High Throughput Technologies for Functional Analysis of Archael Genomics

The specific aims of this project were as follows: (1) to design primers to each predicted open reading frame (ORF) in M. jannaschii and M. thermoautotrophicum to allow the amplification of a unique target sequence that will represent the corresponding coding region on a complete genome chip (2) to amplify each target sequence from M. jannaschii and M. thermoautotrophicum and verify that these PCR products are the expected DNA fragment (3) to establish a relational database that will track the production of target DNAs and the nucleotide sequence used to represent each ORF

Intrinsic DNA curvature in trypanosomes

Trypanosoma cruzi and Trypanosoma brucei are protozoan parasites causing Chagas disease and African sleeping sickness, displaying unique features of cellular and molecular biology. Remarkably, no canonical signals for RNA polymerase II promoters, which drive protein coding genes transcription, have been identified so far. The secondary structure of DNA has long been recognized as a signal in biological processes and more recently, its involvement in transcription initiation in Leishmania was proposed. In order to study whether this feature is conserved in trypanosomatids, we undertook a genome wide search for intrinsic DNA curvature in T. cruzi and T. brucei. Using a region integrated intrinsic curvature (RIIC) scoring that we previously developed, a non-random distribution of sequence-dependent curvature was observed. High RIIC scores were found to be significantly correlated with transcription start sites in T. cruzi, which have been mapped in divergent switch regions, whereas in T. brucei, the high RIIC scores correlated with sites that have been involved not only in RNA polymerase II initiation but also in termination. In addition, we observed regions with high RIIC score presenting in-phase tracts of Adenines, in the subtelomeric regions of the T. brucei chromosomes that harbor the variable surface glycoproteins genes. In both T. cruzi and T. brucei genomes, a link between DNA conformational signals and gene expression was found. High sequence dependent curvature is associated with transcriptional regulation regions. High intrinsic curvature also occurs at the T. brucei chromosome subtelomeric regions where the recombination processes involved in the evasion of the immune host system take place. These findings underscore the relevance of indirect DNA readout in these ancient eukaryotes.https://doi.org/10.1186/s13104-017-2908-

Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium

Microbial consortia constitute a majority of the earth’s biomass, but little is known about how these cooperating communities persist despite competition among community members. Theory suggests that non-random spatial structures contribute to the persistence of mixed communities; when particular structures form, they may provide associated community members with a growth advantage over unassociated members. If true, this has implications for the rise and persistence of multi-cellular organisms. However, this theory is difficult to study because we rarely observe initial instances of non-random physical structure in natural populations. Using two engineered strains of Escherichia coli that constitute a synthetic symbiotic microbial consortium, we fortuitously observed such spatial self-organization. This consortium forms a biofilm and, after several days, adopts a defined layered structure that is associated with two unexpected, measurable growth advantages. First, the consortium cannot successfully colonize a new, downstream environment until it selforganizes in the initial environment; in other words, the structure enhances the ability of the consortium to survive environmental disruptions. Second, when the layered structure forms in downstream environments the consortium accumulates significantly more biomass than it did in the initial environment; in other words, the structure enhances the global productivity of the consortium. We also observed that the layered structure only assembles in downstream environments that are colonized by aggregates from a previous, structured community. These results demonstrate roles for self-organization and aggregation in persistence of multi-cellular communities, and also illustrate a role for the techniques of synthetic biology in elucidating fundamental biological principles

Trypanosoma cruzi mitochondrial maxicircles display species- and strain-specific variation and a conserved element in the non-coding region

