The study of the three protagonists in malaria-the Plasmodium parasite, the Anopheles mosquito, and the human host-is key to developing methods to control and eventually eliminate the disease. Genomic technologies, including the recent development of next-generation sequencing, enable interrogation of this triangle to an unprecedented level of scrutiny, and promise exciting progress toward real-time epidemiology studies and the study of evolutionary adaptation. We discuss the use of genomics by the International Centers of Excellence for Malaria Research, a network of field sites and laboratories in malaria-endemic countries that undertake cutting-edge research, training, and technology transfer in malarious countries of the world.

Interferon-γ (IFN-γ) promotes a population of T-bet(+) CXCR3(+) regulatory T (Treg) cells that limit T helper 1 (Th1) cell-mediated pathology. Our studies demonstrate that interleukin-27 (IL-27) also promoted expression of T-bet and CXCR3 in Treg cells. During infection with Toxoplasma gondii, a similar population emerged that limited T cell responses and was dependent on IFN-γ in the periphery but on IL-27 at mucosal sites. Transfer of Treg cells ameliorated the infection-induced pathology observed in Il27(-/-) mice, and this was dependent on their ability to produce IL-10. Microarray analysis revealed that Treg cells exposed to either IFN-γ or IL-27 have distinct transcriptional profiles. Thus, IFN-γ and IL-27 have different roles in Treg cell biology and IL-27 is a key cytokine that promotes the development of Treg cells specialized to control Th1 cell-mediated immunity at local sites of inflammation.

Unlike most cells, protozoa in the phylum Apicomplexa divide by a distinctive process in which multiple daughters are assembled within the mother (schizogony or endodyogeny), using scaffolding known as the inner membrane complex (IMC). The IMC underlies the plasma membrane during interphase, but new daughters develop in the cytoplasm, as cytoskeletal filaments associate with flattened membrane cisternae (alveolae), which elongate rapidly to encapsulate subcellular organelles. Newly assembled daughters acquire their plasma membrane as they emerge from the mother, leaving behind vestiges of the maternal cell. Although the maternal plasma membrane remains intact throughout this process, the maternal IMC disappears - is it degraded, or recycled to form the daughter IMC? Exploiting fluorescently tagged IMC markers, we have used live-cell imaging, fluorescence recovery after photobleaching (FRAP) and mEos2 photoactivation to monitor the dynamics of IMC biogenesis and turnover during the replication of Toxoplasma gondii tachyzoites. These studies reveal that the formation of the T. gondii IMC involves two distinct steps - de novo assembly during daughter IMC elongation within the mother cell, followed by recycling of maternal IMC membranes after the emergence of daughters from the mother cell.

High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium's minimal information (MIxS) and NCBI's BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.

Ichthyophthirius multifiliis, commonly known as Ich, is a highly pathogenic ciliate responsible for 'white spot', a disease causing significant economic losses to the global aquaculture industry. Options for disease control are extremely limited, and Ich's obligate parasitic lifestyle makes experimental studies challenging. Unlike most well-studied protozoan parasites, Ich belongs to a phylum composed primarily of free-living members. Indeed, it is closely related to the model organism Tetrahymena thermophila. Genomic studies represent a promising strategy to reduce the impact of this disease and to understand the evolutionary transition to parasitism.

Disease progression in response to infection can be strongly influenced by both pathogen burden and infection-induced immunopathology. While current therapeutics focus on augmenting protective immune responses, identifying therapeutics that reduce infection-induced immunopathology are clearly warranted. Despite the apparent protective role for murine CD8⁺ T cells following infection with the intracellular parasite Leishmania, CD8⁺ T cells have been paradoxically linked to immunopathological responses in human cutaneous leishmaniasis. Transcriptome analysis of lesions from Leishmania braziliensis patients revealed that genes associated with the cytolytic pathway are highly expressed and CD8⁺ T cells from lesions exhibited a cytolytic phenotype. To determine if CD8⁺ T cells play a causal role in disease, we turned to a murine model. These studies revealed that disease progression and metastasis in L. braziliensis infected mice was independent of parasite burden and was instead directly associated with the presence of CD8⁺ T cells. In mice with severe pathology, we visualized CD8⁺ T cell degranulation and lysis of L. braziliensis infected cells. Finally, in contrast to wild-type CD8⁺ T cells, perforin-deficient cells failed to induce disease. Thus, we show for the first time that cytolytic CD8⁺ T cells mediate immunopathology and drive the development of metastatic lesions in cutaneous leishmaniasis.

In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available.

