parasite proliferation, differentiation, and probably invasion. To identify optimized new kinase inhibitors for drug development against T. gondii, we screened a library of kinase inhibitor compounds for anti-Toxoplasma activity and host cell cytotoxicity. Pyrimethamine served as a positive control and 0.5% DMSO was used as a negative control. Among the 80 compounds screened, 6 compounds demonstrated ≥ 80% parasite growth inhibition at concentrations at which 5 compounds did not suppress host cell viability, while 3 kinase inhibitors (Bay 11-7082, Tyrphostin AG 1295 and PD-98059) had suppressive effects individually on parasite growth and host cell invasion, but did not strongly induce bradyzoite formation.Electronic supplementary materialThe online version of this article (10.1007/s00436-020-06673-9) contains supplementary material, which is available to authorized users.

Investigation the Effect of Silver Nanoparticles and Bioresonance Wave Radiation on Leishmania major: An In Vitro Study

Introduction: Cutaneous leishmaniasis (CL) is one of the infectious diseases and health problems in tropical regions. Glucantime is commonly used to treat CL and it, not only has some side effects but also observation shows the drug resistance of some of the various Leishmania species. Therefore, the aim of this study was to investigate the effect of silver nanoparticles (AgNPs) and bioresonance waves on Leishmania, in vitro. Materials and Methods: In the present experimental study, Leishmania major promastigotes were cultured in RPMI-1640 supplemented with 10% FBS and 1% penicillin and streptomycin at 23°C. After 6 days, the parasites achieved stationary phases of promastigotes, Then the effects of different concentrations of AgNPs (1, 3, 5, 10 and 25 μg/mL) and different radiation times of bioresonance wave (5 and 20 minutes) were investigated. Herein, the effects of various treatment on parasites proliferation were evaluated with live promastigotes counting after 24, 48 and 72 hours treatment. Results: The parasite count showed that the various concentrations of AgNPs, radiation of bioresonance wave and combination significantly decreased the numbers of live promastigotes over time compared with the control group after 72 hours. The highest antileishmanial activity was seen for AgNPs at concentration of 1 μg/mL when combined with 20 minutes radiation of bioresonance wave (proliferation inhibition: 79.92%). Conclusions: Based on our result, AgNPs and bioresonance waves are potent antileishmanial agents. The authors declare that the more studies should be done.

Molecular Target Validation of Aspartate Transcarbamoylase from Plasmodium falciparum by Torin 2

Malaria is a tropical disease that kills about half a million people around the world annually. Enzymatic reactions within pyrimidine biosynthesis have been proven to be essential for Plasmodium proliferation. Here we report on the essentiality of the second enzymatic step of the pyrimidine biosynthesis pathway, catalyzed by aspartate transcarbamoylase (ATC). Crystallization experiments using a double mutant ofPlasmodium falciparum ATC (PfATC) revealed the importance of the mutated residues for enzyme catalysis. Subsequently, this mutant was employed in protein interference assays (PIAs), which resulted in inhibition of parasite proliferation when parasites transfected with the double mutant were cultivated in medium lacking an excess of nutrients, including aspartate. Addition of 5 or 10 mg/L of aspartate to the minimal medium restored the parasites' normal growth rate. In vitro and whole-cell assays in the presence of the compound Torin 2 showed inhibition of specific activity and parasite growth, respectively. In silico analyses revealed the potential binding mode of Torin 2 to PfATC. Furthermore, a transgenic ATC-overexpressing cell line exhibited a 10-fold increased tolerance to Torin 2 compared with control cultures. Taken together, our results confirm the antimalarial activity of Torin 2, suggesting PfATC as a target of this drug and a promising target for the development of novel antimalarials.