BACKGROUND: The mitochondrial DNA of kinetoplastid flagellates is distinctive in the eukaryotic world due to its massive size, complex form and large sequence content. Comprised of catenated maxicircles that contain rRNA and protein-coding genes and thousands of heterogeneous minicircles encoding small guide RNAs, the kinetoplast network has evolved along with an extreme form of mRNA processing in the form of uridine insertion and deletion RNA editing. Many maxicircle-encoded mRNAs cannot be translated without this post-transcriptional sequence modification. RESULTS: We present the complete sequence and annotation of the Trypanosoma cruzi maxicircles for the CL Brener and Esmeraldo strains. Gene order is syntenic with Trypanosoma brucei and Leishmania tarentolae maxicircles. The non-coding components have strain-specific repetitive regions and a variable region that is unique for each strain with the exception of a conserved sequence element that may serve as an origin of replication, but shows no sequence identity with L. tarentolae or T. brucei. Alternative assemblies of the variable region demonstrate intra-strain heterogeneity of the maxicircle population. The extent of mRNA editing required for particular genes approximates that seen in T. brucei. Extensively edited genes were more divergent among the genera than non-edited and rRNA genes. Esmeraldo contains a unique 236-bp deletion that removes the 5'-ends of ND4 and CR4 and the intergenic region. Esmeraldo shows additional insertions and deletions outside of areas edited in other species in ND5, MURF1, and MURF2, while CL Brener has a distinct insertion in MURF2. CONCLUSION: The CL Brener and Esmeraldo maxicircles represent two of three previously defined maxicircle clades and promise utility as taxonomic markers. Restoration of the disrupted reading frames might be accomplished by strain-specific RNA editing. Elements in the non-coding region may be important for replication, transcription, and anchoring of the maxicircle within the kinetoplast network

Members of a Large Retroposon Family Are Determinants of Post-Transcriptional Gene Expression in Leishmania

Trypanosomatids are unicellular protists that include the human pathogens Leishmania spp. (leishmaniasis), Trypanosoma brucei (sleeping sickness), and Trypanosoma cruzi (Chagas disease). Analysis of their recently completed genomes confirmed the presence of non–long-terminal repeat retrotransposons, also called retroposons. Using the 79-bp signature sequence common to all trypanosomatid retroposons as bait, we identified in the Leishmania major genome two new large families of small elements—LmSIDER1 (785 copies) and LmSIDER2 (1,073 copies)—that fulfill all the characteristics of extinct trypanosomatid retroposons. LmSIDERs are ∼70 times more abundant in L. major compared to T. brucei and are found almost exclusively within the 3′-untranslated regions (3′UTRs) of L. major mRNAs. We provide experimental evidence that LmSIDER2 act as mRNA instability elements and that LmSIDER2-containing mRNAs are generally expressed at lower levels compared to the non-LmSIDER2 mRNAs. The considerable expansion of LmSIDERs within 3′UTRs in an organism lacking transcriptional control and their role in regulating mRNA stability indicate that Leishmania have probably recycled these short retroposons to globally modulate the expression of a number of genes. To our knowledge, this is the first example in eukaryotes of the domestication and expansion of a family of mobile elements that have evolved to fulfill a critical cellular function

Microarray analysis of gene expression induced by sexual contact in Schistosoma mansoni

<p>Abstract</p> <p>Background</p> <p>The parasitic trematode <it>Schistosoma mansoni </it>is one of the major causative agents of Schistosomiasis, a disease that affects approximately 200 million people, mostly in developing countries. Since much of the pathology is associated with eggs laid by the female worm, understanding the mechanisms involved in oogenesis and sexual maturation is an important step towards the discovery of new targets for effective drug therapy. It is known that the adult female worm only develops fully in the presence of a male worm and that the rates of oviposition and maturation of eggs are significantly increased by mating. In order to study gene transcripts associated with sexual maturation and oviposition, we compared the gene expression profiles of sexually mature and immature parasites using DNA microarrays.</p> <p>Results</p> <p>For each experiment, three amplified RNA microarray hybridizations and their dye swaps were analyzed. Our results show that 265 transcripts are differentially expressed in adult females and 53 in adult males when mature and immature worms are compared. Of the genes differentially expressed, 55% are expressed at higher levels in paired females while the remaining 45% are more expressed in unpaired ones and 56.6% are expressed at higher levels in paired male worms while the remaining 43.4% are more expressed in immature parasites. Real-time RT-PCR analysis validated the microarray results. Several new maturation associated transcripts were identified. Genes that were up-regulated in single-sex females were mostly related to energy generation (i.e. carbohydrate and protein metabolism, generation of precursor metabolites and energy, cellular catabolism, and organelle organization and biogenesis) while genes that were down-regulated related to RNA metabolism, reactive oxygen species metabolism, electron transport, organelle organization and biogenesis and protein biosynthesis.</p> <p>Conclusion</p> <p>Our results confirm previous observations related to gene expression induced by sexual maturation in female schistosome worms. They also increase the list of <it>S. mansoni </it>maturation associated transcripts considerably, therefore opening new and exciting avenues for the study of the conjugal biology and development of new drugs against schistosomes.</p

