Regulation of Type II Collagen Synthesis during Osteoarthritis by Prolyl-4-Hydroxylases : Possible Influence of Low Oxygen Levels

Osteoarthritic (OA) chondrocytes are metabolically active, displaying increased synthesis of type II collagen. Here, we show by immunohistochemistry and polymerase chain reaction that in comparison with healthy cartilage, OA articular chondrocytes exhibit increased in vivo synthesis of collagen prolyl-4-hydroxylase type II, a pivotal enzyme in collagen triple helix formation. Exposure of primary human articular chondrocytes to 1% oxygen enhanced accumulation of native type II collagen and stabilized hypoxia-inducible factor-1α (HIF-1α). This effect was abolished by addition of the HIF-1 inhibitor 2-methoxyestradiol. Real-time polymerase chain reaction analyses of mRNAs from these cultures revealed increased transcript levels of both α-subunits of prolyl-4-hydroxylase (P4HA1, ∼2-fold; P4HA2, ∼2.3-fold) and of classical HIF-1 target genes (glucosetransporter-1, ∼2.1-fold; phosphoglyceratekinase-1, ∼2.2-fold). Treatment of hypoxic chondrocytes with 2-methoxyestradiol reduced transcriptional activity of HIF-1 and synthesis of α(II), and to a lesser extent α(I), subunits of collagen prolyl-4-hydroxylases. mRNA levels of type II collagen (Col2A1) and the β-subunit (P4HB) of prolyl-4-hydroxylase, however, displayed only modest changes at 1% oxygen. From these results and our in vivo data, we inferred that besides increased Col2A1 mRNA expression by OA chondrocytes, accelerated posttranslational modification processes might contribute to the increased synthesis and accumula-tion of type II collagen during OA and experimen-tal hypoxia

A genome-scale vector resource enables high-throughput reverse genetic screening in a malaria parasite

The genome-wide identification of gene functions in malaria parasites is hampered by a lack of reverse genetic screening methods. We present a large-scale resource of barcoded vectors with long homology arms for effective modification of the Plasmodium berghei genome. Cotransfecting dozens of vectors into the haploid blood stages creates complex pools of barcoded mutants, whose competitive fitness can be measured during infection of a single mouse using barcode sequencing (barseq). To validate the utility of this resource, we rescreen the P. berghei kinome, using published kinome screens for comparison. We find that several protein kinases function redundantly in asexual blood stages and confirm the targetability of kinases cdpk1, gsk3, tkl3, and PBANKA_082960 by genotyping cloned mutants. Thus, parallel phenotyping of barcoded mutants unlocks the power of reverse genetic screening for a malaria parasite and will enable the systematic identification of genes essential for in vivo parasite growth and transmission

The Alveolin IMC1h Is Required for Normal Ookinete and Sporozoite Motility Behaviour and Host Colonisation in Plasmodium berghei

Alveolins, or inner membrane complex (IMC) proteins, are components of the subpellicular network that forms a structural part of the pellicle of malaria parasites. In Plasmodium berghei, deletions of three alveolins, IMC1a, b, and h, each resulted in reduced mechanical strength and gliding velocity of ookinetes or sporozoites. Using time lapse imaging, we show here that deletion of IMC1h (PBANKA_143660) also has an impact on the directionality and motility behaviour of both ookinetes and sporozoites. Despite their marked motility defects, sporozoites lacking IMC1h were able to invade mosquito salivary glands, allowing us to investigate the role of IMC1h in colonisation of the mammalian host. We show that IMC1h is essential for sporozoites to progress through the dermis in vivo but does not play a significant role in hepatoma cell transmigration and invasion in vitro. Colocalisation of IMC1h with the residual IMC in liver stages was detected up to 30 hours after infection and parasites lacking IMC1h showed developmental defects in vitro and a delayed onset of blood stage infection in vivo. Together, these results suggest that IMC1h is involved in maintaining the cellular architecture which supports normal motility behaviour, access of the sporozoites to the blood stream, and further colonisation of the mammalian host

Efficacy of a Plasmodium vivax Malaria Vaccine Using ChAd63 and Modified Vaccinia Ankara Expressing Thrombospondin-Related Anonymous Protein as Assessed with Transgenic Plasmodium berghei Parasites

Plasmodium vivax is the world's most widely distributed malaria parasite and a potential cause of morbidity and mortality for approximately 2.85 billion people living mainly in Southeast Asia and Latin America. Despite this dramatic burden, very few vaccines have been assessed in humans. The clinically relevant vectors modified vaccinia virus Ankara (MVA) and the chimpanzee adenovirus ChAd63 are promising delivery systems for malaria vaccines due to their safety profiles and proven ability to induce protective immune responses against Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) in clinical trials. Here, we describe the development of new recombinant ChAd63 and MVA vectors expressing P. vivax TRAP (PvTRAP) and show their ability to induce high antibody titers and T cell responses in mice. In addition, we report a novel way of assessing the efficacy of new candidate vaccines against P. vivax using a fully infectious transgenic Plasmodium berghei parasite expressing P. vivax TRAP to allow studies of vaccine efficacy and protective mechanisms in rodents. Using this model, we found that both CD8+ T cells and antibodies mediated protection against malaria using virus-vectored vaccines. Our data indicate that ChAd63 and MVA expressing PvTRAP are good preerythrocytic-stage vaccine candidates with potential for future clinical application

