Diversity and zoogeography of Antarctic deep-sea Munnopsidae (Crustacea, Isopoda, Asellota) from three ANDEEP expeditions

The family Munnopsidae was the most abundant and diverse among 22 isopod families collected by the ANDEEP deep-sea expeditions in 2002 and 2005 in the Atlantic sector of the Southern Ocean. A total of 219 species from 31 genera and eight subfamilies were analysed. Only 20% species were known to science, and 11% of these were reported outside the ANDEEP area mainly from other parts of the SO or the South Atlantic deep sea. One hundred and five species (50%) were rare, occurring at only 1 or 2 stations. Seventy-two percent of all munnopsid specimens belong to the most numerous 25 species with a total abundance of more than 75 specimens; 5 of these species (40% of all specimens) belong to the main genera of the world munnopsid fauna, Eurycope, Disconectes, Betamorpha, and Ilyarachna. About half of all munnopsid specimens and 34% of all species belong to the subfamily Eurycopinae, which is followed in occurrence by the Lipomerinae (19%). Munnopsinae is the poorest represented subfamily (1.5%). The composition of the subfamilies for the munnopsid fauna of the ANDEEP area differs from that of northern faunas. Lipomerinae show a lower percentage (7%) in the North Atlantic and are absent in the Arctic and in the North Pacific. This subfamily is considered as young and having a centre of origin and diversification in the Southern Ocean. The analyses of the taxonomic diversity and the distribution of Antarctic munnopsids and the distribution of the world fauna of all genera of the family revealed that species richness and diversity of the genera are highest in the ANDEEP area. The investigated fauna is characterised also by high percentage of endemic species, the highest richness and diversity of the main munnopsid genera and subfamily Lipomerinae. This supports the hypothesis that the Atlantic sector of SO deep sea may be considered as the main contemporary centre of diversification of the Munnopsidae. It might serve as a diversity pump of species of the Munnopsidae to more northern Atlantic areas via the deep water originating in the Weddell Sea.

Análise filogenética de Leptodeira Fitzinger, 1843 e taxonomia das espécies do clado do sul do complexo Leptodeira annulata/septentrionalis (Serpentes, Dipsadidae)

Leptodeira é um gênero Neotropical de serpentes com nove espécies, dividido em quatro grupos morfológicos: annulata com as espécies L. annulata (L. a. annulata, L. a. ashmeadi, L. a. cussiliris, L. a. pulchriceps, L. a. rhombifera), L. bakeri, L. maculata, L. frenata (L. f. frenata, L. f. malleisi, L. f. yucatanensis), L. uribei; septentrionalis com L. septentrionalis (L. s. septentrionalis, L. s. larcorum, L. s. ornata, L. s. polysticta), L. splendida (L. s. splendida, L. s. bressoni, L. s. ephippiata); nigrofasciata com apenas a espécie L. nigrofasciata; e grupo punctata representado por L. punctata. Na última revisão taxonômica do gênero, várias espécies foram consideradas como subespécies, sendo caracterizadas principalmente com base no padrão de cor. No entanto, recentes análises moleculares não corroboraram o arranjo taxonômico proposto anteriormente. As duas espécies com distribuição geográfica mais ampla e maior número de subespécies, L. annulata e L. septentrionalis, não tiveram monofila validada. Na mais recente proposta filogenética de Leptodeira, a monofilia do complexo L. annulata/septentrionalis foi obtida a partir de caracteres moleculares, sendo recuperados dois clados geograficamente distintos: um clado Norte presente no sul da América do Norte e norte da América Central (L. s. polysticta ((L. rubricata - L. a. rhombifera) ((L. a. cussiliris - L. maculata) L. a. cussiliris))); e outro clado Sul-americano presente no sul da América Central e toda América do Sul ((L. s. ornata–L. a. annulata) (L. s. ornata (L. bakeri (L. a. ashmeadi–L. a. annulata)))). Neste trabalho, apresento uma hipótese filogenética para Leptodeira e reviso taxonomicamente o clado Sul do complexo L. annulata/septentrionalis, apresentando um novo arranjo taxonômico para o grupo. Desta forma, a presente tese está organizada em: Introdução Geral, onde foram apresentadas informações sobre taxonomia e sistemática de Leptodeira, especialmente do complexo L. annulata/septentrionalis; Capítulo 1 intitulado “Análise filogenética de Leptodeira (Serpentes, Dipsadidae)”, com o objetivo de propor uma hipótese filogenética para o gênero, com base em caracteres moleculares; e o Capítulo 2 intitulado “Taxonomia das espécies do clado Sul do complexo L. annulata/septentrionalis (Serpentes, Dipsadidae)”, que teve como objetivo caracterizar morfologicamente os clados obtidos a partir da análise molecular e propor um novo arranjo taxonômico para o grupo.

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite-host interactions during host cell division.

Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton

Summary Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

Theileria annulata promotes Src kinase-dependent host cell polarization by manipulating actin dynamics in podosomes and lamellipodia

Theileria annulata is an intracellular protozoan parasite that infects B cells and macrophages of ruminants. Macrophages infected with T. annulata are de-differentiated and display tumour cell properties and a metastatic behaviour. How parasitized cells adapt their morphology, motility and invasive behaviour has not yet been addressed in detail. In this study, I investigated the regulation of host cell actin dynamics in T. annulata-transformed macrophages and how this affects host cell morphology and motility. T. annulata was found to promote the formation of filamentous-actin-rich podosome-type adhesions (PTAs) and lamellipodia, and to establish a polarized morphology of the infected cell. Characteristic for parasite-dependent host cell polarization is that infected cells display a single, persistent lamellipodium. Src kinases--in particular Hck--are required for the polar extension of this lamellipodium. Hck does so by promoting the clustered assembly of PTAs and accumulation of proteins of the Ezrin, Radixin, Moesin (ERM) family in lamellipodia. Polar accumulation of PTAs and ERM proteins correlates with focal matrix degradation underneath lamellipodia. These findings suggest that T. annulata equips its host cell with properties to adhere and invade. These properties are likely to promote the motile behaviour required for dissemination of infected cells in vivo.

