The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries

Plasmodium falciparum and Toxoplasma gondii are obligate intracellular apicomplexan parasites that rapidly invade and extensively modify host cells. Protein phosphorylation is one mechanism by which these parasites can control such processes. Here we present a phosphoproteome analysis of peptides enriched from schizont stage P. falciparum and T. gondii tachyzoites that are either "intracellular" or purified away from host material. Using liquid chromatography-tandem mass spectrometry, we identified over 5,000 and 10,000 previously unknown phosphorylation sites in P. falciparum and T. gondii, respectively, revealing that protein phosphorylation is an extensively used regulation mechanism both within and beyond parasite boundaries. Unexpectedly, both parasites have phosphorylated tyrosines, and P. falciparum has unusual phosphorylation motifs that are apparently shaped by its A:T-rich genome. This data set provides important information on the role of phosphorylation in the host-pathogen interaction and clues to the evolutionary forces operating on protein phosphorylation motifs in both parasites.     

The biochemical diversity of life near and above 100°C in marine environments

Hyperthermophilic micro-organisms grow at temperatures above 90 °C with a current upper limit of 113 °C. They are a recent discovery in the microbial world and have been isolated mainly from marine geothermal environments, which include both shallow and deep sea hydrothermal vents. By 16S rRNA analyses they are the most slowly evolving of all extant life forms, and all but two of the nearly 20 known genera are classified as Archaea (formerly Archaebacteria). Almost all hyperthermophiles are strict anaerobes. They include species of methanogens, iron-oxidizers and sulphate reducers, but the majority are obligate heterotrophs that depend upon the reduction of elemental sulphur (S°) to hydrogen sulphide for significant growth. The heterotrophs utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S° has been proposed. Two S°-reducing enzymes have been purified from the cytoplasm of one hyperthermophile (T(opt) 100 °C) that is able to grow either with and without S°. However, the mechanisms by which S° reduction is coupled to energy conservation in this organism and in obligate S°-reducing hyperthermophiles is not known. In the heterotrophs, sugar fermentation is achieved by a novel glycolytic pathway involving unusual ADP-dependent kinases and ATP synthetases, and novel oxidoreductases that are ferredoxin- rather than NAD(P)-linked. Similarly, peptide fermentation involves several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Several of these oxido-reductases contain tungsten, an element that is rarely used in biological systems. Tungsten is present in exceedingly low concentrations in normal sea water, but hydrothermal systems contain much higher tungsten concentrations, more than sufficient to support hyperthermophilic life.     

Metabolism in hyperthermophilic microorganisms

Hyperthermophilic microorganisms grow at temperatures of 90 °C and above and are a recent discovery in the microbial world. They are considered to be the most ancient of all extant life forms, and have been isolated mainly from near shallow and deep sea hydrothermal vents. All but two of the nearly twenty known genera are classified asArchaea (formerly archaebacteria). Virtually all of them are strict anaerobes. The majority are obligate heterotrophs that utilize proteinaceous materials as carbon and energy sources, although a few species are also saccharolytic. Most also depend on the reduction of elemental sulfur to hydrogen sulfide (H₂S) for significant growth. Peptide fermentation involves transaminases and glutamate dehydrogenase, together with several unusual ferredoxin-linked oxidoreductases not found in mesophilic organisms. Similarly, a novel pathway based on a partially non-phosphorylated Entner-Doudoroff scheme has been postulated to convert carbohydrates to acetate, H₂ and CO₂, although a more conventional Embden-Meyerhof pathway has also been identified in one saccharolytic species. The few hyperthermophiles known that can assimilate CO₂ do so via a reductive citric acid cycle. Two S^o-reducing enzymes termed sulfhydrogenase and sulfide dehydrogenase have been purified from the cytoplasm of a hyperthermophile that is able to grow either with or without S^o. A scheme for electron flow during the oxidation of carbohydrates and peptides and the reduction of S^o has been proposed. However, the mechanisms by which S^o reduction is coupled to energy conservation in this organism and in obligate S^o-reducing hyperthermophiles is not known.Abbreviations ADH alcohol dehydrogenase (ADH) - AOR aldehyde ferredoxin oxidoreductase - FMOR formate ferredoxin oxidoreductase - FOR formaldehyde ferredoxin oxidoreductase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - GDH glutamate dehydrogenase - GluOR glucose ferredoxin oxidoreductase - KGOR 2-ketoglutarate ferredoxin oxidoreductase - IOR indolepyruvate ferredoxin oxidoreductase - LDH lactate dehydrogenase - MPT molybopterin - POR pyruvate ferredoxin oxidoreductase - PLP pyridoxal-phosphate - PS polysulfide - TPP thiamin pyrophosphate - S^o elemental sulfur - VOR isovalerate ferredoxin oxidoreductase     

