The lectin-specific activity of Toxoplasma gondii microneme proteins 1 and 4 binds Toll-like receptor 2 and 4 N-glycans to regulate innate immune priming.

Infection of host cells by Toxoplasma gondii is an active process, which is regulated by secretion of microneme (MICs) and rhoptry proteins (ROPs and RONs) from specialized organelles in the apical pole of the parasite. MIC1, MIC4 and MIC6 assemble into an adhesin complex secreted on the parasite surface that functions to promote infection competency. MIC1 and MIC4 are known to bind terminal sialic acid residues and galactose residues, respectively and to induce IL-12 production from splenocytes. Here we show that rMIC1- and rMIC4-stimulated dendritic cells and macrophages produce proinflammatory cytokines, and they do so by engaging TLR2 and TLR4. This process depends on sugar recognition, since point mutations in the carbohydrate-recognition domains (CRD) of rMIC1 and rMIC4 inhibit innate immune cells activation. HEK cells transfected with TLR2 glycomutants were selectively unresponsive to MICs. Following in vitro infection, parasites lacking MIC1 or MIC4, as well as expressing MIC proteins with point mutations in their CRD, failed to induce wild-type (WT) levels of IL-12 secretion by innate immune cells. However, only MIC1 was shown to impact systemic levels of IL-12 and IFN-γ in vivo. Together, our data show that MIC1 and MIC4 interact physically with TLR2 and TLR4 N-glycans to trigger IL-12 responses, and MIC1 is playing a significant role in vivo by altering T. gondii infection competency and murine pathogenesis

BMC Med

BACKGROUND: Overall survival (OS) is the gold standard endpoint to assess treatment efficacy in cancer clinical trials. In metastatic breast cancer (mBC), progression-free survival (PFS) is commonly used as an intermediate endpoint. Evidence remains scarce regarding the degree of association between PFS and OS. Our study aimed to describe the individual-level association between real-world PFS (rwPFS) and OS according to first-line treatment in female patients with mBC managed in real-world setting for each BC subtype (defined by status for both hormone-receptor [HR] expression and HER2 protein expression/gene amplification). METHODS: We extracted data from the ESME mBC database (NCT03275311) which gathers deidentified data from consecutive patients managed in 18 French Comprehensive Cancer Centers. Adult women diagnosed with mBC between 2008 and 2017 were included. Endpoints (PFS, OS) were described using the Kaplan-Meier method. Individual-level associations between rwPFS and OS were estimated using the Spearman's correlation coefficient. Analyses were conducted by tumor subtype. RESULTS: 20,033 women were eligible. Median age was 60.0 years. Median follow-up duration was 62.3 months. Median rwPFS ranged from 6.0 months (95% CI 5.8-6.2) for HR-/HER2 - subtype to 13.3 months (36% CI 12.7-14.3) for HR + /HER2 + subtype. Correlation coefficients were highly variable across subtypes and first-line (L1) treatments. Among patients with HR - /HER2 - mBC, correlation coefficients ranged from 0.73 to 0.81, suggesting a strong rwPFS/OS association. For HR + /HER2 + mBC patients, the individual-level associations were weak to strong with coefficients ranging from 0.33 to 0.43 for monotherapy and from 0.67 to 0.78 for combined therapies. CONCLUSIONS: Our study provides comprehensive information on individual-level association between rwPFS and OS for L1 treatments in mBC women managed in real-life practice. Our results could be used as a basis for future research dedicated to surrogate endpoint candidates

Sea ice diatom contributions to Holocene nutrient utilization in East Antarctica

Combined high-resolution Holocene δ30Sidiat and δ13Cdiat paleorecords are presented from theSeasonal Ice Zone, East Antarctica. Both data sets reflect periods of increased nutrient utilization by diatomsduring the Hypsithermal period (circa 7800 to 3500 calendar years (cal years) B.P.), coincident with a higherabundance of open water diatom species (Fragilariopsis kerguelensis), increased biogenic silica productivity(%BSi), and higher regional summer temperatures. The Neoglacial period (after circa 3500 cal years B.P.) isreflected by an increase in sea ice indicative species (Fragilariopsis curta and Fragilariopsis cylindrus,upto50%) along with a decrease in %BSi and δ13Cdiat(< 18‰ to 23‰). However, over this period, δ30Sidiatdata show an increasing trend, to some of the highest values in the Holocene record (average of +0.43‰).Competing hypotheses are discussed to account for the decoupling trend in utilization proxies including ironfertilization, species-dependent fractionation effects, and diatom habitats. Based on mass balance calculations,we highlight that diatom species derived from the semi-enclosed sea ice environment may have a confoundingeffect upon δ30Sidowncorecompositions of the seasonal sea ice zone. A diatom composition, with approximately28% of biogenic silica derived from the sea ice environment (diat-SI) can account for the increased averagecompo sition of δ30Sidiatduring the Neoglacial. These data highlight the significant role sea ice diatoms can playwith relation to their export in sediment records, which has implications on productivity reconstructions fromthe seasonal ice zone

