Future Research Directions in Pneumonia. NHLBI Working Group Report.

Pneumonia is a complex pulmonary disease in need of new clinical approaches. Although triggered by a pathogen, pneumonia often results from dysregulations of host defense that likely precede infection. The coordinated activities of immune resistance and tissue resilience then dictate whether and how pneumonia progresses or resolves. Inadequate or inappropriate host responses lead to more severe outcomes such as acute respiratory distress syndrome and to organ dysfunction beyond the lungs and over extended time frames after pathogen clearance, some of which increase the risk for subsequent pneumonia. Improved understanding of such host responses will guide the development of novel approaches for preventing and curing pneumonia and for mitigating the subsequent pulmonary and extrapulmonary complications of pneumonia. The NHLBI assembled a working group of extramural investigators to prioritize avenues of host-directed pneumonia research that should yield novel approaches for interrupting the cycle of unhealthy decline caused by pneumonia. This report summarizes the working group's specific recommendations in the areas of pneumonia susceptibility, host response, and consequences. Overarching goals include the development of more host-focused clinical approaches for preventing and treating pneumonia, the generation of predictive tools (for pneumonia occurrence, severity, and outcome), and the elucidation of mechanisms mediating immune resistance and tissue resilience in the lung. Specific areas of research are highlighted as especially promising for making advances against pneumonia.

Comparative genomics of the neglected human malaria parasite Plasmodium vivax.

The human malaria parasite Plasmodium vivax is responsible for 25-40% of the approximately 515 million annual cases of malaria worldwide. Although seldom fatal, the parasite elicits severe and incapacitating clinical symptoms and often causes relapses months after a primary infection has cleared. Despite its importance as a major human pathogen, P. vivax is little studied because it cannot be propagated continuously in the laboratory except in non-human primates. We sequenced the genome of P. vivax to shed light on its distinctive biological features, and as a means to drive development of new drugs and vaccines. Here we describe the synteny and isochore structure of P. vivax chromosomes, and show that the parasite resembles other malaria parasites in gene content and metabolic potential, but possesses novel gene families and potential alternative invasion pathways not recognized previously. Completion of the P. vivax genome provides the scientific community with a valuable resource that can be used to advance investigation into this neglected species.

AmoebaDB and MicrosporidiaDB: functional genomic resources for Amoebozoa and Microsporidia species.

AmoebaDB (http://AmoebaDB.org) and MicrosporidiaDB (http://MicrosporidiaDB.org) are new functional genomic databases serving the amoebozoa and microsporidia research communities, respectively. AmoebaDB contains the genomes of three Entamoeba species (E. dispar, E. invadens and E. histolityca) and microarray expression data for E. histolytica. MicrosporidiaDB contains the genomes of Encephalitozoon cuniculi, E. intestinalis and E. bieneusi. The databases belong to the National Institute of Allergy and Infectious Diseases (NIAID) funded EuPathDB (http://EuPathDB.org) Bioinformatics Resource Center family of integrated databases and assume the same architectural and graphical design as other EuPathDB resources such as PlasmoDB and TriTrypDB. Importantly they utilize the graphical strategy builder that affords a database user the ability to ask complex multi-data-type questions with relative ease and versatility. Genomic scale data can be queried based on BLAST searches, annotation keywords and gene ID searches, GO terms, sequence motifs, protein characteristics, phylogenetic relationships and functional data such as transcript (microarray and EST evidence) and protein expression data. Search strategies can be saved within a user's profile for future retrieval and may also be shared with other researchers using a unique strategy web address.

Targeted metagenomics and ecology of globally important uncultured eukaryotic phytoplankton.