Conditional expression of Toxoplasma gondii apical membrane antigen-1 (TgAMA1) demonstrates that TgAMA1 plays a critical role in host cell invasion

Toxoplasma gondii is an obligate intracellular parasite and an important human pathogen. Relatively little is known about the proteins that orchestrate host cell invasion by T. gondii or related apicomplexan parasites (including Plasmodium spp., which cause malaria), due to the difficulty of studying essential genes in these organisms. We have used a recently developed regulatable promoter to create a conditional knockout of T. gondii apical membrane antigen-1 (TgAMA1). TgAMA1 is a transmembrane protein that localizes to the parasite's micronemes, secretory organelles that discharge during invasion. AMA1 proteins are conserved among apicomplexan parasites and are of intense interest as malaria vaccine candidates. We show here that T. gondii tachyzoites depleted of TgAMA1 are severely compromised in their ability to invade host cells, providing direct genetic evidence that AMA1 functions during invasion. The TgAMA1 deficiency has no effect on microneme secretion or initial attachment of the parasite to the host cell, but it does inhibit secretion of the rhoptries, organelles whose discharge is coupled to active host cell penetration. The data suggest a model in which attachment of the parasite to the host cell occurs in two distinct stages, the second of which requires TgAMA1 and is involved in regulating rhoptry secretion.     

Complete genome sequence of Hydrogenobacter thermophilus type strain (TK-6)

Hydrogenobacter thermophilus Kawasumi et al. 1984 is the type species of the genus Hydrogenobacter. H. thermophilus was the first obligate autotrophic organism reported among aerobic hydrogen-oxidizing bacteria. Strain TK-6(T) is of interest because of the unusually efficient hydrogen-oxidizing ability of this strain, which results in a faster generation time compared to other autotrophs. It is also able to grow anaerobically using nitrate as an electron acceptor when molecular hydrogen is used as the energy source, and able to aerobically fix CO(2)via the reductive tricarboxylic acid cycle. This is the fifth completed genome sequence in the family Aquificaceae, and the second genome sequence determined from a strain derived from the original isolate. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 1,742,932 bp long genome with its 1,899 protein-coding and 49 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Cultural, morphological, and physiological characteristics of Thermomonospora fusca (strain 190Th).

The cultural, morphological, and physiological properties of Thermomonospora fusca (strain 190Th) are described. Its physiological properties show that this species is primarily a carbohydrate-degrading actinomycete which can use a wide range of plant sugars and polymeric carbohydrates as sources of carbon and energy. The culture does not use proteins or amino acids for carbon and energy, or as a nitrogen source. A few organic acids are utilized. Ammonia is the preferred nitrogen source. The culture has trace nutrient requirements which include biotin and an undetermined number of amino acids. These and other physiological characteristics are discussed in relation to the roles that T. fusca carries out as a saprophytic bacterium in nature. Its cultural and morphological properties are discussed in relation to the taxonomic status of this species in the literature.     

Genomic determinants of protein folding thermodynamics in prokaryotic organisms

Here we investigate how thermodynamic properties of orthologous proteins are influenced by the genomic environment in which they evolve. We performed a comparative computational study of 21 protein families in 73 prokaryotic species and obtained the following main results. (i) Protein stability with respect to the unfolded state and with respect to misfolding are anticorrelated. There appears to be a trade-off between these two properties, which cannot be optimized simultaneously. (ii) Folding thermodynamic parameters are strongly correlated with two genomic features, genome size and G+C composition. In particular, the normalized energy gap, an indicator of folding efficiency in statistical mechanical models of protein folding, is smaller in proteins of organisms with a small genome size and a compositional bias towards A+T. Such genomic features are characteristic for bacteria with an intracellular lifestyle. We interpret these correlations in light of mutation pressure and natural selection. A mutational bias toward A+T at the DNA level translates into a mutational bias toward more hydrophobic (and in general more interactive) proteins, a consequence of the structure of the genetic code. Increased hydrophobicity renders proteins more stable against unfolding but less stable against misfolding. Proteins with high hydrophobicity and low stability against misfolding occur in organisms with reduced genomes, like obligate intracellular bacteria. We argue that they are fixed because these organisms experience weaker purifying selection due to their small effective population sizes. This interpretation is supported by the observation of a high expression level of chaperones in these bacteria. Our results indicate that the mutational spectrum of a genome and the strength of selection significantly influence protein folding thermodynamics.     

