The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5′ ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct

The role of clathrin in post-golgi trafficking in toxoplasma gondii

Apicomplexan parasites are single eukaryotic cells with a highly polarised secretory system that contains unique secretory organelles (micronemes and rhoptries) that are required for host cell invasion. In contrast, the role of the endosomal system is poorly understood in these parasites. With many typical endocytic factors missing, we speculated that endocytosis depends exclusively on a clathrin-mediated mechanism. Intriguingly, in Toxoplasma gondii we were only able to observe the endogenous clathrin heavy chain 1 (CHC1) at the Golgi, but not at the parasite surface. For the functional characterisation of Toxoplasma gondii CHC1 we generated parasite mutants conditionally expressing the dominant negative clathrin Hub fragment and demonstrate that CHC1 is essential for vesicle formation at the trans-Golgi network. Consequently, the functional ablation of CHC1 results in Golgi aberrations, a block in the biogenesis of the unique secretory microneme and rhoptry organelles, and of the pellicle. However, we found no morphological evidence for clathrin mediating endocytosis in these parasites and speculate that they remodelled their vesicular trafficking system to adapt to an intracellular lifestyle

Expression and Secretion of Endostatin in Thyroid Cancer

Background: In thyroid cancer (TC) endostatin was identified as a powerful negative regulator of tumor angiogenesis in vitro. It is currently being evaluated in phase I trials for antiangiogenic therapy in various solid tumors. The aim of this study was to evaluate endostatin expression in archival TC specimens and its secretion following stimulation with thyrotropin (TSH) and epidermal growth factor (EGF) in TC cell lines. Methods: Tissue microarrays of 44 differentiated and 7 anaplastic TC and their metastasis were immunostained for endostatin protein expression and compared with corresponding non-neoplastic thyroid tissue (NT). In vitro, six differentiated (FTC133, FTC236, HTC, HTCTSHr, XTC, and TPC1) and three anaplastic (C643, Hth74, Kat4.0) TC cell lines were evaluated for basal as well as TSH (1-100 mU/ml) and EGF stimulated (1-100 ng/ml) endostatin. Results: Endostatin was detected in all TC and more than half of the NT. Endostatin expression was more frequent and intense in differentiated as compared to anaplastic TC. In vitro, basal endostatin secretion varied between 33 ± 5 pg/ml (FTC236) and 549 ± 65 pg/ml (TPC1) and was doubled in FTC, when the ''primary'' (FTC133) was compared with the metastasis (FTC236). Some cell lines showed TSH-induced (e.g., 60% in XTC) or EGFinduced (e.g., 120% in TPC1) upregulation of endostatin secretion, while others did not, despite documented receptor expression. Conclusion: This study demonstrates endostatin expression in TC, metastasis and-less frequently and intensely-in NT, suggesting a possible association to tumor progression. In vitro, endostatin secretion of some cell lines is regulated by TSH and EGF, however the individual differences deserve further functional studies. These results support rather tumorspecific than histotype-specific expression and regulation of endostatin in TC

The Plasmodium Export Element Revisited

We performed a bioinformatical analysis of protein export elements (PEXEL) in the putative proteome of the malaria parasite Plasmodium falciparum. A protein family-specific conservation of physicochemical residue profiles was found for PEXEL-flanking sequence regions. We demonstrate that the family members can be clustered based on the flanking regions only and display characteristic hydrophobicity patterns. This raises the possibility that the flanking regions may contain additional information for a family-specific role of PEXEL. We further show that signal peptide cleavage results in a positional alignment of PEXEL from both proteins with, and without, a signal peptide

Merozoite release from Plasmodium falciparum-infected erythrocytes involves the transfer of DiIC16 from infected cell membrane to Maurer’s clefts