Among eukaryotes, four major phytoplankton lineages are responsible for marine photosynthesis; prymnesiophytes, alveolates, stramenopiles, and prasinophytes. Contributions by individual taxa, however, are not well known, and genomes have been analyzed from only the latter two lineages. Tiny "picoplanktonic" members of the prymnesiophyte lineage have long been inferred to be ecologically important but remain poorly characterized. Here, we examine pico-prymnesiophyte evolutionary history and ecology using cultivation-independent methods. 18S rRNA gene analysis showed pico-prymnesiophytes belonged to broadly distributed uncultivated taxa. Therefore, we used targeted metagenomics to analyze uncultured pico-prymnesiophytes sorted by flow cytometry from subtropical North Atlantic waters. The data reveal a composite nuclear-encoded gene repertoire with strong green-lineage affiliations, which contrasts with the evolutionary history indicated by the plastid genome. Measured pico-prymnesiophyte growth rates were rapid in this region, resulting in primary production contributions similar to the cyanobacterium Prochlorococcus. On average, pico-prymnesiophytes formed 25% of global picophytoplankton biomass, with differing contributions in five biogeographical provinces spanning tropical to subpolar systems. Elements likely contributing to success include high gene density and genes potentially involved in defense and nutrient uptake. Our findings have implications reaching beyond pico-prymnesiophytes, to the prasinophytes and stramenopiles. For example, prevalence of putative Ni-containing superoxide dismutases (SODs), instead of Fe-containing SODs, seems to be a common adaptation among eukaryotic phytoplankton for reducing Fe quotas in low-Fe modern oceans. Moreover, highly mosaic gene repertoires, although compositionally distinct for each major eukaryotic lineage, now seem to be an underlying facet of successful marine phytoplankton.

A Rickettsia genome overrun by mobile genetic elements provides insight into the acquisition of genes characteristic of an obligate intracellular lifestyle.

We present the draft genome for the Rickettsia endosymbiont of Ixodes scapularis (REIS), a symbiont of the deer tick vector of Lyme disease in North America. Among Rickettsia species (Alphaproteobacteria: Rickettsiales), REIS has the largest genome sequenced to date (>2 Mb) and contains 2,309 genes across the chromosome and four plasmids (pREIS1 to pREIS4). The most remarkable finding within the REIS genome is the extraordinary proliferation of mobile genetic elements (MGEs), which contributes to a limited synteny with other Rickettsia genomes. In particular, an integrative conjugative element named RAGE (for Rickettsiales amplified genetic element), previously identified in scrub typhus rickettsiae (Orientia tsutsugamushi) genomes, is present on both the REIS chromosome and plasmids. Unlike the pseudogene-laden RAGEs of O. tsutsugamushi, REIS encodes nine conserved RAGEs that include F-like type IV secretion systems similar to that of the tra genes encoded in the Rickettsia bellii and R. massiliae genomes. An unparalleled abundance of encoded transposases (>650) relative to genome size, together with the RAGEs and other MGEs, comprise ~35% of the total genome, making REIS one of the most plastic and repetitive bacterial genomes sequenced to date. We present evidence that conserved rickettsial genes associated with an intracellular lifestyle were acquired via MGEs, especially the RAGE, through a continuum of genomic invasions. Robust phylogeny estimation suggests REIS is ancestral to the virulent spotted fever group of rickettsiae. As REIS is not known to invade vertebrate cells and has no known pathogenic effects on I. scapularis, its genome sequence provides insight on the origin of mechanisms of rickettsial pathogenicity.

Comparative genomic analysis and phylogenetic position of Theileria equi.

BACKGROUND: Transmission of arthropod-borne apicomplexan parasites that cause disease and result in death or persistent infection represents a major challenge to global human and animal health. First described in 1901 as Piroplasma equi, this re-emergent apicomplexan parasite was renamed Babesia equi and subsequently Theileria equi, reflecting an uncertain taxonomy. Understanding mechanisms by which apicomplexan parasites evade immune or chemotherapeutic elimination is required for development of effective vaccines or chemotherapeutics. The continued risk of transmission of T. equi from clinically silent, persistently infected equids impedes the goal of returning the U. S. to non-endemic status. Therefore comparative genomic analysis of T. equi was undertaken to: 1) identify genes contributing to immune evasion and persistence in equid hosts, 2) identify genes involved in PBMC infection biology and 3) define the phylogenetic position of T. equi relative to sequenced apicomplexan parasites. RESULTS: The known immunodominant proteins, EMA1, 2 and 3 were discovered to belong to a ten member gene family with a mean amino acid identity, in pairwise comparisons, of 39%. Importantly, the amino acid diversity of EMAs is distributed throughout the length of the proteins. Eight of the EMA genes were simultaneously transcribed. As the agents that cause bovine theileriosis infect and transform host cell PBMCs, we confirmed that T. equi infects equine PBMCs, however, there is no evidence of host cell transformation. Indeed, a number of genes identified as potential manipulators of the host cell phenotype are absent from the T. equi genome. Comparative genomic analysis of T. equi revealed the phylogenetic positioning relative to seven apicomplexan parasites using deduced amino acid sequences from 150 genes placed it as a sister taxon to Theileria spp. CONCLUSIONS: The EMA family does not fit the paradigm for classical antigenic variation, and we propose a novel model describing the role of the EMA family in persistence. T. equi has lost the putative genes for host cell transformation, or the genes were acquired by T. parva and T. annulata after divergence from T. equi. Our analysis identified 50 genes that will be useful for definitive phylogenetic classification of T. equi and closely related organisms.