Screening the Medicines for Malaria Venture Pathogen Box for invasion and egress inhibitors of the blood stage of Plasmodium falciparum reveals several inhibitory compounds

With emerging resistance to frontline treatments, it is vital that new drugs are identified to target Plasmodium falciparum. One of the most critical processes during parasites asexual lifecycle is the invasion and subsequent egress of red blood cells (RBCs). Many unique parasite ligands, receptors and enzymes are employed during egress and invasion that are essential for parasite proliferation and survival, therefore making these processes druggable targets. To identify potential inhibitors of egress and invasion, we screened the Medicines for Malaria Venture Pathogen Box, a 400 compound library against neglected tropical diseases, including 125 with antimalarial activity. For this screen, we utilised transgenic parasites expressing a bioluminescent reporter, nanoluciferase (Nluc), to measure inhibition of parasite egress and invasion in the presence of the Pathogen Box compounds. At a concentration of 2 µM, we found 15 compounds that inhibited parasite egress by >40% and 24 invasion-specific compounds that inhibited invasion by >90%. We further characterised 11 of these inhibitors through cell-based assays and live cell microscopy, and found two compounds that inhibited merozoite maturation in schizonts, one compound that inhibited merozoite egress, one compound that directly inhibited parasite invasion and one compound that slowed down invasion and arrested ring formation. The remaining compounds were general growth inhibitors that acted during the egress and invasion phase of the cell cycle. We found the sulfonylpiperazine, MMV020291, to be the most invasion-specific inhibitor, blocking successful merozoite internalisation within human RBCs and having no substantial effect on other stages of the cell cycle. This has significant implications for the possible development of an invasion-specific inhibitor as an antimalarial in a combination based therapy, in addition to being a useful tool for studying the biology of the invading parasite.

Activities of Endochin-Like Quinolones Against in vitro Cultured Besnoitia besnoiti Tachyzoites.

Endochin-like quinolones (ELQs) potently inhibit the proliferation of Plasmodium, Toxoplasma, Neospora, and Babesia by targeting the cytochrome b Qo and Qi sites and interfering with oxidative phosphorylation and pyrimidine biosynthesis. The activities of 14 different ELQs were assessed against B. besnoiti tachyzoites grown in human foreskin fibroblasts (HFF) by quantitative real time PCR. The values for 50% proliferation inhibition (IC50) of five ELQs were determined in a 3-days growth assay after an initial screen of 12 ELQs at 0.01, 0.1, and 1 μM. The IC50s of ELQ-121, -136, and -316 were 0.49, 2.36, and 7.97 nM, respectively. The IC50s of ELQs tested against B. besnoiti were higher than IC50s previously observed for P. falciparum and T. gondii. However, the B. besnoiti cytochrome b sequence and the predicted Qo and Qi ELQ binding sites in the Toxoplasma, Neospora, and Besnoitia cytochrome b are virtually identical, suggesting that the differences in ELQ susceptibility are not due to variations in the substrate binding sites. TEM of ELQ-treated parasites primarily demonstrated alterations within the parasite mitochondrion, profound thickening of the nuclear membrane, as well as increased vacuolization within the tachyzoite cytoplasm. Long-term treatment assays of intracellular B. besnoiti with ELQs for up to 20 days followed by the release of drug pressure caused a substantial delay in parasite growth and proliferation while ELQs were present, but parasite proliferation resumed days after ELQs were removed. Interestingly, structural alterations persisted after ELQ removal and parasite proliferation was slowed. These findings provide a basis for further in vivo studies of ELQs as therapeutic options against B. besnoiti infection.

The Putative TCP-1 Chaperonin Is an Important Player Involved in Sialic Acid-Dependent Host Cell Invasion by Toxoplasma gondii.

