Plasmodium falciparum GBP2 Is a Telomere-Associated Protein That Binds to G-Quadruplex DNA and RNA.

In the early-diverging protozoan parasite Plasmodium, few telomere-binding proteins have been identified and several are unique. Plasmodium telomeres, like those of most eukaryotes, contain guanine-rich repeats that can form G-quadruplex structures. In model systems, quadruplex-binding drugs can disrupt telomere maintenance and some quadruplex-binding drugs are potent anti-plasmodial agents. Therefore, telomere-interacting and quadruplex-interacting proteins may offer new targets for anti-malarial therapy. Here, we report that P. falciparum GBP2 is such a protein. It was identified via 'Proteomics of Isolated Chromatin fragments', applied here for the first time in Plasmodium. In vitro, PfGBP2 binds specifically to G-rich telomere repeats in quadruplex form and it can also bind to G-rich RNA. In vivo, PfGBP2 partially colocalises with the known telomeric protein HP1 but is also found in the cytoplasm, probably due to its affinity for RNA. Consistently, its interactome includes numerous RNA-associated proteins. PfGBP2 is evidently a multifunctional DNA/RNA-binding factor in Plasmodium.

Extended Multilocus Sequence Analysis to Describe the Global Population Structure of the Genus Brucella: Phylogeography and Relationship to Biovars.

An extended multilocus sequence analysis (MLSA) scheme applicable to the Brucella, an expanding genus that includes zoonotic pathogens that severely impact animal and human health across large parts of the globe, was developed. The scheme, which extends a previously described nine locus scheme by examining sequences at 21 independent genetic loci in order to increase discriminatory power, was applied to a globally and temporally diverse collection of over 500 isolates representing all 12 known Brucella species providing an expanded and detailed understanding of the population genetic structure of the group. Over 100 sequence types (STs) were identified and analysis of data provided insights into both the global evolutionary history of the genus, suggesting that early emerging Brucella abortus lineages might be confined to Africa while some later lineages have spread worldwide, and further evidence of the existence of lineages with restricted host or geographical ranges. The relationship between biovar, long used as a crude epidemiological marker, and genotype was also examined and showed decreasing congruence in the order Brucella suis > B. abortus > Brucella melitensis. Both the previously described nine locus scheme and the extended 21 locus scheme have been made available at http://pubmlst.org/brucella/ to allow the community to interrogate existing data and compare with newly generated data.

Acanthamoeba interactions with the blood-brain barrier under dynamic fluid flow.

Acanthamoeba granulomatous encephalitis (AGE), caused by Acanthamoeba castellanii, is a fatal infection of immunocompromised individuals. The pathogenesis of blood-brain barrier (BBB) breach remains unknown. Using a novel in vitro BBB infection model under flow conditions, demonstrates that increases in flow rates lead to decreased binding of A. castellanii to host cells. This is a distinct departure from previous findings under static conditions. However, similarly to static conditions binding of A. castellanii to host cells is host mannose dependent. Disruption of the host cell monolayer was independent of amoeba binding, but dependent on secreted serine proteases. For the first time we report the binding dynamics of A. castellanii under physiological conditions, showing that BBB disruption is not directly linked to binding, instead it is reliant on secreted proteases. Our results offer a platform on which therapies designed at modulating physiological parameters can improve the outcome of infection with A. castellanii.

Next generation of non-mammalian blood-brain barrier models to study parasitic infections of the central nervous system.

Transmigration of neuropathogens across the blood-brain barrier is a key step in the development of central nervous system infections, making it a prime target for drug development. The ability of neuropathogens to traverse the blood-brain barrier continues to inspire researchers to understand the specific strategies and molecular mechanisms that allow them to enter the brain. The availability of models of the blood-brain barrier that closely mimic the situation in vivo offers unprecedented opportunities for the development of novel therapeutics.

Comparative performance of SNP typing and 'Bruce-ladder' in the discrimination of Brucella suis and Brucella canis.

Two novel molecular assays, 'Bruce-ladder' and SNP typing, have recently been described designed to differentiate isolates of the genus Brucella, causative organisms of the significant zoonotic disease brucellosis, at the species level. Differentiation of Brucella canis from Brucella suis by molecular approaches can be difficult and here we compare the performance of 'Bruce-ladder' and SNP typing in correctly identifying B. canis isolates. Both assays proved easy to perform but while 'Bruce-ladder' misidentifies a substantial proportion of B. canis isolates as B. suis, all B. canis isolates were correctly identified by SNP typing.

Phylogeny and classification of Cercomonadida (Protozoa, Cercozoa): Cercomonas, Eocercomonas, Paracercomonas, and Cavernomonas gen. nov.

Cercomonads (=Cercomonadida) are biflagellate gliding bacterivorous protozoa, abundant and diverse in soil and freshwater. We establish 56 new species based on 165 cultures, differential interference contrast microscopy, and 18S and ITS2 rDNA sequencing, and a new genus Cavernomonas studied by scanning electron microscopy. We fundamentally revise the phylogeny and classification of cercomonad Cercozoa. We describe 40 Cercomonas species (35 novel), six Eocercomonas (five novel), two Cavernomonas, and 18 Paracercomonas species (14 novel). We obtained additional cercomonad clade A (Cercomonas, Eocercomonas, Cavernomonas) sequences from multiple environmental DNA libraries. The most commonly cultivated genotypes are not the commonest in environmental DNA, suggesting that cercomonad ecology is far more complex than implied by laboratory cultures. Cercomonads have never been isolated from saline environments, although some species can grow in semi-saline media in the laboratory, and environmental DNA libraries regularly detect them in coastal marine sediments. The first ultrastructural study of an anaerobic cercozoan, Paracercomonas anaerobica sp. nov., a highly divergent cercomonad, shows much simpler ciliary roots than in clade A cercomonads, a ciliary hub-lattice and axosome, and mitochondria with tubular cristae, consistent with it being only facultatively anaerobic. We also describe Agitata tremulans gen. et sp. nov., previously misidentified as Cercobodo (=Dimastigamoeba) agilis Moroff.