A Multilocus Sequence Typing System (MLST) reveals a high level of diversity and a genetic component to Entamoeba histolytica virulence.

BACKGROUND: The outcome of an Entamoeba histolytica infection is variable and can result in either asymptomatic carriage, immediate or latent disease (diarrhea/dysentery/amebic liver abscess). An E. histolytica multilocus genotyping system based on tRNA gene-linked arrays has shown that genetic differences exist among parasites isolated from patients with different symptoms however, the tRNA gene-linked arrays cannot be located in the current assembly of the E. histolytica Reference genome (strain HM-1:IMSS) and are highly variable. RESULTS: To probe the population structure of E. histolytica and identify genetic markers associated with clinical outcome we identified in E. histolytica positive samples selected single nucleotide polymorphisms (SNPs) by multiplexed massive parallel sequencing. Profile SNPs were selected which, compared to the reference strain HM-1:IMSS sequence, changed an encoded amino acid at the SNP position, and were present in independent E. histolytica isolates from different geographical origins. The samples used in this study contained DNA isolated from either xenic strains of E. histolytica trophozoites established in culture or E. histolytica positive clinical specimens (stool and amebic liver abscess aspirates). A record of the SNPs present at 16 loci out of the original 21 candidate targets was obtained for 63 of the initial 84 samples (63% of asymptomatically colonized stool samples, 80% of diarrheal stool, 73% of xenic cultures and 84% of amebic liver aspirates). The sequences in all the 63 samples both passed sequence quality control metrics and also had the required greater than 8X sequence coverage for all 16 SNPs in order to confidently identify variants. CONCLUSIONS: Our work is in agreement with previous findings of extensive diversity among E. histolytica isolates from the same geographic origin. In phylogenetic trees, only four of the 63 samples were able to group in two sets of two with greater than 50% confidence. Two SNPs in the cylicin-2 gene (EHI_080100/XM_001914351) were associated with disease (asymptomatic/diarrhea p = 0.0162 or dysentery/amebic liver abscess p = 0.0003). This study demonstrated that there are genetic differences between virulent and avirulent E. histolytica strains and that this approach has the potential to define genetic changes that influence infection outcomes.

Pathema: a clade-specific bioinformatics resource center for pathogen research.

Pathema (http://pathema.jcvi.org) is one of the eight Bioinformatics Resource Centers (BRCs) funded by the National Institute of Allergy and Infectious Disease (NIAID) designed to serve as a core resource for the bio-defense and infectious disease research community. Pathema strives to support basic research and accelerate scientific progress for understanding, detecting, diagnosing and treating an established set of six target NIAID Category A-C pathogens: Category A priority pathogens; Bacillus anthracis and Clostridium botulinum, and Category B priority pathogens; Burkholderia mallei, Burkholderia pseudomallei, Clostridium perfringens and Entamoeba histolytica. Each target pathogen is represented in one of four distinct clade-specific Pathema web resources and underlying databases developed to target the specific data and analysis needs of each scientific community. All publicly available complete genome projects of phylogenetically related organisms are also represented, providing a comprehensive collection of organisms for comparative analyses. Pathema facilitates the scientific exploration of genomic and related data through its integration with web-based analysis tools, customized to obtain, display, and compute results relevant to ongoing pathogen research. Pathema serves the bio-defense and infectious disease research community by disseminating data resulting from pathogen genome sequencing projects and providing access to the results of inter-genomic comparisons for these organisms.