Host cell invasion by Toxoplasma gondii is crucial for the survival and proliferation of parasite. The process of T. gondii tachyzoite invasion requires interaction between parasite proteins and receptors on the surface of host cells. Sialic acid is one of the important receptors for host cell invasion by T. gondii. However, the parasite-derived proteins interacting with sialic acid have not been well characterized. In this study, a novel protein named putative TCP-1 chaperonin (TGME49_318410) in T. gondii (TgTCP-1) was targeted and characterized. TgTCP-1 protein colocalized with MIC3 protein, which could be secreted from T. gondii tachyzoites, and this protein showed a specific binding activity to sialic acid, and DC and Vero cells in vitro. The binding of TgTCP-1 protein to DC and Vero cells were inhibited by either pre-incubation with free sialic acid or neuraminidase treatment of the cells. Moreover, a significant reduction of T. gondii invasion in Vero cells was observed after pre-incubation of the cells with recombinant TgTCP-1 protein. These results illustrated that TgTCP-1 is an important molecule involved in sialic acid-dependent host cell invasion by T. gondii.

Targeting Plasmodium with constrained peptides and peptidomimetics.

Malaria remains a worldwide health concern with an estimated quarter of a billion people infected and nearly half a million deaths annually. Malaria is caused by a parasite infection from Plasmodium strains which are transmitted from mosquitoes into the human host. Although several small molecule inhibitors have been found to target the early stages of transmission and prevent parasite proliferation, multiple drug resistant parasite strains have emerged and drug resistance remains a major hurdle. As an alternative to small molecule inhibition, several peptide-based therapeutics have been explored for their potential as antimalarial compounds. Chemically constrained peptides or peptidomimetics were developed to target large binding interfaces of parasite-based proteins that have historically been difficult to selectively inhibit using small molecules. Here, we review ongoing research aimed at developing constrained peptides targeting protein-protein interactions pertinent to malaria pathogenesis. These targets include Falcipain-2, the J domain of CDPK1, myosin A tail domain interacting protein, the PKA signaling pathway, and an unclear signaling pathway involving angiotensin-derived peptides. Diverse synthetic methods were also used for each target. Merging parasite biology with synthetic strategies may provide new opportunities to develop alternative methods for uncovering novel antimalarials and may offer an alternate source for targeting drug-resistant parasite strains.

Plasmodium Condensin Core Subunits SMC2/SMC4 Mediate Atypical Mitosis and Are Essential for Parasite Proliferation and Transmission

SummaryCondensin is a multi-subunit protein complex regulating chromosome condensation and segregation during cell division. In Plasmodium spp., the causative agent of malaria, cell division is atypical and the role of condensin is unclear. Here we examine the role of SMC2 and SMC4, the core subunits of condensin, during endomitosis in schizogony and endoreduplication in male gametogenesis. During early schizogony, SMC2/SMC4 localize to a distinct focus, identified as the centromeres by NDC80 fluorescence and chromatin immunoprecipitation sequencing (ChIP-seq) analyses, but do not form condensin I or II complexes. In mature schizonts and during male gametogenesis, there is a diffuse SMC2/SMC4 distribution on chromosomes and in the nucleus, and both condensin I and condensin II complexes form at these stages. Knockdown of smc2 and smc4 gene expression reveals essential roles in parasite proliferation and transmission. The condensin core subunits (SMC2/SMC4) form different complexes and may have distinct functions at various stages of the parasite life cycle.

Characterization of the apicoplast-localized enzyme TgUroD in Toxoplasma gondii reveals a key role of the apicoplast in heme biosynthesis

Apicomplexan parasites such as Toxoplasma gondii possess an unusual heme biosynthesis pathway whose enzymes localize to the mitochondrion, cytosol, or apicoplast, a nonphotosynthetic plastid present in most apicomplexans. To characterize the involvement of the apicoplast in the T. gondii heme biosynthesis pathway, we investigated the role of the apicoplast-localized enzyme uroporphyrinogen III decarboxylase (TgUroD). We found that TgUroD knockdown impaired parasite proliferation, decreased free heme levels in the parasite, and decreased the abundance of heme-containing c-type cytochrome proteins in the parasite mitochondrion. We validated the effects of heme loss on mitochondrial cytochromes by knocking down cytochrome c/c1 heme lyase 1 (TgCCHL1), a mitochondrial enzyme that catalyzes the covalent attachment of heme to c-type cytochromes. TgCCHL1 depletion reduced parasite proliferation and decreased the abundance of c-type cytochromes. We further sought to characterize the overall importance of TgUroD and TgCCHL1 for both mitochondrial and general parasite metabolism. TgUroD depletion decreased cellular ATP levels, mitochondrial oxygen consumption, and extracellular acidification rates. By contrast, depletion of TgCCHL1 neither diminished ATP levels in the parasite nor impaired extracellular acidification rate, but resulted in specific defects in mitochondrial oxygen consumption. Together, our results indicate that the apicoplast has a key role in heme biology in T. gondii and is important for both mitochondrial and general parasite metabolism. Our study highlights the importance of heme and its synthesis in these parasites.