Merozoite release from infected erythrocytes is a complex process, which is still not fully understood. Such process was characterised at ultra-structural level in this work by labelling erythrocyte membrane with a fluorescent lipid probe and subsequent photo-conversion into an electron-dense precipitate. A lipophilic DiIC16 probe was inserted into the infected erythrocyte surface and the transport of this phospholipid analogue through the erythrocyte membrane was followed up during 48 h of the asexual erythrocyte cycle. The lipid probe was transferred from infected erythrocyte membranes to Maurer’s clefts during merozoite release, thereby indicating that these membranes remained inside host cells after parasite release. Fluorescent structures were never observed inside infected erythrocytes preceding merozoite exit and merozoites released from infected erythrocyte were not fluorescent. However, specific precipitated material was localised bordering the parasitophorous vacuole membrane and tubovesicular membranes when labelled non-infected erythrocytes were invaded by merozoites. It was revealed that lipids were interchangeable from one membrane to another, passing from infected erythrocyte membrane to Maurer’s clefts inside the erythrocyte ghost, even after merozoite release. Maurer’s clefts became photo-converted following merozoite release, suggesting that these structures were in close contact with infected erythrocyte membrane during merozoite exit and possibly played some role in malarial parasite exit from the host cell

The crystal structure of the Sgt1-Skp1 complex: the link between Hsp90 and both SCF E3 ubiquitin ligases and kinetochores

The essential cochaperone Sgt1 recruits Hsp90 chaperone activity to a range of cellular factors including SCF E3 ubiquitin ligases and the kinetochore in eukaryotes. In these pathways Sgt1 interacts with Skp1, a small protein that heterodimerizes with proteins containing the F-box motif. We have determined the crystal structure of the interacting domains of Saccharomyces cerevisiae Sgt1 and Skp1 at 2.8 Å resolution and validated the interface in the context of the full-length proteins in solution. The BTB/POZ domain of Skp1 associates with Sgt1 via the concave surface of its TPR domain using residues that are conserved in humans. Dimerization of yeast Sgt1 occurs via an insertion that is absent from monomeric human Sgt1. We identify point mutations that disrupt dimerization and Skp1 binding in vitro and find that the interaction with Skp1 is an essential function of Sgt1 in yeast. Our data provide a structural rationale for understanding the phenotypes of temperature-sensitive Sgt1 mutants and for linking Skp1-associated proteins to Hsp90-dependent pathways

Biophysics of Malarial Parasite Exit from Infected Erythrocytes

Upon infection and development within human erythrocytes, P. falciparum induces alterations to the infected RBC morphology and bio-mechanical properties to eventually rupture the host cells through parasitic and host derived proteases of cysteine and serine families. We used previously reported broad-spectrum inhibitors (E64d, EGTA-AM and chymostatin) to inhibit these proteases and impede rupture to analyze mechanical signatures associated with parasite escape. Treatment of late-stage iRBCs with E64d and EGTA-AM prevented rupture, resulted in no major RBC cytoskeletal reconfiguration but altered schizont morphology followed by dramatic re-distribution of three-dimensional refractive index (3D-RI) within the iRBC. These phenotypes demonstrated several-fold increased iRBC membrane flickering. In contrast, chymostatin treatment showed no 3D-RI changes and caused elevated fluctuations solely within the parasitophorous vacuole. We show that E64d and EGTA-AM supported PV breakdown and the resulting elevated fluctuations followed non-Gaussian pattern that resulted from direct merozoite impingement against the iRBC membrane. Optical trapping experiments highlighted reduced deformability of the iRBC membranes upon rupture-arrest, more specifically in the treatments that facilitated PV breakdown. Taken together, our experiments provide novel mechanistic interpretations on the role of parasitophorous vacuole in maintaining the spherical schizont morphology, the impact of PV breakdown on iRBC membrane fluctuations leading to eventual parasite escape and the evolution of membrane stiffness properties of host cells in which merozoites were irreversibly trapped, recourse to protease inhibitors. These findings provide a comprehensive, previously unavailable, body of information on the combined effects of biochemical and biophysical factors on parasite egress from iRBCs.Singapore. Agency for Science, Technology and ResearchSingapore-MIT AllianceGlobal Enterprise for Micro-Mechanics and Molecular MedicineNational University of SingaporeNational Institutes of Health (U.S.) (Grant R01 HL094270-01A1)National Institutes of Health (U.S.) (Grant 1-R01-GM076689-01)National Institutes of Health (U.S.) (P41-RR02594-18-24