NIH Workshop on HIV-Associated Comorbidities, Coinfections, and Complications: Summary and Recommendation for Future Research.

BACKGROUND: With potent antiretroviral therapy and simplified regimens, people living with HIV (PWH) are achieving near-normal lifespans but not necessarily a normal health span or healthy aging. PWH have a higher than expected risk of developing a number of non-AIDS comorbidities, coinfections, and complications (CCC), often against a background of stigma, poverty, and isolation. SETTING: To gain a better understanding of research needs for HIV-associated CCC, the NIH convened a 2-day workshop (HIV-associated CCC, or HIV ACTION). METHODS: A cross-institute NIH planning committee identified 6 key research areas: epidemiology and population research, pathogenesis and basic science research, clinical research, implementation science research, syndemics research and international research in low and middle income countries. Investigators were selected to lead working groups (WGs) to assess the state-of-the-art and identify 3-5 priority areas in each field before the workshop. A 2-day program at the NIH was developed which included presentations by invited experts and WG members. RESULTS: Over 400 participants attended the workshop. After general and individual WG discussions, the most pressing gaps, questions, or proposed action items were identified. Priority lists of pressing research issues were presented by cochairs of each WG. A detailed report is posted at the NHLBI website. This article reports the streamlined priority list and a summary of WG discussions to inform investigators of current priorities in the field. CONCLUSION: Collaborative efforts of many disciplines are needed to improve the health and wellbeing of PWH. Several common themes emerged across WG representing potential priorities for investigators and recommendations for the NIH.

Understanding the Host in the Management of Pneumonia. An Official American Thoracic Society Workshop Report.

Pneumonia causes a significant burden of disease worldwide. Although all populations are at risk of pneumonia, those at extremes of age and those with immunosuppressive disorders, underlying respiratory disease, and critical illness are particularly vulnerable. Although clinical practice guidelines addressing the management and treatment of pneumonia exist, few of the supporting studies focus on the crucial contributions of the host in pneumonia pathogenesis and recovery. Such essential considerations include the host risk factors that lead to susceptibility to lung infections; biomarkers reflecting the host response and the means to pursue host-directed pneumonia therapy; systemic effects of pneumonia on the host; and long-term health outcomes after pneumonia. To address these gaps, the Pneumonia Working Group of the Assembly on Pulmonary Infection and Tuberculosis led a workshop held at the American Thoracic Society meeting in May 2018 with overarching objectives to foster attention, stimulate research, and promote funding for short-term and long-term investigations into the host contributions to pneumonia. The workshop involved participants from various disciplines with expertise in lung infection, pneumonia, sepsis, immunocompromised patients, translational biology, data science, genomics, systems biology, and clinical trials. This workshop report summarizes the presentations and discussions and important recommendations for future clinical pneumonia studies. These recommendations include establishing consensus disease and outcome definitions, improved phenotyping, development of clinical study networks, standardized data and biospecimen collection and protocols, and development of innovative trial designs.

Reassociation kinetics-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus.

BACKGROUND: The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic. To selectively obtain gene-enriched regions of this tick's genome, Cot filtration was performed, and Cot-filtered DNA was sequenced via 454 FLX pyrosequencing. RESULTS: The sequenced Cot-filtered genomic DNA was assembled with an EST-based gene index of 14,586 unique entries where each EST served as a potential "seed" for scaffold formation. The new sequence assembly extended the lengths of 3,913 of the 14,586 gene index entries. Over half of the extensions corresponded to extensions of over 30 amino acids. To survey the repetitive elements in the tick genome, the complete sequences of five BAC clones were determined. Both Class I and II transposable elements were found. Comparison of the BAC and Cot filtration data indicates that Cot filtration was highly successful in filtering repetitive DNA out of the genomic DNA used in 454 sequencing. CONCLUSION: Cot filtration is a very useful strategy to incorporate into genome sequencing projects on organisms with large genome sizes and which contain high percentages of repetitive, difficult to assemble, genomic DNA. Combining the Cot selection approach with 454 sequencing and assembly with a pre-existing EST database as seeds resulted in extensions of 27% of the members of the EST database.

Deconstructing Syndemics: The Many Layers of Clustering Multi-Comorbidities in People Living with HIV.

