Characterization of a MOB1 Homolog in the Apicomplexan Parasite Toxoplasma gondii.

Monopolar spindle One Binder1 (MOB1) proteins are conserved components of the tumor-suppressing Hippo pathway, regulating cellular processes such as cytokinesis. Apicomplexan parasites present a life cycle that relies on the parasites' ability to differentiate between stages and regulate their proliferation; thus, Hippo signaling pathways could play an important role in the regulation of the apicomplexan life cycle. Here, we report the identification of one MOB1 protein in the apicomplexan Toxoplasma gondii. To characterize the function of MOB1, we generated gain-of-function transgenic lines with a ligand-controlled destabilization domain, and loss-of-function clonal lines obtained through CRISPR/Cas9 technology. Contrary to what has been characterized in other eukaryotes, MOB1 is not essential for cytokinesis in T. gondii. However, this picture is complex since we found MOB1 localized between the newly individualized daughter nuclei at the end of mitosis. Moreover, we detected a significant delay in the replication of overexpressing tachyzoites, contrasting with increased replication rates in knockout tachyzoites. Finally, using the proximity-biotinylation method, BioID, we identified novel members of the MOB1 interactome, a probable consequence of the observed lack of conservation of some key amino acid residues. Altogether, the results point to a complex evolutionary history of MOB1 roles in apicomplexans, sharing properties with other eukaryotes but also with divergent features, possibly associated with their complex life cycle.

Peptidylprolyl isomerase C (Ppic) regulates invariant Natural Killer T cell (iNKT) differentiation in mice.

Peptidyl-prolyl cis-trans isomerase C (Ppic) is expressed in several bone marrow (BM) hematopoietic progenitors and in T-cell precursors. Since the expression profile of Ppic in the hematoimmune system was suggestive that it could play a role in hematopoiesis and/or T lymphocyte differentiation, we sought to test that hypothesis in vivo. Specifically, we generated a Ppic-deficient mouse model by targeting the endogenous locus by CRISPR/Cas9 and tested the requirement of Ppic in hematopoiesis. Several immune cell lineages covering BM progenitors, lymphocyte precursors, as well as mature cells at the periphery were analyzed. While most lineages were unaffected, invariant NKT (iNKT) cells were reduced in percentage and absolute cell numbers in the Ppic-deficient thymus. This affected the most mature stages in the thymus, S2 and S3, and the phenotype was maintained at the periphery. Additionally, immature transitional T1 and T2 B lymphocytes were increased in the Ppic-deficient spleen, but the phenotype was lost in mature B lymphocytes. In sum, our data show that Ppic is dispensable for myeloid cells, platelets, erythrocytes, αß, and γδ T lymphocytes in vivo in the steady state, while being involved in B- and iNKT cell differentiation.

Immunophenotype of Gastric Tumors Unveils a Pleiotropic Role of Regulatory T Cells in Tumor Development.

Gastric cancer (GC) patients display increased regulatory T cell (Tregs) numbers in peripheral blood and among tumor-infiltrating lymphocytes. Nevertheless, the role of Tregs in GC progression remains controversial. Here, we sought to explore the impact of Tregs in GCs with distinct histology, and whether Tregs can directly influence tumor cell behavior and GC development. We performed a comprehensive immunophenotyping of 82 human GC cases, through an integrated analysis of multispectral immunofluorescence detection of T cells markers and patient clinicopathological data. Moreover, we developed 3D in vitro co-cultures with Tregs and tumor cells that were followed by high-throughput and light-sheet imaging, and their biological features studied with conventional/imaging flow cytometry and Western blotting. We showed that Tregs located at the tumor nest were frequent in intestinal-type GCs but did not associate with increased levels of effector T cells. Our in vitro results suggested that Tregs preferentially infiltrated intestinal-type GC spheroids, induced the expression of IL2Rα and activation of MAPK signaling pathway in tumor cells, and promoted spheroid growth. Accumulation of Tregs in intestinal-type GCs was increased at early stages of the stomach wall invasion and in the absence of vascular and perineural invasion. In this study, we proposed a non-immunosuppressive mechanism through which Tregs might directly modulate GC cells and thereby promote tumor growth. Our findings hold insightful implications for therapeutic strategies targeting intestinal-type GCs and other tumors with similar immune context.

Targeting of the mitochondrion by dinuclear thiolato-bridged arene ruthenium complexes in cancer cells and in the apicomplexan parasite Neospora caninum.

