The Golgi apparatus in parasitic protists

This review summarizes the current reports on the Golgi apparatus of parasitic protists. Numerous recent publications have demonstrated that studies on intracellular traffic in parasites essentially advanced our knowledge on the Golgi structure and function, which has been traditionally based on research on yeast and mammalian cultured cells. It has been reported that the parasitic lifestyle determines the functional and structural peculiarities of the secretory systems in unrelated groups of unicellular parasites that make them different from those in mammalian and yeast cells. This review covers the best-studied protists, predominantly those of high medical importance, belonging to the following taxa: Parabasalia (Trichomonas), Diplomonada (Giardia), Entamoebidae (Entamoeba), parasitic Alveolata of the phyllum Apicomplexa (Toxoplasma, Plasmodium), and Kinetoplastida (Trypanosoma, Leishmania). The morphology of the Golgi organelle in eukaryotes from various taxonomic groups has been compared. Within three of the six highest taxa of Eukaryota (Adl et al., 2005) a minimum of eight groups are represented by species lacking Golgi dictiosomes. However, biochemical and/or molecular (genomic) evidence indicate that an organelle with the functions of the Golgi was present in every lineage of eukaryotes studied thus far. Loss of the Golgi organelle is a secondary event as proven by identification of Golgi genes in the genomes of Golgi-lacking lineages. The loss might have occurred independently several times in evolution. Neither the number of stacks, nor the size of the organelle correlates with the intensity of secretion or the position of the species on the evolutionary tree (in terms of presumably early/lately diverged lineages).     

Endosymbiotic bacteria as a source of carotenoids in whiteflies

Although carotenoids serve important biological functions, animals are generally unable to synthesize these pigments and instead obtain them from food. However, many animals, such as sap-feeding insects, may have limited access to carotenoids in their diet, and it was recently shown that aphids have acquired the ability to produce carotenoids by lateral transfer of fungal genes. Whiteflies also contain carotenoids but show no evidence of the fungus-derived genes found in aphids. Because many sap-feeding insects harbour intracellular bacteria, it has long been hypothesized that these endosymbionts could serve as an alternative source of carotenoid biosynthesis. We sequenced the genome of the obligate bacterial endosymbiont Portiera from the whitefly Bemisia tabaci. The genome exhibits typical signatures of obligate endosymbionts in sap-feeding insects, including extensive size reduction (358.2 kb) and enrichment for genes involved in essential amino acid biosynthesis. Unlike other sequenced insect endosymbionts, however, Portiera has bacterial homologues of the fungal carotenoid biosynthesis genes in aphids. Therefore, related lineages of sap-feeding insects appear to have convergently acquired the same functional trait by distinct evolutionary mechanisms—bacterial endosymbiosis versus fungal lateral gene transfer.     

Endosymbiotic microorganisms in Adelges (Sacchiphantes) viridis (Insecta,Hemiptera, Adelgoidea: Adelgidae): Molecular characterization,ultrastructure and transovarial transmission

The aim of this paper was to identify endosymbiotic microorganisms living in the body cavity of a Polish population of an aphid, Adelges (Sacchiphantes) viridis, as well as to describe their ultrastructure and mode of transmission between generations. Molecular data (amplification and sequencing of 16S rRNA genes) indicated that endosymbionts of A. (S.) viridis are Betaproteobacteria of the species “Candidatus Vallotia virida”. Endosymbiotic bacteria are rod-shaped and localized in the cytoplasm of specific cells, termed bacteriocytes, of host insects. Endosymbionts sharing the same bacteriocytes differ in the density of their cytoplasm. There are two morphotypes of endosymbiotic bacteria: with electron-dense cytoplasm and electron-translucent cytoplasm. Since only bacteria containing electron-dense cytoplasm were observed in the binary fusion stage, differences in density of the cytoplasm are probably due to changes in the cytoskeleton of bacteria during division. Endosymbionts of A. (S.) viridis are transovarially (i.e. via oocytes) transmitted from the mother to the offspring.     