Multiple avirulence paralogues in cereal powdery mildew fungi may contribute to parasite fitness and defeat of plant resistance

Powdery mildews, obligate biotrophic fungal parasites on a wide range of important crops, can be controlled by plant resistance (R) genes, but these are rapidly overcome by parasite mutants evading recognition. It is unknown how this rapid evolution occurs without apparent loss of parasite fitness. R proteins recognize avirulence (AVR) molecules from parasites in a gene-for-gene manner and trigger defense responses. We identify AVR(a10) and AVR(k1) of barley powdery mildew fungus, Blumeria graminis f sp hordei (Bgh), and show that they induce both cell death and inaccessibility when transiently expressed in Mla10 and Mlk1 barley (Hordeum vulgare) varieties, respectively. In contrast with other reported fungal AVR genes, AVR(a10) and AVR(k1) encode proteins that lack secretion signal peptides and enhance infection success on susceptible host plant cells. AVR(a10) and AVR(k1) belong to a large family with >30 paralogues in the genome of Bgh, and homologous sequences are present in other formae speciales of the fungus infecting other grasses. Our findings imply that the mildew fungus has a repertoire of AVR genes, which may function as effectors and contribute to parasite virulence. Multiple copies of related but distinct AVR effector paralogues might enable populations of Bgh to rapidly overcome host R genes while maintaining virulence.     

The SAR11 group of alpha-proteobacteria is not related to the origin of mitochondria

Although free living, members of the successful SAR11 group of marine alpha-proteobacteria contain a very small and A+T rich genome, two features that are typical of mitochondria and related obligate intracellular parasites such as the Rickettsiales. Previous phylogenetic analyses have suggested that Candidatus Pelagibacter ubique, the first cultured member of this group, is related to the Rickettsiales+mitochondria clade whereas others disagree with this conclusion. In order to determine the evolutionary position of the SAR11 group and its relationship to the origin of mitochondria, we have performed phylogenetic analyses on the concatenation of 24 proteins from 5 mitochondria and 71 proteobacteria. Our results support that SAR11 group is not the sistergroup of the Rickettsiales+mitochondria clade and confirm that the position of this group in the alpha-proteobacterial tree is strongly affected by tree reconstruction artefacts due to compositional bias. As a consequence, genome reduction and bias toward a high A+T content may have evolved independently in the SAR11 species, which points to a different direction in the quest for the closest relatives to mitochondria and Rickettsiales. In addition, our analyses raise doubts about the monophyly of the newly proposed Pelagibacteraceae family.     

Theileria equi RAP-1a and RAP-1b proteins contain immunoreactive epitopes and are suitable candidates for vaccine and diagnostics development

Theileria equi is an obligate intracellular protozoan parasite that causes severe hemolytic anaemia in most equid species. Similar to other apicomplexan parasites, T. equi contains rhoptries whose contents have been implicated in host cell invasion and formation of the parasitophorous vacuole that is crucial for survival of the species within cells. Despite their importance, the composition of T. equi rhoptries and their role(s) in host cell invasion remain unexplored. To gain insight into these issues, we evaluated the expression, immunogenicity, and functional roles of two T. equi rhoptry-associated proteins abbreviated as RAP-1a and RAP-1b. The full-length RAP-1a protein was expressed to perform the analysis but our efforts to express the full-length RAP-1b protein failed due to an unknown reason. We therefore generated synthetic immunogenic peptides that map onto the N- and C-termini of the RAP-1b protein as an alternative approach. Our findings show that both proteins are expressed in the extracellular and intra-erythrocytic merozoite stages of T. equi. Serological analyses show that T. equi-infected horses mount antibody responses that recognise both proteins and correlate with a decrease in T. equi load in both acutely and persistently infected horses. In vitro neutralisation studies show that the T. equi RAP-1a protein contains neutralisation-sensitive epitopes as antibodies developed against the protein significantly inhibited the parasites from invading equine erythrocytes. Conversely, antibodies developed against the RAP-1b synthetic peptides did not neutralise parasite invasion, showing that the protein regions on which the peptides were based are not required for T. equi invasion. Overall, the data shows that T. equi rhoptries and their contents are involved in invasion of host cells and supports T. equi RAP-1 proteins as candidates for developing novel serodiagnosis tools and vaccines.     