The HIV epidemic has dramatically changed over the past 30 years; there are now fewer newly infected people (especially children), fewer AIDS-related deaths, and more people with HIV (PWH) receiving treatment. However, the HIV epidemic is far from over. Despite the tremendous advances in anti-retroviral therapies (ART) and the implementation of ART regimens, HIV incidence (number of new infections over a defined period of time) and prevalence (the burden of HIV infection) in certain regions of the world and socio-economic groups are still on the rise. HIV continues to disproportionally affect highly marginalized populations that constitute higher-risk and stigmatized groups, underserved and/or neglected populations. In addition, it is not uncommon for PWH to suffer enhanced debilitating conditions resulting from the synergistic interactions of both communicable diseases (CDs) and non-communicable diseases (NCDs). While research utilizing only a comorbidities framework has advanced our understanding of the biological settings of the co-occurring conditions from a molecular and mechanistic view, harmful interactions between comorbidities are often overlooked, particularly under adverse socio-economical and behavioral circumstances, likely prompting disease clustering in PWH. Synergistic epidemics (syndemics) research aims to capture these understudied interactions: the mainly non-biological aspects that are central to interpret disease clustering in the comorbidities/multi-morbidities only framework. Connecting population-level clustering of social and health problems through syndemic interventions has proved to be a critical knowledge gap that will need to be addressed in order to improve prevention and care strategies and bring us a step closer to ending the HIV epidemic.

Genome wide survey, discovery and evolution of repetitive elements in three Entamoeba species.

BACKGROUND: Identification and mapping of repetitive elements is a key step for accurate gene prediction and overall structural annotation of genomes. During the assembly and annotation of three highly repetitive amoeba genomes, Entamoeba histolytica, Entamoeba dispar, and Entamoeba invadens, we performed comparative sequence analysis to identify and map all class I and class II transposable elements in their sequences. RESULTS: Here, we report the identification of two novel Entamoeba-specific repeats: ERE1 and ERE2; ERE1 is spread across the three genomes and associated with different repeats in a species-specific manner, while ERE2 is unique to E. histolytica. We also report the identification of two novel subfamilies of LINE and SINE retrotransposons in E. dispar and provide evidence for how the different LINE and SINE subfamilies evolved in these species. Additionally, we found a putative transposase-coding gene in E. histolytica and E. dispar related to the mariner transposon Hydargos from E. invadens. The distribution of transposable elements in these genomes is markedly skewed with a tendency of forming clusters. More than 70% of the three genomes have a repeat density below their corresponding average value indicating that transposable elements are not evenly distributed. We show that repeats and repeat-clusters are found at syntenic break points between E. histolytica and E. dispar and hence, could work as recombination hot spots promoting genome rearrangements. CONCLUSION: The mapping of all transposable elements found in these parasites shows that repeat coverage is up to three times higher than previously reported. LINE, ERE1 and mariner elements were present in the common ancestor to the three Entamoeba species while ERE2 was likely acquired by E. histolytica after its separation from E. dispar. We demonstrate that E. histolytica and E. dispar share their entire repertoire of LINE and SINE retrotransposons and that Eh_SINE3/Ed_SINE1 originated as a chimeric SINE from Eh/Ed_SINE2 and Eh_SINE1/Ed_SINE3. Our work shows that transposable elements are organized in clusters, frequently found at syntenic break points providing insights into their contribution to chromosome instability and therefore, to genomic variation and speciation in these parasites.

Membrane Trafficking Modulation during Entamoeba Encystation.

Entamoeba histolytica is an intestinal parasite that infects 50-100 million people and causes up to 55,000 deaths annually. The transmissive form of E. histolytica is the cyst, with a single infected individual passing up to 45 million cysts per day, making cyst production an attractive target for infection control. Lectins and chitin are secreted to form the cyst wall, although little is known about the underlying membrane trafficking processes supporting encystation. As E. histolytica does not readily form cysts in vitro, we assessed membrane trafficking gene expression during encystation in the closely related model Entamoeba invadens. Genes involved in secretion are up-regulated during cyst formation, as are some trans-Golgi network-to-endosome trafficking genes. Furthermore, endocytic and general trafficking genes are up-regulated in the mature cyst, potentially preserved as mRNA in preparation for excystation. Two divergent dynamin-related proteins found in Entamoeba are predominantly expressed during cyst formation. Phylogenetic analyses indicate that they are paralogous to, but quite distinct from, classical dynamins found in human, suggesting that they may be potential drug targets to block encystation. The membrane-trafficking machinery is clearly regulated during encystation, providing an additional facet to understanding this crucial parasitic process.