Genome of the host-cell transforming parasite Theileria annulata compared with T. parva

Theileria annulata and T. parva are closely related protozoan parasites that cause lymphoproliferative diseases of cattle. We sequenced the genome of T. annulata and compared it with that of T. parva to understand the mechanisms underlying transformation and tropism. Despite high conservation of gene sequences and synteny, the analysis reveals unequally expanded gene families and species-specific genes. We also identify divergent families of putative secreted polypeptides that may reduce immune recognition, candidate regulators of host-cell transformation, and a Theileria-specific protein domain [frequently associated in Theileria (FAINT)] present in a large number of secreted proteins.

Alteration of host cell phenotype by Theileria annulata and Theileria parva: mining for manipulators in the parasite genomes

The apicomplexan parasites Theileria annulata and Theileria parva cause severe lymphoproliferative disorders in cattle. Disease pathogenesis is linked to the ability of the parasite to transform the infected host cell (leukocyte) and induce uncontrolled proliferation. It is known that transformation involves parasite dependent perturbation of leukocyte signal transduction pathways that regulate apoptosis, division and gene expression, and there is evidence for the translocation of Theileria DNA binding proteins to the host cell nucleus. However, the parasite factors responsible for the inhibition of host cell apoptosis, or induction of host cell proliferation are unknown. The recent derivation of the complete genome sequence for both T. annulata and T. parva has provided a wealth of information that can be searched to identify molecules with the potential to subvert host cell regulatory pathways. This review summarizes current knowledge of the mechanisms used by Theileria parasites to transform the host cell, and highlights recent work that has mined the Theileria genomes to identify candidate manipulators of host cell phenotype.

Proteomic analysis of the Theileria annulata schizont

The apicomplexan parasite, Theileria annulata, is the causative agent of tropical theileriosis, a devastating lymphoproliferative disease of cattle. The schizont stage transforms bovine leukocytes and provides an intriguing model to study host/pathogen interactions. The genome of T. annulata has been sequenced and transcriptomic data are rapidly accumulating. In contrast, little is known about the proteome of the schizont, the pathogenic, transforming life cycle stage of the parasite. Using one-dimensional (1-D) gel LC-MS/MS, a proteomic analysis of purified T. annulata schizonts was carried out. In whole parasite lysates, 645 proteins were identified. Proteins with transmembrane domains (TMDs) were under-represented and no proteins with more than four TMDs could be detected. To tackle this problem, Triton X-114 treatment was applied, which facilitates the extraction of membrane proteins, followed by 1-D gel LC-MS/MS. This resulted in the identification of an additional 153 proteins. Half of those had one or more TMD and 30 proteins with more than four TMDs were identified. This demonstrates that Triton X-114 treatment can provide a valuable additional tool for the identification of new membrane proteins in proteomic studies. With two exceptions, all proteins involved in glycolysis and the citric acid cycle were identified. For at least 29% of identified proteins, the corresponding transcripts were not present in the existing expressed sequence tag databases. The proteomics data were integrated into the publicly accessible database resource at EuPathDB (www.eupathdb.org) so that mass spectrometry-based protein expression evidence for T. annulata can be queried alongside transcriptional and other genomics data available for these parasites.

Recruitment of EB1, a master regulator of microtubule dynamics, to the surface of the Theileria annulata schizont

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton.

Cell cycle-dependent phosphorylation of Theileria annulata schizont surface proteins.

The invasion of Theileria sporozoites into bovine leukocytes is rapidly followed by the destruction of the surrounding host cell membrane, allowing the parasite to establish its niche within the host cell cytoplasm. Theileria infection induces host cell transformation, characterised by increased host cell proliferation and invasiveness, and the activation of anti-apoptotic genes. This process is strictly dependent on the presence of a viable parasite. Several host cell kinases, including PI3-K, JNK, CK2 and Src-family kinases, are constitutively activated in Theileria-infected cells and contribute to the transformed phenotype. Although a number of host cell molecules, including IkB kinase and polo-like kinase 1 (Plk1), are recruited to the schizont surface, very little is known about the schizont molecules involved in host-parasite interactions. In this study we used immunofluorescence to detect phosphorylated threonine (p-Thr), serine (p-Ser) and threonine-proline (p-Thr-Pro) epitopes on the schizont during host cell cycle progression, revealing extensive schizont phosphorylation during host cell interphase. Furthermore, we established a quick protocol to isolate schizonts from infected macrophages following synchronisation in S-phase or mitosis, and used mass spectrometry to detect phosphorylated schizont proteins. In total, 65 phosphorylated Theileria proteins were detected, 15 of which are potentially secreted or expressed on the surface of the schizont and thus may be targets for host cell kinases. In particular, we describe the cell cycle-dependent phosphorylation of two T. annulata surface proteins, TaSP and p104, both of which are highly phosphorylated during host cell S-phase. TaSP and p104 are involved in mediating interactions between the parasite and the host cell cytoskeleton, which is crucial for the persistence of the parasite within the dividing host cell and the maintenance of the transformed state.