The Genome of the Obligate Intracellular Parasite Trachipleistophora hominis: New Insights into Microsporidian Genome Dynamics and Reductive Evolution

The dynamics of reductive genome evolution for eukaryotes living inside other eukaryotic cells are poorly understood compared to well-studied model systems involving obligate intracellular bacteria. Here we present 8.5 Mb of sequence from the genome of the microsporidian Trachipleistophora hominis, isolated from an HIV/AIDS patient, which is an outgroup to the smaller compacted-genome species that primarily inform ideas of evolutionary mode for these enormously successful obligate intracellular parasites. Our data provide detailed information on the gene content, genome architecture and intergenic regions of a larger microsporidian genome, while comparative analyses allowed us to infer genomic features and metabolism of the common ancestor of the species investigated. Gene length reduction and massive loss of metabolic capacity in the common ancestor was accompanied by the evolution of novel microsporidian-specific protein families, whose conservation among microsporidians, against a background of reductive evolution, suggests they may have important functions in their parasitic lifestyle. The ancestor had already lost many metabolic pathways but retained glycolysis and the pentose phosphate pathway to provide cytosolic ATP and reduced coenzymes, and it had a minimal mitochondrion (mitosome) making Fe-S clusters but not ATP. It possessed bacterial-like nucleotide transport proteins as a key innovation for stealing host-generated ATP, the machinery for RNAi, key elements of the early secretory pathway, canonical eukaryotic as well as microsporidian-specific regulatory elements, a diversity of repetitive and transposable elements, and relatively low average gene density. Microsporidian genome evolution thus appears to have proceeded in at least two major steps: an ancestral remodelling of the proteome upon transition to intracellular parasitism that involved reduction but also selective expansion, followed by a secondary compaction of genome architecture in some, but not all, lineages.     

Complete genome sequence of Treponema succinifaciens type strain (6091)

Treponema succinifaciens Cwyk and Canale-Parola 1981 is of interest because this strictly anaerobic, apathogenic member of the genus Treponema oxidizes carbohydrates and couples the Embden-Meyerhof pathway via activity of a pyruvate-formate lyase to the production of acetyl-coenzyme A and formate. This feature separates this species from most other anaerobic spirochetes. The genome of T. succinifaciens 6091(T) is only the second completed and published type strain genome from the genus Treponema in the family Spirochaetaceae. The 2,897,425 bp long genome with one plasmid harbors 2,723 protein-coding and 63 RNA genes and is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

The genome sequence of the obligately chemolithoautotrophic, facultatively anaerobic bacterium Thiobacillus denitrificans

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, beta-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by beta-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.     

Toxoplasma gondii: the model apicomplexan

Toxoplasma gondii is an obligate intracellular protozoan parasite which is a significant human and veterinary pathogen. Other members of the phylum Apicomplexa are also important pathogens including Plasmodium species (i.e. malaria), Eimeria species, Neospora, Babesia, Theileria and Cryptosporidium. Unlike most of these organisms, T. gondii is readily amenable to genetic manipulation in the laboratory. Cell biology studies are more readily performed in T. gondii due to the high efficiency of transient and stable transfection, the availability of many cell markers, and the relative ease with which the parasite can be studied using advanced microscopic techniques. Thus, for many experimental questions, T. gondii remains the best model system to study the biology of the Apicomplexa. Our understanding of the mechanisms of drug resistance, the biology of the apicoplast, and the process of host cell invasion has been advanced by studies in T. gondii. Heterologous expression of apicomplexan proteins in T. gondii has frequently facilitated further characterisation of proteins that could not be easily studied. Recent studies of Apicomplexa have been complemented by genome sequencing projects that have facilitated discovery of surprising differences in cell biology and metabolism between Apicomplexa. While results in T. gondii will not always be applicable to other Apicomplexa, T. gondii remains an important model system for understanding the biology of apicomplexan parasites.     