Proteins interacting with PfEMP1 in P. falciparum infected erythrocyte

During infection in humans, P. falciparum invades and refurbishes the red blood cells (RBCs) in order to persist and proliferate. Parasite survival depends on expression of a parasite encoded cytoadherence ligand at the surface of the infected RBC called PfEMP1 (P. falciparum erythrocyte membrane protein 1). PfEMP1 comprises a N-terminal extracellular part, which mediates binding to receptors at the host endothelium. It also contains a C-terminal part which is conserved among all PfEMP1 molecules. This acidic terminal segment (ATS) anchors PfEMP1 to the membrane of infected RBCs and has a role for trafficking. To bring proteins to the RBC surface, P. falciparum builds its own trafficking machinery de novo since the erythrocytes do not contain secretory organelles. These new structures include parasite derived compartments called Maurer's clefts (MCs) located at the RBC periphery. The resident MCs protein MAHRP1 (membrane-associated histidine-rich protein 1) has been shown to be critical for PfEMP1 trafficking. A MAHRP1 KO strain had no PfEMP1 on the RBC surface but accumulated PfEMP1 within the parasite. MAHRP1 is a 28.9kDa protein with 249 amino acids containing a N-terminal, a transmembrane, and a C-terminal domain comprising histidine-rich repeats. To re-establish PfEMP1 trafficking, the MAHRP1 KO parasite line was complemented with various truncated HA-tagged fragments of MAHRP1. Immunofluorescent assays (IFAs) showed that the Cterminal domain with the histidine-rich repeats was not essential for PfEMP1 trafficking. ${MAHRP1}_{1-130}$ and ${MAHRP1}_{1-169}$ were exported to MCs and restored trafficking of PfEMP1. Shorter MAHRP1 fragments were not exported to MCs and PfEMP1 transport remained impaired. New transfection constructs were designed including the C-terminal part to identify important domain(s) of MAHRP1 for PfEMP1 trafficking. To identify other proteins interacting with PfEMP1, the ATS domain of PfEMP1 was recombinantly expressed and used in pull-down experiments. In these experiments using parasite lysates, PF14_0377 was identified as a potential interaction partner. This protein has been described as putative, vesicular-associated membrane protein. To subsequently assess the interaction(s) and the localization(s), PF14_0377 was cloned and transfected in 3D7 for IFAs. Due to time restrictions all these new transfectants could not be analyzed completel

Does protein phosphorylation govern host cell entry and egress by the Apicomplexa?

Members of the phylum Apicomplexa are responsible for a wide range of diseases in humans and animals. The absence of an effective vaccine or safe curing drugs and the continuous emergence of resistant parasites to available treatments impose a high demand on the identification of novel targets for intervention against the apicomplexans. Protein kinases are considered attractive potential therapeutic targets not only against cancers but also to combat infectious diseases. The scope and aim of this review is to report on the recent progress in dissecting the impact of protein phosphorylation in regulating motility and invasion

Toxoplasma gondii myosin F, an essential motor for centrosomes positioning and apicoplast inheritance

Members of the Apicomplexa phylum possess an organelle surrounded by four membranes, originating from the secondary endosymbiosis of a red alga. This so-called apicoplast hosts essential metabolic pathways. We report here that apicoplast inheritance is an actin-based process. Concordantly, parasites depleted in either profilin or actin depolymerizing factor, or parasites overexpressing the FH2 domain of formin 2, result in loss of the apicoplast. The class XXII myosin F (MyoF) is conserved across the phylum and localizes in the vicinity of the Toxoplasma gondii apicoplast during division. Conditional knockdown of TgMyoF severely affects apicoplast turnover, leading to parasite death. This recapitulates the phenotype observed upon perturbation of actin dynamics that led to the accumulation of the apicoplast and secretory organelles in enlarged residual bodies. To further dissect the mode of action of this motor, we conditionally stabilized the tail of MyoF, which forms an inactive heterodimer with endogenous TgMyoF. This dominant negative mutant reveals a central role of this motor in the positioning of the two centrosomes prior to daughter cell formation and in apicoplast segregation

Assessment of phosphorylation in Toxoplasma glideosome assembly and function

Members of the phylum Apicomplexa possess a highly conserved molecular motor complex anchored in the parasite pellicle and associated with gliding motility, invasion and egress from infected cells. This machinery, called the glideosome, is structured around the acylated gliding-associated protein GAP45 that recruits the motor complex composed of myosin A and two associated myosin light chains (TgMLC1 and TgELC1). This motor is presumably firmly anchored to the inner membrane complex underneath the plasma membrane via an interaction with two integral membrane proteins, GAP50 and GAP40. To determine if the previously mapped phosphorylation sites on TgGAP45 and TgMLC1 have a direct significance for glideosome assembly and function, a series of phospho-mimetic and phospho-null mutants were generated. Neither the overexpression nor the allelic replacement of TgMLC1 with phospho-mutants impacted on glideosome assembly and parasite motility. TgGAP45 phosphorylation mutants were functionally investigated using a complementation strategy in a TgGAP45 inducible knockout background. The loss of interaction with TgGAP50 by one previously reported GAP45-mutant appeared to depend only on the presence of a remaining competing wild type copy of TgGAP45. Accordingly, this mutant displayed no phenotype in complementation experiments. Unexpectedly, GAP45 lacking the region encompassing the cluster of twelve phosphorylation sites did not impact on its dual function in motor recruitment and pellicle integrity. Despite the extensive phosphorylation of TgMLC1 and TgGAP45, this post-translational modification does not appear to be critical for the assembly and function of the glideosome