A Knockout Screen of ApiAP2 Genes Reveals Networks of Interacting Transcriptional Regulators Controlling the Plasmodium Life Cycle

A family of apicomplexa-specific proteins containing AP2 DNA-binding domains (ApiAP2s) was identified in malaria parasites. This family includes sequence-specific transcription factors that are key regulators of development. However, functions for the majority of ApiAP2 genes remain unknown. Here, a systematic knockout screen in Plasmodium berghei identified ten ApiAP2 genes that were essential for mosquito transmission: four were critical for the formation of infectious ookinetes, and three were required for sporogony. We describe non-essential functions for AP2-O and AP2-SP proteins in blood stages, and identify AP2-G2 as a repressor active in both asexual and sexual stages. Comparative transcriptomics across mutants and developmental stages revealed clusters of co-regulated genes with shared cis promoter elements, whose expression can be controlled positively or negatively by different ApiAP2 factors. We propose that stage-specific interactions between ApiAP2 proteins on partly overlapping sets of target genes generate the complex transcriptional network that controls the Plasmodium life cycle

Localisation of IMC1h in ookinetes and sporozoites.

<p>A. Single optical section of a mature IMC1h-HA ookinete immunostained for HA, P28 and the nucleus stained with Hoechst. The HA tagged IMC1h localizes to the peripheral edge of the ookinete. Both the anterior and posterior ends are spared by IMC1h-HA whereas P28 covers the whole surface of the ookinete. Scale bar = 1 µm. B. Representative TEM section of an IMC1h-HA ookinete immunogold-labelled for HA showing the pellicle. Gold particles (white arrowheads) localise at the IMC. IMC = inner membrane complex, MT = microtubules, SPN = subpellicular network, PM = plasma membrane. Scale bar = 100 nm. C. Salivary gland IMC1h-HA sporozoite immunostained for HA and GFP. The nucleus is stained with Hoechst. Single optical section is shown revealing the localisation of IMC1h-HA to the cell periphery. The IMC1h-HA is distributed over the full length of the sporozoite. Scale bar = 5 µm.</p

Comparative genomics in Chlamydomonas and Plasmodium identifies an ancient nuclear envelope protein family essential for sexual reproduction in protists, fungi, plants, and vertebrates

Fertilization is a crucial yet poorly characterized event in eukaryotes. Our previous discovery that the broadly conserved protein HAP2 (GCS1) functioned in gamete membrane fusion in the unicellular green alga Chlamydomonas and the malaria pathogen Plasmodium led us to exploit the rare biological phenomenon of isogamy in Chlamydomonas in a comparative transcriptomics strategy to uncover additional conserved sexual reproduction genes. All previously identified Chlamydomonas fertilization-essential genes fell into related clusters based on their expression patterns. Out of several conserved genes in a minus gamete cluster, we focused on Cre06.g280600, an ortholog of the fertilization-related Arabidopsis GEX1. Gene disruption, cell biological, and immunolocalization studies show that CrGEX1 functions in nuclear fusion in Chlamydomonas. Moreover, CrGEX1 and its Plasmodium ortholog, PBANKA_113980, are essential for production of viable meiotic progeny in both organisms and thus for mosquito transmission of malaria. Remarkably, we discovered that the genes are members of a large, previously unrecognized family whose first-characterized member, KAR5, is essential for nuclear fusion during yeast sexual reproduction. Our comparative transcriptomics approach provides a new resource for studying sexual development and demonstrates that exploiting the data can lead to the discovery of novel biology that is conserved across distant taxa

Characteristics of sporozoite motility in Matrigel.

<p>A. Speed of WT (n = 15) and IMC1h-KO (n = 11) sporozoites. B. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041409#s2" target="_blank">Results</a> of the manual assignment of movement patterns of WT (n = 15) and IMC1h-KO (n = 11) sporozoites. M = Meandering, CW = clock-wise, and CCW = counter clock-wise. C. Reconstruction of tracked sporozoite paths from high-magnification movies revealing movement patterns (two frames per second for 100 s). WT sporozoites (upper panel) mainly display a circular mode of motility while IMC1h-KO sporozoites (lower panel) mainly show a meandering mode of motility. Scale bar = 20 µm. D. Brightfield time-lapse image sequences showing motile WT and IMC1h-KO sporozoites. Scale bar = 10 µm.</p