Macrophage metallothioneins participate in the antileishmanial activity of antimonials

Host cell functions that participate in the pharmacokinetics and pharmacodynamics (PK/PD) of drugs against intracellular pathogen infections are critical for drug efficacy. In this study, we investigated whether macrophage mechanisms of xenobiotic detoxification contribute to the elimination of intracellular Leishmania upon exposure to pentavalent antimonials (SbV). Primary macrophages from patients with cutaneous leishmaniasis (CL) (n=6) were exposed ex vivo to L. V. panamensis infection and SbV, and transcriptomes were generated. Seven metallothionein (MT) genes, potent scavengers of heavy metals and central elements of the mammalian cell machinery for xenobiotic detoxification, were within the top 20 up-regulated genes. To functionally validate the participation of MTs in drug-mediated killing of intracellular Leishmania, tandem knockdown (KD) of MT2-A and MT1-E, MT1-F, and MT1-X was performed using a pan-MT shRNA approach in THP-1 cells. Parasite survival was unaffected in tandem-KD cells, as a consequence of strong transcriptional upregulation of MTs by infection and SbV, overcoming the KD effect. Gene silencing of the metal transcription factor-1 (MTF-1) abrogated expression of MT1 and MT2-A genes, but not ZnT-1. Upon exposure to SbV, intracellular survival of Leishmania in MTF-1KD cells was significantly enhanced. Results from this study highlight the participation of macrophage MTs in Sb-dependent parasite killing

The genetic map and comparative analysis with the physical map of Trypanosoma brucei

Trypanosoma brucei is the causative agent of African sleeping sickness in humans and contributes to the debilitating disease ‘Nagana’ in cattle. To date we know little about the genes that determine drug resistance, host specificity, pathogenesis and virulence in these parasites. The availability of the complete genome sequence and the ability of the parasite to undergo genetic exchange have allowed genetic investigations into this parasite and here we report the first genetic map of T.brucei for the genome reference stock TREU 927, comprising of 182 markers and 11 major linkage groups, that correspond to the 11 previously identified chromosomes. The genetic map provides 90% probability of a marker being 11 cM from any given locus. Its comparison to the available physical map has revealed the average physical size of a recombination unit to be 15.6 Kb/cM. The genetic map coupled with the genome sequence and the ability to undertake crosses presents a new approach to identifying genes relevant to the disease and its prevention in this important pathogen through forward genetic analysis and positional cloning

Non-Invasive Cytology Brush PCR Diagnostic Testing in Mucosal Leishmaniasis: Superior Performance to Conventional Biopsy with Histopathology

Traditional methods of diagnosing mucosal leishmaniasis (ML), such as biopsy with histopathology, are insensitive and require collection of an invasive diagnostic specimen. species identification was performed by PCR-based assays of positive specimens. (n = 3).Use of commercial grade cytology brush PCR for diagnosis of ML is sensitive, rapid, well tolerated, and carries none of the risks of invasive diagnostic procedures such as biopsy. Further optimization is required for adequate species identification. Further evaluation of this method in field and other settings is warranted