Toxoplasma gondii Recruits Factor H and C4b-Binding Protein to Mediate Resistance to Serum Killing and Promote Parasite Persistence in vivo.

Regulating complement is an important step in the establishment of infection by microbial pathogens. Toxoplasma gondii actively resists complement-mediated killing in non-immune human serum (NHS) by inactivating C3b, however the precise molecular basis is unknown. Here, a flow cytometry-based C3b binding assay demonstrated that Type II strains had significantly higher levels of surface-bound C3b than Type I strains. However, both strains efficiently inactivated C3b and were equally resistant to serum killing, suggesting that resistance is not strain-dependent. Toxoplasma activated both the lectin (LP) and alternative (AP) pathways, and the deposition of C3b was both strain and lectin-dependent. A flow cytometry-based lectin binding assay identified strain-specific differences in the level and heterogeneity of surface glycans detected. Specifically, increased lectin-binding by Type II strains correlated with higher levels of the LP recognition receptor mannose binding lectin (MBL). Western blot analyses demonstrated that Toxoplasma recruits both classical pathway (CP) and LP regulator C4b-binding proteins (C4BP) and AP regulator Factor H (FH) to the parasite surface to inactivate bound C3b–iC3b and C3dg and limit formation of the C5b-9 attack complex. Blocking FH and C4BP contributed to increased C5b-9 formation in vitro. However, parasite susceptibility in vitro was only impacted when FH was blocked, indicating that down regulation of the alternative pathway by FH may be more critical for parasite resistance. Infection of C3 deficient mice led to uncontrolled parasite growth, acute mortality, and reduced antibody production, indicating that both the presence of C3, and the ability of the parasite to inactivate C3, was protective. Taken together, our results establish that Toxoplasma regulation of the complement system renders mice resistant to acute infection by limiting parasite proliferation in vivo, but susceptible to chronic infection, with all mice developing transmissible cysts to maintain its life cycle.

The outcome of arginase activity inhibition in BALB/c mice hosting Leishmania tropica.

Two species of Leishmania (L), L.tropica and L.major, are among the main causative agents of cutaneous leishmaniasis. Arginase (ARG) is an essential enzyme for cell growth, thus an attractive drug target. In this study, we tried to survey the inhibitory impact of ARG by nor-NOHA (N-ω-hydroxy-L-nor-arginine) on in vivo infection caused by L.tropica. BALB/c mice were inoculated with L.tropicaEGFP-LUC (Ltrop) or L.majorEGFP-LUC (Lmj) and then were treated by nor-NOHA. ARG inhibitor only indicated a delay in generation of a cutaneous lesion in inoculated footpad with nor-NOHA-Ltrop and nor-NOHA-Lmj. ARG activity has been significantly reduced in nor-NOHA-Ltrop group. In this group, ARG activity inhibition correlated with increased levels of nitric oxide (NO). In both inoculated mice with Ltrop or Lmj, parasite load showed a significant decrease at later steps during the CL course post-treatment. In vivo bioluminescence intensity did not show any ARG's inhibitory effect on treated-Ltrop. The findings verified that the ARG activity may partially control the L.tropica infection in BALB/c mice through reduction of parasite proliferation and parasite killing through NO generation. This effect is dose-dependent.