Receptor-mediated endocytosis in an apicomplexan parasite (Toxoplasma gondii)

Endocytosis mechanisms are poorly known in apicomplexan parasites. Here, we show that extracellular tachyzoites of Toxoplasma gondii bind and internalize heparin-like sulfated glycans in a specific, saturable manner. Discrete binding of the glycan occurs at the anterior third of the tachyzoite, where it is rapidly concentrated inside single tubulo vesicular compartments that become multiple with time. The compound is held for several hours intracellularly with no apparent exocytosis or acidification. Incubation in the continuous presence of fluorescein isothiocyanate-conjugated heparin enhances the binding and internalization of this ligand by live tachyzoites. Two tachyzoite surface polypeptides exhibit strong binding and specificity for heparin, making them candidate receptors. Uptake of fluid-phase endocytic tracers occurs via nonspecific pinocytosis in the same region of the parasite cell, but with much lower efficiency. These observations show that extracellular tachyzoites can acquire molecules through both receptor-specific and fluid-phase endocytic mechanisms. Understanding the physiological relevance of these processes for the extracellular and intracellular stages of T. gondii may bring about direct targeting of the parasite by drug delivery into the tachyzoites.     

Creating a knowledge translation trainee collaborative: from conceptualization to lessons learned in the first year

Background

Patient involvement in healthcare represents the means by which to achieve a healthcare system that is responsive to patient needs and values. Characterization and evaluation of strategies for involving patients in their healthcare may benefit from a knowledge translation (KT) approach. The purpose of this knowledge synthesis is to develop a conceptual framework for patient-mediated KT interventions.

Methods

A preliminary conceptual framework for patient-mediated KT interventions was compiled to describe intended purpose, recipients, delivery context, intervention, and outcomes. A realist review will be conducted in consultation with stakeholders from the arthritis and cancer fields to explore how these interventions work, for whom, and in what contexts. To identify patient-mediated KT interventions in these fields, we will search MEDLINE, the Cochrane Library, and EMBASE from 1995 to 2010; scan references of all eligible studies; and examine five years of tables of contents for journals likely to publish quantitative or qualitative studies that focus on developing, implementing, or evaluating patient-mediated KT interventions. Screening and data collection will be performed independently by two individuals.

Conclusions

The conceptual framework of patient-mediated KT options and outcomes could be used by healthcare providers, managers, educationalists, patient advocates, and policy makers to guide program planning, service delivery, and quality improvement and by us and other researchers to evaluate existing interventions or develop new interventions. By raising awareness of options for involving patients in improving their own care, outcomes based on using a KT approach may lead to greater patient-centred care delivery and improved healthcare outcomes.     

The catalytic aspartate is protonated in the Michaelis complex formed between trypsin and an in vitro evolved substrate-like inhibitor: a refined mechanism of serine protease action

The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the O_δ2–C_γ bond appears to be a double bond, with O_δ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on O_δ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mF_obs − DF_calc density above 2.5 σ next to O_δ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pK_a of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pK_a histidine theory.     

Plasmodium falciparum Rosetting Epitopes Converge in the SD3-Loop of PfEMP1-DBL1α

The ability of Plasmodium falciparum parasitized RBC (pRBC) to form rosettes with normal RBC is linked to the virulence of the parasite and RBC polymorphisms that weaken rosetting confer protection against severe malaria. The adhesin PfEMP1 mediates the binding and specific antibodies prevent sequestration in the micro-vasculature, as seen in animal models. Here we demonstrate that epitopes targeted by rosette disrupting antibodies converge in the loop of subdomain 3 (SD3) which connects the h6 and h7 α-helices of PfEMP1-DBL1α. Both monoclonal antibodies and polyclonal IgG, that bound to epitopes in the SD3-loop, stained the surface of pRBC, disrupted rosettes and blocked direct binding of recombinant NTS-DBL1α to RBC. Depletion of polyclonal IgG raised to NTS-DBL1α on a SD3 loop-peptide removed the anti-rosetting activity. Immunizations with recombinant subdomain 1 (SD1), subdomain 2 (SD2) or SD3 all generated antibodies reacting with the pRBC-surface but only the sera of animals immunized with SD3 disrupted rosettes. SD3-sequences were found to segregate phylogenetically into two groups (A/B). Group A included rosetting sequences that were associated with two cysteine-residues present in the SD2-domain while group B included those with three or more cysteines. Our results suggest that the SD3 loop of PfEMP1-DBL1α is an important target of anti-rosetting activity, clarifying the molecular basis of the development of variant-specific rosette disrupting antibodies.     