Refining Current Scientific Priorities and Identifying New Scientific Gaps in HIV-Related Heart, Lung, Blood, and Sleep Research.

The National Heart, Lung, and Blood Institute (NHLBI) AIDS Program's goal is to provide direction and support for research and training programs in areas of HIV-related heart, lung, blood, and sleep (HLBS) diseases. To better define NHLBI current HIV-related scientific priorities and with the goal of identifying new scientific priorities and gaps in HIV-related HLBS research, a wide group of investigators gathered for a scientific NHLBI HIV Working Group on December 14-15, 2015, in Bethesda, MD. The core objectives of the Working Group included discussions on: (1) HIV-related HLBS comorbidities in the antiretroviral era; (2) HIV cure; (3) HIV prevention; and (4) mechanisms to implement new scientific discoveries in an efficient and timely manner so as to have the most impact on people living with HIV. The 2015 Working Group represented an opportunity for the NHLBI to obtain expert advice on HIV/AIDS scientific priorities and approaches over the next decade.

Evolution of non-LTR retrotransposons in the trypanosomatid genomes: Leishmania major has lost the active elements.

The ingi and L1Tc non-LTR retrotransposons--which constitute the ingi clade--are abundant in the genome of the trypanosomatid species Trypanosoma brucei and Trypanosoma cruzi, respectively. The corresponding retroelements, however, are not present in the genome of a closely related trypanosomatid, Leishmania major. To study the evolution of non-LTR retrotransposons in trypanosomatids, we have analyzed all ingi/L1Tc elements and highly degenerate ingi/L1Tc-related sequences identified in the recently completed T. brucei, T. cruzi and L. major genomes. The coding sequences of 242 degenerate ingi/L1Tc-related elements (DIREs) in all three genomes were reconstituted by removing the numerous frame shifts. Three independent phylogenetic analyses conducted on the conserved domains encoded by these elements show that all DIREs, including the 52 L. major DIREs, form a monophyletic group belonging to the ingi clade. This indicates that the trypanosomatid ancestor contained active mobile elements that have been retained in the Trypanosoma species, but were lost from L. major genome, where only remnants (DIRE) are detectable. All 242 DIREs analyzed group together according to their species origin with the exception of 11 T. cruzi DIREs which are close to the T. brucei ingi/DIRE families. Considering the absence of known horizontal transfer between the African T. brucei and the South-American T. cruzi, this suggests that this group of elements evolved at a lower rate when compared to the other trypanosomatid elements. Interestingly, the only nucleotide sequence conserved between ingi and L1Tc (the first 79 residues) is also present at the 5'-extremity of all the full length DIREs and suggests a possible role for this conserved motif, as well as for DIREs.

The sequence and analysis of Trypanosoma brucei chromosome II.

We report here the sequence of chromosome II from Trypanosoma brucei, the causative agent of African sleeping sickness. The 1.2-Mb pairs encode about 470 predicted genes organised in 17 directional clusters on either strand, the largest cluster of which has 92 genes lined up over a 284-kb region. An analysis of the GC skew reveals strand compositional asymmetries that coincide with the distribution of protein-coding genes, suggesting these asymmetries may be the result of transcription-coupled repair on coding versus non-coding strand. A 5-cM genetic map of the chromosome reveals recombinational 'hot' and 'cold' regions, the latter of which is predicted to include the putative centromere. One end of the chromosome consists of a 250-kb region almost exclusively composed of RHS (pseudo)genes that belong to a newly characterised multigene family containing a hot spot of insertion for retroelements. Interspersed with the RHS genes are a few copies of truncated RNA polymerase pseudogenes as well as expression site associated (pseudo)genes (ESAGs) 3 and 4, and 76 bp repeats. These features are reminiscent of a vestigial variant surface glycoprotein (VSG) gene expression site. The other end of the chromosome contains a 30-kb array of VSG genes, the majority of which are pseudogenes, suggesting that this region may be a site for modular de novo construction of VSG gene diversity during transposition/gene conversion events.

