From TgO/GABA-AT, GABA, and T-263 Mutant to Conception of Toxoplasma.

Toxoplasma gondii causes morbidity, mortality, and disseminates widely via cat sexual stages. Here, we find T. gondii ornithine aminotransferase (OAT) is conserved across phyla. We solve TgO/GABA-AT structures with bound inactivators at 1.55 Å and identify an inactivator selective for TgO/GABA-AT over human OAT and GABA-AT. However, abrogating TgO/GABA-AT genetically does not diminish replication, virulence, cyst-formation, or eliminate cat's oocyst shedding. Increased sporozoite/merozoite TgO/GABA-AT expression led to our study of a mutagenized clone with oocyst formation blocked, arresting after forming male and female gametes, with "Rosetta stone"-like mutations in genes expressed in merozoites. Mutations are similar to those in organisms from plants to mammals, causing defects in conception and zygote formation, affecting merozoite capacitation, pH/ionicity/sodium-GABA concentrations, drawing attention to cyclic AMP/PKA, and genes enhancing energy or substrate formation in TgO/GABA-AT-related-pathways. These candidates potentially influence merozoite's capacity to make gametes that fuse to become zygotes, thereby contaminating environments and causing disease.

Limited Impact of the Protein Corona on the Cellular Uptake of PEGylated Zein Micelles by Melanoma Cancer Cells.

The formation of a protein layer "corona" on the nanoparticle surface upon entry into a biological environment was shown to strongly influence the interactions with cells, especially affecting the uptake of nanomedicines. In this work, we present the impact of the protein corona on the uptake of PEGylated zein micelles by cancer cells, macrophages, and dendritic cells. Zein was successfully conjugated with poly(ethylene glycol) (PEG) of varying chain lengths (5K and 10K) and assembled into micelles. Our results demonstrate that PEGylation conferred stealth effects to the zein micelles. The presence of human plasma did not impact the uptake levels of the micelles by melanoma cancer cells, regardless of the PEG chain length used. In contrast, it decreased the uptake by macrophages and dendritic cells. These results therefore make PEGylated zein micelles promising as potential drug delivery systems for cancer therapy.

An Exaggerated Immune Response in Female BALB/c Mice Controls Initial Toxoplasma gondii Multiplication but Increases Mortality and Morbidity Relative to Male Mice.

Studies indicate that female mice are more susceptible to T. gondii infection, as defined by higher mortality rates in comparison to male mice. However, whether this is due to an inability to control initial parasite multiplication or due to detrimental effects of the immune system has not been determined. Therefore, the following studies were undertaken to determine the influence of sex on early parasite multiplication and the immune response during T. gondii infection and to correlate this with disease outcome. Early parasite replication was studied through applying an in vivo imaging system (IVIS) with luciferase expressing T. gondii. In parallel immunological events were studied by cytometric bead array to quantify key immunological mediators. The results confirmed the previous findings that female mice are more susceptible to acute infection, as determined by higher mortality rates and weight loss compared with males. However, conflicting with expectations, female mice had lower parasite burdens during the acute infection than male mice. Female mice also exhibited significantly increased production of Monocyte Chemoattractant Protein-1 (MCP-1), Interferon (IFN)-γ, and Tumour Necrosis Factor (TNF)-α than male mice. MCP-1 was found to be induced by T. gondii in a dose dependent manner suggesting that the observed increased levels detected in female mice was due to a host-mediated sex difference rather than due to parasite load. However, MCP-1 was not affected by physiological concentration of estrogen or testosterone, indicating that MCP-1 differences observed between the sexes in vivo are due to an as yet unidentified intermediary factor that in turn influences MCP-1 levels. These results suggest that a stronger immune response in female mice compared with male mice enhances their ability to control parasite replication but increases their morbidity and mortality.

Multi-Omics Studies Demonstrate Toxoplasma gondii-Induced Metabolic Reprogramming of Murine Dendritic Cells.

Toxoplasma gondii is capable of actively invading almost any mammalian cell type including phagocytes. Early events in phagocytic cells such as dendritic cells are not only key to establishing parasite infection, but conversely play a pivotal role in initiating host immunity. It is now recognized that in addition to changes in canonical immune markers and mediators, alteration in metabolism occurs upon activation of phagocytic cells. These metabolic changes are important for supporting the developing immune response, but can affect the availability of nutrients for intracellular pathogens including T. gondii. However, the interaction of T. gondii with these cells and particularly how infection changes their metabolism has not been extensively investigated. Herein, we use a multi-omics approach comprising transcriptomics and metabolomics validated with functional assays to better understand early events in these cells following infection. Analysis of the transcriptome of T. gondii infected bone marrow derived dendritic cells (BMDCs) revealed significant alterations in transcripts associated with cellular metabolism, activation of T cells, inflammation mediated chemokine and cytokine signaling pathways. Multivariant analysis of metabolomic data sets acquired through non-targeted liquid chromatography mass spectroscopy (LCMS) identified metabolites associated with glycolysis, the TCA cycle, oxidative phosphorylation and arginine metabolism as major discriminants between control uninfected and T. gondii infected cells. Consistent with these observations, glucose uptake and lactate dehydrogenase activity were upregulated in T. gondii infected BMDC cultures compared with control BMDCs. Conversely, BMDC mitochondrial membrane potential was reduced in T. gondii-infected cells relative to mitochondria of control BMDCs. These changes to energy metabolism, similar to what has been described following LPS stimulation of BMDCs and macrophages are often termed the Warburg effect. This metabolic reprogramming of cells has been suggested to be an important adaption that provides energy and precursors to facilitate phagocytosis, antigen processing and cytokine production. Other changes to BMDC metabolism are evident following T. gondii infection and include upregulation of arginine degradation concomitant with increased arginase-1 activity and ornithine and proline production. As T. gondii is an arginine auxotroph the resultant reduced cellular arginine levels are likely to curtail parasite multiplication. These results highlight the complex interplay of BMDCs and parasite metabolism within the developing immune response and the consequences for adaptive immunity and pathogen clearance.