Interrelationships of chromalveolates within a broadly sampled tree of photosynthetic protists

The Chromalveolata “supergroup” is a massive assemblage of single-celled and multicellular protists such as ciliates and kelps that remains to be substantiated in molecular trees. Recent multigene analyses place chromalveolates into two major clades, the SAR (Stramenopiles, Alveolata, and Rhizaria) and the Cryptophyta + Haptophyta. Here we determined 69 new sequences from different chromalveolates to study the interrelationships of its constituent phyla. We included in our trees, the novel groups Telonemia and Katablepharidophyta that have previously been described as chromalvoleate allies. The best phylogenetic resolution resulted from a 6-protein (actin, α-tubulin, β-tubulin, cytosolic HSP70, BIP HSP70, HSP90) and a 5-protein (lacking HSP90) alignment that validated the SAR and cryptophyte + haptophyte clades with the inclusion of telonemids in the former and katablepharids in the latter. We assessed the Plastidophila hypothesis that is based on EF2 data and suggest this grouping may be explained by horizontal gene transfers involving the EF2 gene rather than indicating host relationships.     

Endosymbiosis of Aphids with Microorganisms: a Model Case of Dynamic Endosymbiotic Evolution

Abstract Almost all aphids harbor prokaryotic intracellular symbionts in the cytoplasm of mycetocytes, huge cells in the abdomen specialized for this purpose. The aphids and their intracellular symbionts are in close mutualistic association and unable to live without their partner. The intracellular symbionts of various aphids are of a single origin; they are descendants of a prokaryote that was acquired by the common ancestor of the present aphids. The date of establishment of the symbiotic association is estimated to be 160–280 million years ago using 16S rRNA molecular clock calibrated by aphid fossils. Molecular phylogeny indicates that the intracellular symbiont belongs to a group of gut bacteria, suggesting the possibility that it was derived from a gut microbe of aphids. While the in-tracellular symbionts are universal and highly conserved amongst aphids, other types of symbiotic microorganisms are also present. In various aphids, bacterial “secondary” intracellular symbionts are found in addition to the standard symbionts. They are thought to be acquired many times in various lineages independently. Some Cerataphidini aphids do not have intracellular symbiotic system but harbor yeast-like extracellular symbionts in the hemocoel. In a lineage of this group, symbiont replacement from intracellular prokaryote to extracellular yeast must have occurred. The diversity of the endosymbiotic system of aphids illuminates a dynamic aspect of endosymbiotic evolution.     

Rewiring and regulation of cross-compartmentalized metabolism in protists

Plastid acquisition, endosymbiotic associations, lateral gene transfer, organelle degeneracy or even organelle loss influence metabolic capabilities in many different protists. Thus, metabolic diversity is sculpted through the gain of new metabolic functions and moderation or loss of pathways that are often essential in the majority of eukaryotes. What is perhaps less apparent to the casual observer is that the sub-compartmentalization of ubiquitous pathways has been repeatedly remodelled during eukaryotic evolution, and the textbook pictures of intermediary metabolism established for animals, yeast and plants are not conserved in many protists. Moreover, metabolic remodelling can strongly influence the regulatory mechanisms that control carbon flux through the major metabolic pathways. Here, we provide an overview of how core metabolism has been reorganized in various unicellular eukaryotes, focusing in particular on one near universal catabolic pathway (glycolysis) and one ancient anabolic pathway (isoprenoid biosynthesis). For the example of isoprenoid biosynthesis, the compartmentalization of this process in protists often appears to have been influenced by plastid acquisition and loss, whereas for glycolysis several unexpected modes of compartmentalization have emerged. Significantly, the example of trypanosomatid glycolysis illustrates nicely how mathematical modelling and systems biology can be used to uncover or understand novel modes of pathway regulation.     

The origins of parasitism in the protists

The origins of parasitism among the protists are, like the group itself, polyphyletic. Probably the majority of present-day parasitic forms evolved from free-living ancestors which were ingested as part of the food of their hosts, though origins from ectoparasitic forms and via a phase of facultative parasitism are possibilities, particularly among the ciliated protozoa and (for ectoparasitism) the Kinetoplasta. Sporozoan parasites most probably developed via a stage which was ingested and became adapted to life in the host's gut. Further developments in parasitism involved deeper penetration into the host's tissues and the adoption of more than one host in the life cycle, thus avoiding entirely the potentially hazardous phase of existence outside the host.     