Structure of the germline genome of Tetrahymena thermophila and relationship to the massively rearranged somatic genome.

The germline genome of the binucleated ciliate Tetrahymena thermophila undergoes programmed chromosome breakage and massive DNA elimination to generate the somatic genome. Here, we present a complete sequence assembly of the germline genome and analyze multiple features of its structure and its relationship to the somatic genome, shedding light on the mechanisms of genome rearrangement as well as the evolutionary history of this remarkable germline/soma differentiation. Our results strengthen the notion that a complex, dynamic, and ongoing interplay between mobile DNA elements and the host genome have shaped Tetrahymena chromosome structure, locally and globally. Non-standard outcomes of rearrangement events, including the generation of short-lived somatic chromosomes and excision of DNA interrupting protein-coding regions, may represent novel forms of developmental gene regulation. We also compare Tetrahymena's germline/soma differentiation to that of other characterized ciliates, illustrating the wide diversity of adaptations that have occurred within this phylum.

Transcriptome analysis of encystation in Entamoeba invadens.

Encystation is an essential differentiation process for the completion of the life cycle of a group of intestinal protozoa including Entamoeba histolytica, the causative agent of intestinal and extraintestinal amebiasis. However, regulation of gene expression during encystation is poorly understood. To comprehensively understand the process at the molecular level, the transcriptomic profiles of E. invadens, which is a related reptilian species that causes an invasive disease similar to that of E. histolytica, was investigated during encystation. Using a custom-generated Affymetrix platform microarray, we performed time course (0.5, 2, 8, 24, 48, and 120 h) gene expression analysis of encysting E. invadens. ANOVA analysis revealed that a total of 1,528 genes showed ≥3 fold up-regulation at one or more time points, relative to the trophozoite stage. Of these modulated genes, 8% (116 genes) were up-regulated at the early time points (0.5, 2 and 8h), while 63% (962 genes) were up-regulated at the later time points (24, 48, and 120 h). Twenty nine percent (450 genes) are either up-regulated at 2 to 5 time points or constitutively up-regulated in both early and late stages. Among the up-regulated genes are the genes encoding transporters, cytoskeletal proteins, proteins involved in vesicular trafficking (small GTPases), Myb transcription factors, cysteine proteases, components of the proteasome, and enzymes for chitin biosynthesis. This study represents the first kinetic analysis of gene expression during differentiation from the invasive trophozoite to the dormant, infective cyst stage in Entamoeba. Functional analysis on individual genes and their encoded products that are modulated during encystation may lead to the discovery of targets for the development of new chemotherapeutics that interfere with stage conversion of the parasite.

The genome and transcriptome of the enteric parasite Entamoeba invadens, a model for encystation.

BACKGROUND: Several eukaryotic parasites form cysts that transmit infection. The process is found in diverse organisms such as Toxoplasma, Giardia, and nematodes. In Entamoeba histolytica this process cannot be induced in vitro, making it difficult to study. In Entamoeba invadens, stage conversion can be induced, but its utility as a model system to study developmental biology has been limited by a lack of genomic resources. We carried out genome and transcriptome sequencing of E. invadens to identify molecular processes involved in stage conversion. RESULTS: We report the sequencing and assembly of the E. invadens genome and use whole transcriptome sequencing to characterize changes in gene expression during encystation and excystation. The E. invadens genome is larger than that of E. histolytica, apparently largely due to expansion of intergenic regions; overall gene number and the machinery for gene regulation are conserved between the species. Over half the genes are regulated during the switch between morphological forms and a key signaling molecule, phospholipase D, appears to regulate encystation. We provide evidence for the occurrence of meiosis during encystation, suggesting that stage conversion may play a key role in recombination between strains. CONCLUSIONS: Our analysis demonstrates that a number of core processes are common to encystation between distantly related parasites, including meiosis, lipid signaling and RNA modification. These data provide a foundation for understanding the developmental cascade in the important human pathogen E. histolytica and highlight conserved processes more widely relevant in enteric pathogens.