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of Toxoplasma, whose definitive host is the dog. Both species are tissue-dwelling Coccidia and members of the phylum Apicomplexa; they share many common features, but Neospora neither infects humans nor shares the same wide host range as Toxoplasma, rather it shows a striking preference for highly efficient vertical transmission in cattle. These species therefore provide a remarkable opportunity to investigate mechanisms of host restriction, transmission strategies, virulence and zoonotic potential. We sequenced the genome of N. caninum and transcriptomes of the invasive stage of both species, undertaking an extensive comparative genomics and transcriptomics analysis. We estimate that these organisms diverged from their common ancestor around 28 million years ago and find that both genomes and gene expression are remarkably conserved. However, in N. caninum we identified an unexpected expansion of surface antigen gene families and the divergence of secreted virulence factors, including rhoptry kinases. Specifically we show that the rhoptry kinase ROP18 is pseudogenised in N. caninum and that, as a possible consequence, Neospora is unable to phosphorylate host immunity-related GTPases, as Toxoplasma does. This defense strategy is thought to be key to virulence in Toxoplasma. We conclude that the ecological niches occupied by these species are influenced by a relatively small number of gene products which operate at the host-parasite interface and that the dominance of vertical transmission in N. caninum may be associated with the evolution of reduced virulence in this species.

Toxoplasma gondii gives rise to toxoplasmosis, among the most prevalent parasitic diseases of animals and man. Transformation of the tachzyoite stage into the latent bradyzoite-cyst form underlies chronic disease and leads to a lifetime risk of recrudescence in individuals whose immune system becomes compromised. Given the importance of tissue cyst formation, there has been intensive focus on the development of methods to study bradyzoite differentiation, although the molecular basis for the developmental switch is still largely unknown.

The OrthoMCL database (http://orthomcl.cbil.upenn.edu) houses ortholog group predictions for 55 species, including 16 bacterial and 4 archaeal genomes representing phylogenetically diverse lineages, and most currently available complete eukaryotic genomes: 24 unikonts (12 animals, 9 fungi, microsporidium, Dictyostelium, Entamoeba), 4 plants/algae and 7 apicomplexan parasites. OrthoMCL software was used to cluster proteins based on sequence similarity, using an all-against-all BLAST search of each species' proteome, followed by normalization of inter-species differences, and Markov clustering. A total of 511,797 proteins (81.6% of the total dataset) were clustered into 70,388 ortholog groups. The ortholog database may be queried based on protein or group accession numbers, keyword descriptions or BLAST similarity. Ortholog groups exhibiting specific phyletic patterns may also be identified, using either a graphical interface or a text-based Phyletic Pattern Expression grammar. Information for ortholog groups includes the phyletic profile, the list of member proteins and a multiple sequence alignment, a statistical summary and graphical view of similarities, and a graphical representation of domain architecture. OrthoMCL software, the entire FASTA dataset employed and clustering results are available for download. OrthoMCL-DB provides a centralized warehouse for orthology prediction among multiple species, and will be updated and expanded as additional genome sequence data become available.

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein-attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii-infected cells in lipoate-deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate-deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.

To better understand the initiation of CD8(+) T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8(+) T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8(+) T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8(+) T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8(+) T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis.

The non-photosynthetic plastid - or apicoplast - of Toxoplasma gondii and other apicomplexan parasites is an essential organelle and promising drug target. Most apicoplast proteins are encoded in the nucleus and targeted into the organelle through the apicoplast's four membranes courtesy of a bipartite N-terminal leader sequence comprising of an endomembrane signal peptide followed by a plastid transit peptide. Apicoplast transit peptides, like plant plastid transit peptides, have no primary consensus, are variable in length and may be distinguishable only by a relative depletion of negative charged residues and consequent enrichment in basic residues. In this study we examine the role of charged residues within an apicoplast transit peptide in T. gondii by point mutagenesis. We demonstrate that positive charged residues, combined with the absence of negatively charged amino acids, are essential for apicoplast transit peptide fidelity, as also observed in P. falciparum. Furthermore, we show that positive charge is more important at the transit peptide's N-terminus than its C-terminus, and that the nature of the positive residue and the exact position of the N-terminal positive charge are not important. These results suggest that a simple, rule-based prediction for T. gondii transit peptides, similar to that successfully implemented for P. falciparum should help to identify apicoplast proteins and facilitate the identification of drug targets in this important human pathogen.

VectorBase (VectorBase.org) is part of the VEuPathDB Bioinformatics Resource Center, providing free online access to multi-omics and population biology data, focusing on arthropod vectors and invertebrates of importance to human health. VectorBase includes genomics and functional genomics data from bed bugs, biting midges, body lice, kissing bugs, mites, mosquitoes, sand flies, ticks, tsetse flies, stable flies, house flies, fruit flies, and a snail intermediate host. Tools include the Search Strategy system and MapVEu, enabling users to interrogate and visualize diverse 'omics and population-level data using a graphical interface (no programming experience required). Users can also analyze their own private data, such as transcriptomic sequences, exploring their results in the context of other publicly-available information in the database. Help Desk: help@vectorbase.org.