萤火虫荧光素酶基因cDNA真核表达载体在细菌中的高效表达

将荧火虫荧光素酶基因cDNA真核表达载体pGL2导入大肠杆菌HB101、鼠伤寒杆菌LB5000和X4064中,它在这些原核细胞中均有较好的表达效果。它在大肠杆菌HB101的表达量是该基因cDNA原核表达载体pUHF12-1的表达量的30倍。采用计算机PC／GENE程序包分析,pGL2的SV40早期启动子序列中含有SV40基因组HindⅢB片断中一段长为49bp的DNA序列,这一序列可能就是SV40基因组HindⅢB片断的原核增强子功能的决定序列,正是该序列使pGL2在细菌中获得了高效表达。     

Molecular phylogeny of protists: origin and evolution of the Dinoflagellates

Abstract

Molecular sequence data have become prominent tools for phylogenetic relationship inference, particularly useful in the analysis of highly diverse taxonomic orders. Ribosomal RNA sequences provide markers that can be used in the study of phylogeny, because their function and structure have been conserved to a large extent throughout the evolutionary history of organisms. These sequences are inferred from cloned or enzymatically amplified gene sequences, or determined by direct RNA sequencing. The first step of the phylogenetic interpretation of nucleic acid sequence variations implies proper alignment of corresponding sequences from various organisms. Best alignment based on similarity criteria is greatly reinforced, in the case of ribosomal RNAs, by secondary structure homologies. Distance matrix methods to infer evolutionary trees are based on the assumption that the phylogenetic distance between each pair of organisms is proportional to the number of nucleotide substitution events. Computed tree inference methods usually take into consideration the possibility of unequal mutation rates among lineages. Divergence times can be estimated on the tree, provided that at least one lineage has been dated by fossil records. We have utilized this approach based on ribosomal RNA sequence comparison to investigate the phylogenetic relationship between dinoflagellated and other eukaryote protists, and to refine controverse phylogenies of the class Dinophycae.     

Association between Wolbachia and Spiroplasma within Drosophila neotestacea: an emerging symbiotic mutualism?

Interspecific mutualism can evolve when specific lineages of different species tend to be associated with each other from one generation to the next. Different maternally transmitted endosymbionts occurring within the same cytoplasmic lineage fulfil this requirement. Drosophila neotestacea is infected with maternally transmitted Wolbachia and Spiroplasma, which are cotransmitted at high frequency in natural populations. Molecular phylogenetic evidence indicates that both endosymbionts have been present in D. neotestacea for considerable evolutionary periods. Thus, conditions are suitable for the evolution of mutualism between them. In support of this possibility, there is a significant positive association between Wolbachia and Spiroplasma infection in many samples of D. neotestacea from natural populations. Theoretically, such a positive association can result from either mutualism between these endosymbionts or recent spread. Collections from present‐day populations suggest that recent spread and mutualism have both operated to generate the positive association between Wolbachia and Spiroplasma. If selection acts on the combination of these two endosymbionts, they may be in the early stages of evolution of a more complex, cooperative association.     

抗菌肽的基因工程研究进展

近年来细菌耐药性问题日趋严峻,寻找新型抗生素已迫在眉睫。抗菌肽是生物体产生的一种阳离子短肽,具有天然的抗菌活性。由于抗菌肽具有与传统抗生素不同的作用机制,不产生耐药性,因而具有重要的临床应用价值。但实践表明,抗菌肽的开发并非易事。针对近年来抗菌肽开发的基因工程策略和实践,尤其是大肠杆菌表达系统和酵母表达系统,进行了简要综述。     

Nonhomogeneous model of sequence evolution indicates independent origins of primary endosymbionts within the enterobacteriales (gamma-Proteobacteria)

