An exported protein-interacting complex involved in the trafficking of virulence determinants in Plasmodium-infected erythrocytes

The malaria parasite, Plasmodium falciparum, displays the P. falciparum erythrocyte membrane protein 1 (PfEMP1) on the surface of infected red blood cells (RBCs). We here examine the physical organization of PfEMP1 trafficking intermediates in infected RBCs and determine interacting partners using an epitope-tagged minimal construct (PfEMP1B). We show that parasitophorous vacuole (PV)-located PfEMP1B interacts with components of the PTEX (Plasmodium Translocon of EXported proteins) as well as a novel protein complex, EPIC (Exported Protein-Interacting Complex). Within the RBC cytoplasm PfEMP1B interacts with components of the Maurer\u27s clefts and the RBC chaperonin complex. We define the EPIC interactome and, using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in altered knob morphology, reduced cell rigidity and decreased binding to CD36. Accordingly, we show that deletion of the Plasmodium berghei homologue of PV1 is associated with attenuation of parasite virulence in vivo

Expansion of Kuravirus-like Phage Sequences within the Past Decade, including Escherichia Phage YF01 from Japan, Prompt the Creation of Three New Genera

Bacteriophages, viruses that infect bacteria, are currently receiving significant attention amid an ever-growing global antibiotic resistance crisis. In tandem, a surge in the availability and affordability of next-generation and third-generation sequencing technologies has driven the deposition of a wealth of phage sequence data. Here, we have isolated a novel Escherichia phage, YF01, from a municipal wastewater treatment plant in Yokohama, Japan. We demonstrate that the YF01 phage shares a high similarity to a collection of thirty-five Escherichia and Shigella phages found in public databases, six of which have been previously classified into the Kuravirus genus by the International Committee on Taxonomy of Viruses (ICTV). Using modern phylogenetic approaches, we demonstrate that an expansion and reshaping of the current six-membered Kuravirus genus is required to accommodate all thirty-six member phages. Ultimately, we propose the creation of three additional genera, Vellorevirus, Jinjuvirus, and Yesanvirus, which will allow a more organized approach to the addition of future Kuravirus-like phages

A network of protein interactions involved in the trafficking of PfEMP1 in Plasmodium falciparum-infected erythrocytes

© 2016 Dr. Steven Adam BatinovicPlasmodium falciparum is the most virulent human malaria parasite. Parasites invade red blood cells (RBCs) and extensively modify the structure and morphology of their host cell. As part of these modifications, the parasite establishes multi-protein virulence complexes that assemble within knob-like structures under the surface of the RBC plasma membrane. These structures allow infected RBCs to cytoadhere and sequester within the microvasculature of the host, effectively bypassing human clearance mechanisms that occur in the spleen. The most prominent members of these virulence complexes are protein members of the P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family. The trafficking of PfEMP1 from the parasite to the host cell and its correct display on the RBC surface involves a complement of both host and parasite-derived trafficking machineries that are generated de novo during the approximate 48-hour lifecycle of the parasite inside its host RBC. In this work we examine the physical organisation of PfEMP1 trafficking intermediates in infected RBCs and determine interacting protein partners using an epitope-tagged minimal construct (PfEMP1B). Known and novel interacting-proteins were identified across multiple parasite and host compartments, consistent with our current knowledge of the PfEMP1 trafficking pathway. We show that PV-located PfEMP1B interacts directly with components of the Plasmodium Translocon of EXported proteins (PTEX) as well as a novel protein complex we refer to as the Exported Protein-Interacting Complex (EPIC). We define the EPIC interactome and using an inducible knockdown approach, show that depletion of one of its components, the parasitophorous vacuolar protein-1 (PV1), results in attenuation of infected RBC cytoadherence to endothelial cell ligands. Within the RBC cytoplasm, we show that PfEMP1B interacts with components of the Maurer’s clefts, and suggest a role for membranous tether structures in the transfer of PfEMP1 to the infected RBC surface. We also demonstrate a direct interaction between PfEMP1B and the RBC chaperonin complex, TCP-1 Ring Complex (TRiC), which may represent a novel mechanism for parasite recruitment of host factors in the export of parasite proteins such as PfEMP1. Ultimately, understanding and targeting the export of parasite virulence proteins may ultimately allow us to ablate parasite virulence in vivo