Acanthamoeba Activates Macrophages Predominantly through Toll-Like Receptor 4- and MyD88-Dependent Mechanisms To Induce Interleukin-12 (IL-12) and IL-6.

Acanthamoeba castellanii is a ubiquitous free-living amoeba with a worldwide distribution that can occasionally infect humans, causing particularly severe infections in immunocompromised individuals. Dissecting the immunology of Acanthamoeba infections has been considered problematic due to the very low incidence of disease, despite the high exposure rates. While macrophages are acknowledged as playing a significant role in Acanthamoeba infections, little is known about how this facultative parasite influences macrophage activity. Therefore, in this study we investigated the effects of Acanthamoeba on the activation of resting macrophages. Consequently, murine bone marrow-derived macrophages were cocultured with trophozoites of either the laboratory Neff strain or a clinical isolate of A. castellaniiIn vitro real-time imaging demonstrated that trophozoites of both strains often established evanescent contact with macrophages. Both Acanthamoeba strains induced a proinflammatory macrophage phenotype characterized by the significant production of interleukin-12 (IL-12) and IL-6. However, macrophages cocultured with the clinical isolate of Acanthamoeba produced significantly less IL-12 and IL-6 than the Neff strain. The utilization of macrophages derived from MyD88-, TRIF-, Toll-like receptor 2 (TLR2)-, TLR4-, and TLR2/4-deficient mice indicated that Acanthamoeba-induced proinflammatory cytokine production was through MyD88-dependent, TRIF-independent, TLR4-induced events. This study shows for the first time the involvement of TLRs expressed on macrophages in the recognition of and response to Acanthamoeba trophozoites.

New paradigms for understanding and step changes in treating active and chronic, persistent apicomplexan infections.

Toxoplasma gondii, the most common parasitic infection of human brain and eye, persists across lifetimes, can progressively damage sight, and is currently incurable. New, curative medicines are needed urgently. Herein, we develop novel models to facilitate drug development: EGS strain T. gondii forms cysts in vitro that induce oocysts in cats, the gold standard criterion for cysts. These cysts highly express cytochrome b. Using these models, we envisioned, and then created, novel 4-(1H)-quinolone scaffolds that target the cytochrome bc1 complex Qi site, of which, a substituted 5,6,7,8-tetrahydroquinolin-4-one inhibits active infection (IC50, 30 nM) and cysts (IC50, 4 µM) in vitro, and in vivo (25 mg/kg), and drug resistant Plasmodium falciparum (IC50, <30 nM), with clinically relevant synergy. Mutant yeast and co-crystallographic studies demonstrate binding to the bc1 complex Qi site. Our results have direct impact on improving outcomes for those with toxoplasmosis, malaria, and ~2 billion persons chronically infected with encysted bradyzoites.

MAP kinase phosphatase-2 plays a key role in the control of infection with Toxoplasma gondii by modulating iNOS and arginase-1 activities in mice.

The dual specific phosphatase, MAP kinase phosphatase-2 (MKP-2) has recently been demonstrated to negatively regulate macrophage arginase-1 expression, while at the same time to positively regulate iNOS expression. Consequently, MKP-2 is likely to play a significant role in the host interplay with intracellular pathogens. Here we demonstrate that MKP-2(-/-) mice on the C57BL/6 background have enhanced susceptibility compared with wild-type counterparts following infection with type-2 strains of Toxoplasma gondii as measured by increased parasite multiplication during acute infection, increased mortality from day 12 post-infection onwards and increased parasite burdens in the brain, day 30 post-infection. MKP-2(-/-) mice did not, however, demonstrate defective type-1 responses compared with MKP-2(+/+) mice following infection although they did display significantly reduced serum nitrite levels and enhanced tissue arginase-1 expression. Early resistance to T. gondii in MKP-2(+/+), but not MKP-2(-/-), mice was nitric oxide (NO) dependent as infected MKP-2(+/+), but not MKP-2(-/-) mice succumbed within 10 days post-infection with increased parasite burdens following treatment with the iNOS inhibitor L-NAME. Conversely, treatment of infected MKP-2(-/-) but not MKP-2(+/+) mice with nor-NOHA increased parasite burdens indicating a protective role for arginase-1 in MKP-2(-/-) mice. In vitro studies using tachyzoite-infected bone marrow derived macrophages and selective inhibition of arginase-1 and iNOS activities confirmed that both iNOS and arginase-1 contributed to inhibiting parasite replication. However, the effects of arginase-1 were transient and ultimately the role of iNOS was paramount in facilitating long-term inhibition of parasite multiplication within macrophages.