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.     

Differential Recognition of P. falciparum VAR2CSA Domains by Naturally Acquired Antibodies in Pregnant Women from a Malaria Endemic Area

Background

Plasmodium falciparum infected red blood cells (iRBC) express variant surface antigens (VSA) of which VAR2CSA is involved in placental sequestration and causes pregnancy-associated malaria (PAM). Primigravidae are most susceptible to PAM whereas antibodies associated with protection are often present at higher levels in multigravid women. However, HIV co-infection with malaria has been shown to alter this parity-dependent acquisition of immunity, with more severe symptoms as well as more malaria episodes in HIV positive women versus HIV negative women of a similar parity.

Methods

Using VAR2CSA DBL-domains expressed on the surface of CHO-745 cells we quantified levels of DBL-domain specific IgG in sera from pregnant Malawian women by flow cytometry. Dissociations constants of DBL5ε specific antibodies were determined using a surface plasmon resonance technique, as an indication of antibody affinities.

Results

VAR2CSA DBL5ε was recognized in a gender and parity-dependent manner with anti-DBL5ε IgG correlating significantly with IgG levels to VSA-PAM on the iRBC surface. HIV positive women had lower levels of anti-DBL5ε IgG than HIV negative women of similar parity. In primigravidae, antibodies in HIV positive women also showed significantly lower affinity to VAR2CSA DBL5ε.

Conclusions

Pregnant women from a malaria-endemic area had increased levels of anti-DBL5ε IgG by parity, indicating this domain of VAR2CSA to be a promising vaccine candidate against PAM. However, it is important to consider co-infection with HIV, as this seems to change the properties of antibody response against malaria. Understanding the characteristics of antibody response against VAR2CSA is undoubtedly imperative in order to design a functional and efficient vaccine against PAM.     

Inference of the Xenopus tropicalis embryonic regulatory network and spatial gene expression patterns

Background

During embryogenesis, signaling molecules produced by one cell population direct gene regulatory changes in neighboring cells and influence their developmental fates and spatial organization. One of the earliest events in the development of the vertebrate embryo is the establishment of three germ layers, consisting of the ectoderm, mesoderm and endoderm. Attempts to measure gene expression in vivo in different germ layers and cell types are typically complicated by the heterogeneity of cell types within biological samples (i.e., embryos), as the responses of individual cell types are intermingled into an aggregate observation of heterogeneous cell types. Here, we propose a novel method to elucidate gene regulatory circuits from these aggregate measurements in embryos of the frog Xenopus tropicalis using gene network inference algorithms and then test the ability of the inferred networks to predict spatial gene expression patterns.

Results

We use two inference models with different underlying assumptions that incorporate existing network information, an ODE model for steady-state data and a Markov model for time series data, and contrast the performance of the two models. We apply our method to both control and knockdown embryos at multiple time points to reconstruct the core mesoderm and endoderm regulatory circuits. Those inferred networks are then used in combination with known dorsal-ventral spatial expression patterns of a subset of genes to predict spatial expression patterns for other genes. Both models are able to predict spatial expression patterns for some of the core mesoderm and endoderm genes, but interestingly of different gene subsets, suggesting that neither model is sufficient to recapitulate all of the spatial patterns, yet they are complementary for the patterns that they do capture.

Conclusion

The presented methodology of gene network inference combined with spatial pattern prediction provides an additional layer of validation to elucidate the regulatory circuits controlling the spatial-temporal dynamics in embryonic development.