Standard methods of phylogenetic reconstruction are based on models that assume homogeneity of nucleotide composition among taxa. However, this assumption is often violated in biological data sets. In this study, we examine possible effects of nucleotide heterogeneity among lineages on the phylogenetic reconstruction of a bacterial group that spans a wide range of genomic nucleotide contents: obligately endosymbiotic bacteria and free-living or commensal species in the gamma-Proteobacteria. We focus on AT-rich primary endosymbionts to better understand the origins of obligately intracellular lifestyles. Previous phylogenetic analyses of this bacterial group point to the importance of accounting for base compositional variation in estimating relationships, particularly between endosymbiotic and free-living taxa. Here, we develop an approach to compare susceptibility of various phylogenetic reconstruction methods to the effects of nucleotide heterogeneity. First, we identify candidate trees of gamma-Proteobacteria groEL and 16S rRNA using approaches that assume homogeneous and stationary base composition, including Bayesian, maximum likelihood, parsimony, and distance methods. We then create permutations of the resulting candidate trees by varying the placement of the AT-rich endosymbiont Buchnera. These permutations are evaluated under the nonhomogeneous and nonstationary maximum likelihood model of Galtier and Gouy, which allows equilibrium base content to vary among examined lineages. Our results show that commonly used phylogenetic methods produce incongruent trees of the Enterobacteriales, and that the placement of Buchnera is especially unstable. However, under a nonhomogeneous model, various groEL and 16S rRNA phylogenies that separate Buchnera from other AT-rich endosymbionts (Blochmannia and Wigglesworthia) have consistently and significantly higher likelihood scores. Blochmannia and Wigglesworthia appear to have evolved from secondary endosymbionts, and represent an origin of primary endosymbiosis that is independent from Buchnera. This application of a nonhomogeneous model offers a computationally feasible way to test specific phylogenetic hypotheses for taxa with heterogeneous and nonstationary base composition.     

Cospeciation of termite gut flagellates and their bacterial endosymbionts: Trichonympha species and 'Candidatus Endomicrobium trichonymphae'

Symbiotic flagellates play a major role in the digestion of lignocellulose in the hindgut of lower termites. Many termite gut flagellates harbour a distinct lineage of bacterial endosymbionts, so-called Endomicrobia, which belong to the candidate phylum Termite Group 1. Using an rRNA-based approach, we investigated the phylogeny of Trichonympha , the predominant flagellates in a wide range of termite species, and of their Endomicrobia symbionts. We found that Trichonympha species constitute three well-supported clusters in the Parabasalia tree. Endomicrobia were detected only in the apical lineage (Cluster I), which comprises flagellates present in the termite families Termopsidae and Rhinotermitidae, but apparently absent in the basal lineages (Clusters II and III) consisting of flagellates from other termite families and from the wood-feeding cockroach, Cryptocercus punctulatus . The endosymbionts of Cluster I form a monophyletic group distinct from many other lineages of Endomicrobia and seem to have cospeciated with their flagellate host. The distribution pattern of the symbiotic pairs among different termite species indicates that cospeciation of flagellates and endosymbionts is not simply the result of a spatial separation of the flagellate lineages in different termite species, but that Endomicrobia are inherited among Trichonympha species by vertical transmission. We suggest extending the previously proposed candidatus name ' Endomicrobium trichonymphae ' to all Endomicrobia symbionts of Trichonympha species, and estimate that the acquisition by an ancestor of Trichonympha Cluster I must have occurred about 40–70 million years ago, long after the flagellates entered the termites.     

Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase–polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.     

Biodiversity and evolution in host-parasite associations

Evolutionary relationships in heterospecific associations (parasitoidism, parasitism, commensalism and mutualism) are analysed through a game theory model defined in terms of fitness of hosts and parasites. In front of the game solutions (i.e. ESS) which present a great diversity of evolutionary patterns, we envisage co-evolution between hosts and parasites through the evolution of its two fundamental parameters (i.e. host's resistance and parasite's virulence). We then discuss the reciprocal influence of hosts and parasites on their respective biodiversity.