Expansion of <i>Kuravirus</i>-like Phage Sequences within the Past Decade, including <i>Escherichia</i> Phage YF01 from Japan, Prompt the Creation of Three New Genera

Bacteriophages, viruses that infect bacteria, are currently receiving significant attention amid an ever-growing global antibiotic resistance crisis. In tandem, a surge in the availability and affordability of next-generation and third-generation sequencing technologies has driven the deposition of a wealth of phage sequence data. Here, we have isolated a novel Escherichia phage, YF01, from a municipal wastewater treatment plant in Yokohama, Japan. We demonstrate that the YF01 phage shares a high similarity to a collection of thirty-five Escherichia and Shigella phages found in public databases, six of which have been previously classified into the Kuravirus genus by the International Committee on Taxonomy of Viruses (ICTV). Using modern phylogenetic approaches, we demonstrate that an expansion and reshaping of the current six-membered Kuravirus genus is required to accommodate all thirty-six member phages. Ultimately, we propose the creation of three additional genera, Vellorevirus, Jinjuvirus, and Yesanvirus, which will allow a more organized approach to the addition of future Kuravirus-like phages

Isolation and Characterisation of the Bundooravirus Genus and Phylogenetic Investigation of the Salasmaviridae Bacteriophages

Bacillus is a highly diverse genus containing over 200 species that can be problematic in both industrial and medical settings. This is mainly attributed to Bacillus sp. being intrinsically resistant to an array of antimicrobial compounds, hence alternative treatment options are needed. In this study, two bacteriophages, PumA1 and PumA2 were isolated and characterized. Genome nucleotide analysis identified the two phages as novel at the DNA sequence level but contained proteins similar to phi29 and other related phages. Whole genome phylogenetic investigation of 34 phi29-like phages resulted in the formation of seven clusters that aligned with recent ICTV classifications. PumA1 and PumA2 share high genetic mosaicism and form a genus with another phage named WhyPhy, more recently isolated from the United States of America. The three phages within this cluster are the only candidates to infect B. pumilus. Sequence analysis of B. pumilus phage resistant mutants revealed that PumA1 and PumA2 require polymerized and peptidoglycan bound wall teichoic acid (WTA) for their infection. Bacteriophage classification is continuously evolving with the increasing phages’ sequences in public databases. Understanding phage evolution by utilizing a combination of phylogenetic approaches provides invaluable information as phages become legitimate alternatives in both human health and industrial processes

Characterization of Novel Lytic Bacteriophages of Achromobacter marplantensis Isolated from a Pneumonia Patient

Achromobacter spp. are becoming increasingly associated with lung infections in patients suffering from cystic fibrosis (CF). A. marplatensis, which is closely related to A. xylosoxidans, has been isolated from the lungs of CF patients and other human infections. This article describes the isolation, morphology and characterization of two lytic bacteriophages specific for an A. marplatensis strain isolated from a pneumonia patient. This host strain was the causal agent of hospital acquired pneumonia&ndash;the first clinical report of such an occurrence. Full genome sequencing revealed bacteriophage genomes ranging in size from 45901 to 46,328 bp. Transmission electron microscopy revealed that the two bacteriophages AMA1 and AMA2 belonged to the Siphoviridae family. Host range analysis showed that their host range did not extend to A. xylosoxidans. The possibility exists for future testing of such bacteriophages in the control of Achromobacter infections such as those seen in CF and other infections of the lungs. The incidence of antibiotic resistance in this genus highlights the importance of seeking adjuncts and alternatives in CF and other lung infections

Tyroviruses are a new group of temperate phages that infect Bacillus species in soil environments worldwide