Rhoptry organelles of the apicomplexa: Their role in host cell invasion and intracellular survival

Members of the phylum Apicomplexa are obligate intracellular parasites that invade erythrocytes, lymphocytes, macrophages or cells of the alimentary canal in various vertebrate species. Organelles within the apical complex of invasive stages facilitate host cell invasion. Parasites in this phylum cause some of the most debilitating diseases of medical and veterinary importance. These include malaria, toxoplasmosis, babesiosis, theileriosis (East Coast fever), and coccidiosis in poultry and livestock. In recent years, opportunistic infections caused by Cryptosporidium parvum, and recrudescent Toxoplasma gondii infections in AIDS patients have prompted intensified efforts in understanding the biology of these parasites. In this review, Tobili Sam-Yellowe examines the unifying and variant molecular features of rhoptry proteins, and addresses the role of multigene families in organelle function: the biogenesis of the rhoptries will also be examined, in an attempt to understand the sequence of events leading to successful packaging, modification and processing of proteins within the organelle.     

Complete Genome Sequence of the Hydrogenotrophic, Methanogenic Archaeon Methanoculleus bourgensis Strain MS2T, Isolated from a Sewage Sludge Digester

Methanoculleus bourgensis, of the order Methanomicrobiales, is a dominant methanogenic archaeon in many biogas-producing reactor systems fed with renewable primary products. It is capable of synthesizing methane via the hydrogenotrophic pathway utilizing hydrogen and carbon dioxide or formate as the substrates. Here we report the complete and finished genome sequence of M. bourgensis strain MS2(T), isolated from a sewage sludge digester.     

Fermentation metabolism of the unicellular cyanobacterium Cyanothece PCC 7822

The hydrogenase-catalyzed hydrogen production exhibited by the unicellular cyanobacterium Cyanothece 7822 during anoxic incubation in the dark is a result of the fermentative degradation of carbon reserves. Simultaneously with hydrogen production, evolution of carbon dioxide was detected, and excretion of ethanol, lactate, formate and acetate was demonstrated. The fermentation balance indicates that carbohydrates are fermented via a branched pathway, in which both the pentose phosphate pathway and glycolysis appear to be involved. It is proposed that the physiological function of hydrogen production is the introduction of protons as terminal electron acceptors. This removal of reducing equivalents might give rise to continuation of the pyruvate decarboxylation and consequently of the acetate formation, thereby increasing the efficiency of fermentative energy generation.     

Complete Genome Sequence of Desulfurococcus fermentans,a Hyperthermophilic Cellulolytic Crenarchaeon Isolated from a Freshwater Hot Spring in Kamchatka,Russia

Desulfurococcus fermentans is the first known cellulolytic archaeon. This hyperthermophilic and strictly anaerobic crenarchaeon produces hydrogen from fermentation of various carbohydrates and peptides without inhibition by accumulating hydrogen. The complete genome sequence reported here suggested that D. fermentans employs membrane-bound hydrogenases and novel glycohydrolases for hydrogen production from cellulose.     

Comparative analysis of gene content evolution in phytoplasmas and mycoplasmas

Phytoplasmas and mycoplasmas are two groups of important pathogens in the bacterial class Mollicutes. Because of their economical and clinical importance, these obligate pathogens have attracted much research attention. However, difficulties involved in the empirical study of these bacteria, particularly the fact that phytoplasmas have not yet been successfully cultivated outside of their hosts despite decades of attempts, have greatly hampered research progress. With the rapid advancements in genome sequencing, comparative genome analysis provides a new approach to facilitate our understanding of these bacteria. In this study, our main focus is to investigate the evolution of gene content in phytoplasmas, mycoplasmas, and their common ancestor. By using a phylogenetic framework for comparative analysis of 12 complete genome sequences, we characterized the putative gains and losses of genes in these obligate parasites. Our results demonstrated that the degradation of metabolic capacities in these bacteria has occurred predominantly in the common ancestor of Mollicutes, prior to the evolutionary split of phytoplasmas and mycoplasmas. Furthermore, we identified a list of genes that are acquired by the common ancestor of phytoplasmas and are conserved across all strains with complete genome sequences available. These genes include several putative effectors for the interactions with hosts and may be good candidates for future functional characterization.