Design, synthesis, and biological activity of diaryl ether inhibitors of Toxoplasma gondii enoyl reductase.

Triclosan is a potent inhibitor of Toxoplasma gondii enoyl reductase (TgENR), which is an essential enzyme for parasite survival. In view of triclosan's poor druggability, which limits its therapeutic use, a new set of B-ring modified analogs were designed to optimize its physico-chemical properties. These derivatives were synthesized and evaluated by in vitro assay and TgENR enzyme assay. Some analogs display improved solubility, permeability and a comparable MIC50 value to that of triclosan. Modeling of these inhibitors revealed the same overall binding mode with the enzyme as triclosan, but the B-ring modifications have additional interactions with the strongly conserved Asn130.

T cell hypo-responsiveness against Leishmania major in MAP kinase phosphatase (MKP) 2 deficient C57BL/6 mice does not alter the healer disease phenotype.

We have recently demonstrated that MAP kinase phosphatase 2 (MKP-2) deficient C57BL/6 mice, unlike their wild-type counterparts, are unable to control infection with the protozoan parasite Leishmania mexicana. Increased susceptibility was associated with elevated Arginase-1 levels and reduced iNOS activity in macrophages as well as a diminished T(H)1 response. By contrast, in the present study footpad infection of MKP-2(-/-) mice with L. major resulted in a healing response as measured by lesion size and parasite numbers similar to infected MKP-2(+/+) mice. Analysis of immune responses following infection demonstrated a reduced T(H)1 response in MKP-2(-/-) mice with lower parasite specific serum IgG2b levels, a lower frequency of IFN-γ and TNF-α producing CD4(+) and CD8(+) T cells and lower antigen stimulated spleen cell IFN-γ production than their wild-type counterparts. However, infected MKP-2(-/-) mice also had similarly reduced levels of antigen induced spleen and lymph node cell IL-4 production compared with MKP-2(+/+) mice as well as reduced levels of parasite-specific IgG1 in the serum, indicating a general T cell hypo-responsiveness. Consequently the overall T(H)1/T(H)2 balance was unaltered in MKP-2(-/-) compared with wild-type mice. Although non-stimulated MKP-2(-/-) macrophages were more permissive to L. major growth than macrophages from MKP-2(+/+) mice, reflecting their reduced iNOS and increased Arginase-1 expression, LPS/IFN-γ activation was equally effective at controlling parasite growth in MKP-2(-/-) and MKP-2(+/+) macrophages. Consequently, in the absence of any switch in the T(H)1/T(H)2 balance in MKP-2(-/-) mice, no significant change in disease phenotype was observed.

Toxoplasma gondii HLA-B*0702-restricted GRA7(20-28) peptide with adjuvants and a universal helper T cell epitope elicits CD8(+) T cells producing interferon-γ and reduces parasite burden in HLA-B*0702 mice.

The ability of CD8(+) T cells to act as cytolytic effectors and produce interferon-γ (IFN-γ) was demonstrated to mediate resistance to Toxoplasma gondii in murine models because of the recognition of peptides restricted by murine major histocompatibility complex (MHC) class I molecules. However, no T gondii-specific HLA-B07-restricted peptides were proven protective against T gondii. Recently, 2 T gondii-specific HLA-B*0702-restricted T cell epitopes, GRA7(20-28) (LPQFATAAT) and GRA3(27-35) (VPFVVFLVA), displayed high-affinity binding to HLA-B*0702 and elicited IFN-γ from peripheral blood mononuclear cells of seropositive HLA-B*07 persons. Herein, these peptides were evaluated to determine whether they could elicit IFN-γ in splenocytes of HLA-B*0702 transgenic mice when administered with adjuvants and protect against subsequent challenge. Peptide-specific IFN-γ-producing T cells were identified by enzyme-linked immunosorbent spot and proliferation assays utilizing splenic T lymphocytes from human lymphocyte antigen (HLA) transgenic mice. When HLA-B*0702 mice were immunized with one of the identified epitopes, GRA7(20-28) in conjunction with a universal CD4(+) T cell epitope (PADRE) and adjuvants (CD4(+) T cell adjuvant, GLA-SE, and TLR2 stimulatory Pam(2)Cys for CD8(+) T cells), this immunization induced CD8(+) T cells to produce IFN-γ and protected mice against high parasite burden when challenged with T gondii. This work demonstrates the feasibility of bioinformatics followed by an empiric approach based on HLA binding to test this biologic activity for identifying protective HLA-B*0702-restricted T gondii peptides and adjuvants that elicit protective immune responses in HLA-B*0702 mice.