Abstract Background Bacteriophages are widely considered to be highly abundant and genetically diverse, with their role in the evolution and virulence of many pathogens becoming increasingly clear. Less attention has been paid on phages preying on Bacillus, despite the potential for some of its members, such as Bacillus anthracis, to cause serious human disease. Results We have isolated five phages infecting the causative agent of anthrax, Bacillus anthracis. Using modern phylogenetic approaches we place these five new Bacillus phages, as well as 21 similar phage genomes retrieved from publicly available databases and metagenomic datasets into the Tyrovirus group, a newly proposed group named so due to the conservation of three distinct tyrosine recombinases. Genomic analysis of these large phages (~ 160–170 kb) reveals their DNA packaging mechanism and genomic features contributing to virion morphogenesis, host cell lysis and phage DNA replication processes. Analysis of the three tyrosine recombinases suggest Tyroviruses undergo a prophage lifecycle that may involve both host integration and plasmid stages. Further we show that Tyroviruses rely on divergent invasion mechanisms, with a subset requiring host S-layer for infection. Conclusions Ultimately, we expand upon our understanding on the classification, phylogeny, and genomic organisation of a new and substantial phage group that prey on critically relevant Bacillus species. In an era characterised by a rapidly evolving landscape of phage genomics the deposition of future Tyroviruses will allow the further unravelling of the global spread and evolutionary history of these Bacillus phages

Molecular characterisation of a novel pathogenic avipoxvirus from the Australian magpie (Gymnorhina tibicen)

Avipoxviruses are significant pathogens infecting a wide range of wild and domestic bird species globally. Here, we describe a novel genome sequence of magpiepox virus (MPPV) isolated from an Australian magpie. In the present study, histopathologically confirmed cutaneous pox lesions were used for transmission electron microscopic analysis, which demonstrated brick-shaped virions with regular spaced thread-like ridges, indicative of likely infectious particles. Subsequent analysis of the recovered MPPV genome positioned phylogenetically to a distinct sub-clade with the recently isolated avipoxvirus genome sequences from shearwater and canary bird species, and demonstrates a high degree of sequence similarity with CNPV (96.14%) and SWPV-2 (95.87%). The novel MPPV complete genome is missing 19 genes with a further 41 genes being truncated/fragmented compared to SWPV-2 and contains nine predicted unique genes. This is the first avipoxvirus complete genome sequence that infects Australian magpie

Haemoglobin degradation underpins the sensitivity of early ring stage <i>Plasmodium falciparum </i>to artemisinins

Current first-line artemisinin antimalarials are threatened by the emergence of resistant Plasmodium falciparum. Decreased sensitivity is evident in the initial (early ring) stage of intraerythrocytic development, meaning that it is crucial to understand the action of artemisinins at this stage. Here, we examined the roles of iron (Fe) ions and haem in artemisinin activation in early rings using Fe ion chelators and a specific haemoglobinase inhibitor (E64d). Quantitative modelling of the antagonism accounted for its complex dependence on the chemical features of the artemisinins and on the drug exposure time, and showed that almost all artemisinin activity in early rings (>80%) is due to haem-mediated activation. The surprising implication that haemoglobin uptake and digestion is active in early rings is supported by identification of active haemoglobinases (falcipains) at this stage. Genetic down-modulation of the expression of the two main cysteine protease haemoglobinases, falcipains 2 and 3, renders early ring stage parasites resistant to artemisinins. This confirms the important role of haemoglobin-degrading falcipains in artemisinin activation, and shows that changes in the rate of artemisinin activation could mediate high-level artemisinin resistance

Proteomic analysis reveals novel proteins associated with the Plasmodium protein exporter PTEX and a loss of complex stability upon truncation of the core PTEX component, PTEX150

The Plasmodium translocon for exported proteins (PTEX) has been established as the machinery responsible for the translocation of all classes of exported proteins beyond the parasitophorous vacuolar membrane of the intraerythrocytic malaria parasite. Protein export, particularly in the asexual blood stage, is crucial for parasite survival as exported proteins are involved in remodelling the host cell, an essential process for nutrient uptake, waste removal and immune evasion. Here, we have truncated the conserved C-terminus of one of the essential PTEX components, PTEX150, in Plasmodium falciparum in an attempt to create mutants of reduced functionality. Parasites tolerated C-terminal truncations of up to 125 amino acids with no reduction in growth, protein export or the establishment of new permeability pathways. Quantitative proteomic approaches however revealed a decrease in other PTEX subunits associating with PTEX150 in truncation mutants, suggesting a role for the C-terminus of PTEX150 in regulating PTEX stability. Our analyses also reveal three previously unreported PTEX-associated proteins, namely PV1, Pf113 and Hsp70-x (respective PlasmoDB numbers; PF3D7_1129100, PF3D7_1420700 and PF3D7_0831700) and demonstrate that core PTEX proteins exist in various distinct multimeric forms outside the major complex