A library of 18 dinuclear-thiolato bridged arene ruthenium complexes, some of which with demonstrated activity against cancer cells, was screened for activity against a transgenic Neospora caninum strain that constitutively expresses beta-galactosidase. Initial assessments were done at concentrations of 2500, 250, 25 and 2.5 nM, and 5 compounds were further evaluated with regard to their half maximal proliferation-inhibiting concentration (IC50). Among those, [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-p-CH3)3]Cl (1), [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-p-But)3]Cl (2) and [(η6-p-MeC6H4Pri)2Ru2(µ2-SCH2C6H4-p-But)2(µ2-SC6H4-p-OH)]BF4 (9) inhibited N. caninum proliferation with low C50 values of 15, 5 and 1 nM, respectively, while [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-p-OH)3]Cl (3) and [(η6-p-MeC6H4Pri)2Ru2(µ2-SC6H4-p-mco)3]Cl (5, mco = 4-methylcoumarinyl) were less active (IC50 = 280 and 108 nM, respectively). These compounds did not affect human foreskin fibroblast (HFF) host cells at dosages of 5 µM and above, but impaired proliferation of the human ovarian carcinoma cell line A2780 (IC50 values of 130 nM (1), 30 nM (2), 530 nM (3), 7730 nM (5), 130 nM (9)). A2780 cancer cells were treated with complexes 1, 2, and 5, and biodistribution analysis using inductively coupled plasma mass spectrometry (ICP-MS) showed that most of the drugs accumulated in the mitochondrial fractions. Transmission electron microscopy showed that the parasite mitochondrion is the primary target also in N. caninum tachyzoites, but these compounds, when applied at 200 nM for 15 days in vitro, did not act parasiticidal. Complexes 1, 2 and 9 applied orally at 2 and 10 mg kg-1 day-1 during 5 days in a neosporosis mouse model did not reduce parasite load and did not limit parasite dissemination to the central nervous system. In accordance with these results, ICP-MS carried out on different organs of mice orally administrated with complexes 1 and 9, demonstrated that the drugs were readily absorbed, and after 3 and 48 h, were mainly detected in liver and kidney, but were largely absent from the brain. Thus, dinuclear thiolato-bridged arene ruthenium complexes exhibit interesting activities against N. caninum in vitro, but further modifications of these promising molecules are required to improve their bioavailability and pharmacokinetic properties in order to exert a pronounced and selective effect against N. caninum in vivo.

Neospora caninum in non-pregnant and pregnant mouse models: cross-talk between infection and immunity.

Neospora caninum is a cyst-forming coccidian which causes abortion in cattle, with a high economic impact globally. Vaccination is considered to be the most cost-effective strategy to control and prevent bovine neosporosis. However, there is no commercial vaccine available to date. To investigate this disease under laboratory conditions, mouse models were developed, and they have been efficiently used as an initial proof-of-concept platform to investigate different immunogenic formulations. We here provide a detailed review on the current knowledge on immunity against neosporosis in non-pregnant as well as pregnant mice, and present a general overview of the most relevant parameters that may be responsible for protective immunity, which in turn could be relevant for vaccine development. Despite the considerable differences in immunity between cattle and mice, it is essential to understand how mice respond immunologically to Neospora caninum infection and how this response influences congenital infection and offspring survival. In this context, pregnant mouse models play a key role, and allow correlation of the outcome of congenital neosporosis with specific immune mechanisms which could also be relevant in cattle.

Characterization of the Activities of Dinuclear Thiolato-Bridged Arene Ruthenium Complexes against Toxoplasma gondii.

The in vitro effects of 18 dinuclear thiolato-bridged arene ruthenium complexes (1 monohiolato compound, 4 dithiolato compounds, and 13 trithiolato compounds), originally designed as anticancer agents, on the apicomplexan parasite Toxoplasma gondii grown in human foreskin fibroblast (HFF) host cells were studied. Some trithiolato compounds exhibited antiparasitic efficacy at concentrations of 250 nM and below. Among those, complex 1 and complex 2 inhibited T. gondii proliferation with 50% inhibitory concentrations (IC50s) of 34 and 62 nM, respectively, and they did not affect HFFs at dosages of 200 µM or above, resulting in selectivity indices of >23,000. The IC50s of complex 9 were 1.2 nM for T. gondii and above 5 µM for HFFs. Transmission electron microscopy detected ultrastructural alterations in the matrix of the parasite mitochondria at the early stages of treatment, followed by a more pronounced destruction of tachyzoites. However, none of the three compounds applied at 250 nM for 15 days was parasiticidal. By affinity chromatography using complex 9 coupled to epoxy-activated Sepharose followed by mass spectrometry, T. gondii translation elongation factor 1α and two ribosomal proteins, RPS18 and RPL27, were identified to be potential binding proteins. In conclusion, organometallic ruthenium complexes exhibit promising activities against Toxoplasma, and the potential mechanisms of action of these compounds as well as their prospective applications for the treatment of toxoplasmosis are discussed.

Immune response profile elicited by the model antigen ovalbumin expressed in fusion with the bacterial OprI lipoprotein.

The use of immunogenic formulations targeting pattern recognition receptors towards modulation of immune responses is a promising strategy to develop better vaccines against infectious and malignant diseases. Molecules targeting TLR2 offer interesting properties that are relevant for vaccine development, including the possibility to covalently attach the lipidic ligands to the antigens. However, the type of immune response elicited by these formulations is still controversial. In this work, we used the model antigen ovalbumin (OVA) expressed in fusion with the bacterial lipoprotein OprI in order to characterize the immunomodulatory properties of this TLR ligand. Murine bone marrow-derived dendritic cells stimulated with OprI-OVA fusion lipoprotein produced high levels of the pro-inflammatory cytokines TNF-α and IL-6 and also IL-10, IL-12(p70) and IL-27, while TGF-ß and IL-23 were not detected. Using OT-II and OT-I mice, an enhancement of MHC class II and class I antigen presentation was observed for the OVA antigen in fusion with OprI. Mice immunized by intraperitoneal route with this fusion lipoprotein in prime-boost protocols developed strong specific antibody responses including IgG1, IgG2c, IgG2b, IgG3 and IgE. These results, together with data obtained by restimulation of splenocytes from the immunized mice, point to an immune response profile that does not correspond to a strict Th1 or Th2 polarization. Finally, in a challenge experiment using a melanoma syngeneic mouse model (B16-OVA), prophylactic inoculation with OprI fused with the unrelated antigen eGFP increased the tumor growth, while the fusion with the tumor-associated antigen OVA delayed the tumor growth and increased mice survival.

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations.

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.