EuPathDB (http://eupathdb.org) resources include 11 databases supporting eukaryotic pathogen genomic and functional genomic data, isolate data and phylogenomics. EuPathDB resources are built using the same infrastructure and provide a sophisticated search strategy system enabling complex interrogations of underlying data. Recent advances in EuPathDB resources include the design and implementation of a new data loading workflow, a new database supporting Piroplasmida (i.e. Babesia and Theileria), the addition of large amounts of new data and data types and the incorporation of new analysis tools. New data include genome sequences and annotation, strand-specific RNA-seq data, splice junction predictions (based on RNA-seq), phosphoproteomic data, high-throughput phenotyping data, single nucleotide polymorphism data based on high-throughput sequencing (HTS) and expression quantitative trait loci data. New analysis tools enable users to search for DNA motifs and define genes based on their genomic colocation, view results from searches graphically (i.e. genes mapped to chromosomes or isolates displayed on a map) and analyze data from columns in result tables (word cloud and histogram summaries of column content). The manuscript herein describes updates to EuPathDB since the previous report published in NAR in 2010.

The new release of SchistoDB (http://SchistoDB.net) provides a rich resource of genomic data for key blood flukes (genus Schistosoma) which cause disease in hundreds of millions of people worldwide. SchistoDB integrates whole-genome sequence and annotation of three species of the genus and provides enhanced bioinformatics analyses and data-mining tools. A simple, yet comprehensive web interface provided through the Strategies Web Development Kit is available for the mining and visualization of the data. Genomic scale data can be queried based on BLAST searches, annotation keywords and gene ID searches, gene ontology terms, sequence motifs, protein characteristics and phylogenetic relationships. Search strategies can be saved within a user's profile for future retrieval and may also be shared with other researchers using a unique web address.

Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators.

Web sites associated with the Eukaryotic Pathogen Bioinformatics Resource Center (EuPathDB.org) have recently introduced a graphical user interface, the Strategies WDK, intended to make advanced searching and set and interval operations easy and accessible to all users. With a design guided by usability studies, the system helps motivate researchers to perform dynamic computational experiments and explore relationships across data sets. For example, PlasmoDB users seeking novel therapeutic targets may wish to locate putative enzymes that distinguish pathogens from their hosts, and that are expressed during appropriate developmental stages. When a researcher runs one of the approximately 100 searches available on the site, the search is presented as a first step in a strategy. The strategy is extended by running additional searches, which are combined with set operators (union, intersect or minus), or genomic interval operators (overlap, contains). A graphical display uses Venn diagrams to make the strategy's flow obvious. The interface facilitates interactive adjustment of the component searches with changes propagating forward through the strategy. Users may save their strategies, creating protocols that can be shared with colleagues. The strategy system has now been deployed on all EuPathDB databases, and successfully deployed by other projects. The Strategies WDK uses a configurable MVC architecture that is compatible with most genomics and biological warehouse databases, and is available for download at code.google.com/p/strategies-wdk. Database URL: www.eupathdb.org.

Toxoplasma gondii is a common parasite of animals that also causes a zoonotic infection in humans. Previous studies have revealed a strongly clonal population structure that is shared between North America and Europe, while South American strains show greater genetic diversity and evidence of sexual recombination. The common inheritance of a monomorphic version of chromosome Ia (referred to as ChrIa*) among three clonal lineages from North America and Europe suggests that inheritance of this chromosome might underlie their recent clonal expansion. To further examine the diversity and distribution of ChrIa, we have analyzed additional strains with greater geographic diversity. Our findings reveal that the same haplotype of ChrIa* is found in the clonal lineages from North America and Europe and in older lineages in South America, where sexual recombination is more common. Although lineages from all three continents harbor the same conserved ChrIa* haplotype, strains from North America and Europe are genetically separate from those in South America, and these respective geographic regions show limited evidence of recent mixing. Genome-wide, array-based profiling of polymorphisms provided evidence for an ancestral flow from particular older southern lineages that gave rise to the clonal lineages now dominant in the north. Collectively, these data indicate that ChrIa* is widespread among nonclonal strains in South America and has more recently been associated with clonal expansion of specific lineages in North America and Europe. These findings have significant implications for the spread of genetic loci influencing transmission and virulence in pathogen populations.

Epidemiological evidence suggests that chronic infections impair immune responses to unrelated pathogens and vaccines. The underlying mechanisms, however, are unclear and distinguishing effects on priming versus development of immunological memory has been challenging. We investigated whether bystander chronic infections impact differentiation of memory CD8(+) T cells, the hallmark of protective immunity against intracellular pathogens. Chronic bystander infections impaired development of memory CD8(+) T cells in several mouse models and humans. These effects were independent of initial priming and were associated with chronic inflammatory signatures. Chronic inflammation negatively impacted the number of bystander CD8(+) T cells and their memory development. Distinct underlying mechanisms of altered survival and differentiation were revealed with the latter regulated by the transcription factors T-bet and Blimp-1. Thus, exposure to prolonged bystander inflammation impairs the effector to memory transition. These data have relevance for immunity and vaccination during persisting infections and chronic inflammation.