Systems analysis of host-parasite interactions.

Parasitic diseases caused by protozoan pathogens lead to hundreds of thousands of deaths per year in addition to substantial suffering and socioeconomic decline for millions of people worldwide. The lack of effective vaccines coupled with the widespread emergence of drug-resistant parasites necessitates that the research community take an active role in understanding host-parasite infection biology in order to develop improved therapeutics. Recent advances in next-generation sequencing and the rapid development of publicly accessible genomic databases for many human pathogens have facilitated the application of systems biology to the study of host-parasite interactions. Over the past decade, these technologies have led to the discovery of many important biological processes governing parasitic disease. The integration and interpretation of high-throughput -omic data will undoubtedly generate extraordinary insight into host-parasite interaction networks essential to navigate the intricacies of these complex systems. As systems analysis continues to build the foundation for our understanding of host-parasite biology, this will provide the framework necessary to drive drug discovery research forward and accelerate the development of new antiparasitic therapies

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

Transcriptomic analysis of diplomonad parasites reveals a trans-spliced intron in a helicase gene in Giardia

ABSTRACT Background. The mechanisms by which DNA sequences are expressed is the central preoccupation of molecular genetics. Recently, ourselves and others reported that in the diplomonad protist Giardia lamblia, the coding regions of several mRNAs are produced by ligation of independent RNA species expressed from distinct genomic loci. Such trans-splicing of introns was found to affect nearly as many genes in this organism as does classical cis-splicing of introns. These findings raised questions about the incidence of intron trans-splicing both across the G. lamblia transcriptome and across diplomonad diversity in general, however a dearth of transcriptomic data at the time prohibited systematic study of these questions. Methods. I leverage newly available transcriptomic data from G. lamblia and the related diplomonad Spironucleus salmonicida to search for trans-spliced introns. My computational pipeline recovers all four previously reported trans-spliced introns in G. lamblia, suggesting good sensitivity. Results. Scrutiny of thousands of potential cases revealed only a single additional trans-spliced intron in G. lamblia, in the p68 helicase gene, and no cases in S. salmonicida. The p68 intron differs from the previously reported trans-spliced introns in its high degree of streamlining: the core features of G. lamblia trans-spliced introns are closely packed together, revealing striking economy in the implementation of a seemingly inherently uneconomical molecular mechanism. Discussion. These results serve to circumscribe the role of trans-splicing in diplomonads both in terms of the number of genes effected and taxonomically. Future work should focus on the molecular mechanisms, evolutionary origins and phenotypic implications of this intriguing phenomenon

Alternative splicing and trans-splicing events revealed by analysis of the Bombyx mori transcriptome

Alternative splicing and trans-splicing events have not been systematically studied in the silkworm Bombyx mori. Here, the silkworm transcriptome was analyzed by RNA-seq. We identified 320 novel genes, modified 1140 gene models, and found thousands of alternative splicing and 58 trans-splicing events. Studies of three SR proteins show that both their alternative splicing patterns and mRNA products are conserved from insect to human, and one isoform of Srsf6 with a retained intron is expressed sex-specifically in silkworm gonads. Trans-splicing of mod(mdg4) in silkworm was experimentally confirmed. We identified integrations from a common 5′-gene with 46 newly identified alternative 3′-exons that are located on both DNA strands over a 500-kb region. Other trans-splicing events in B. mori were predicted by bioinformatic analysis, in which 12 events were confirmed by RT-PCR, six events were further validated by chimeric SNPs, and two events were confirmed by allele-specific RT-PCR in F 1 hybrids from distinct silkworm lines of JS and L10, indicating that trans-splicing is more widespread in insects than previously thought. Analysis of the B. mori transcriptome by RNA-seq provides valuable information of regulatory alternative splicing events. The conservation of splicing events across species and newly identified trans-splicing events suggest that B. mori is a good model for future studies. Published by Cold Spring Harbor Laboratory Press. Copyrigh

Identification and comparison of non-coding RNAs and ribonucleoprotein complexes in several diverse protist species

Many different classes of non-coding (nc)RNAs are found in species throughout the tree of life, each of which performs important cellular functions. The objective of my thesis was to identify and characterize ncRNAs and their associated protein complexes, with particular focus on small nucleolar RNAs (snoRNA), in unicellular eukaryotes whose genomes have undergone significant reduction or expansion. To do this I utilized the unicellular eukaryotes Giardia lamblia, Giardia muris and Euglena gracilis. A diRNP containing RNase P and snoRNA domains that targets tRNAMet 2ʹ-O-methylation and the U3 snoRNA were discovered and characterized in two Giardia species. Unique binding/assembly properties were determined for C/D snoRNP proteins in G. lamblia. A large collection of E. gracilis snoRNAs were discovered leading to a better understanding Ψ-guide snoRNA structure and evolution. These findings highlight the diversity of ncRNA features that exist in less well studied eukaryotic species, and their unique functions

Characterization of mRNA Polyadenylation in the Apicomplexa

Messenger RNA polyadenylation is a universal aspect of gene expression in eukaryotes. In well-established model organisms, this process is mediated by a conserved complex of 15–20 subunits. To better understand this process in apicomplexans, a group of unicellular parasites that causes serious disease in humans and livestock, a computational and high throughput sequencing study of the polyadenylation complex and poly(A) sites in several species was conducted. BLAST-based searches for orthologs of the human polyadenylation complex yielded clear matches to only two—poly(A) polymerase and CPSF73—of the 19 proteins used as queries in this analysis. As the human subunits that recognize the AAUAAA polyadenylation signal (PAS) were not immediately obvious, a computational analysis of sequences adjacent to experimentally-determined apicomplexan poly(A) sites was conducted. The results of this study showed that there exists in apicomplexans an A-rich region that corresponds in position to the AAUAAA PAS. The set of experimentally-determined sites in one species, Sarcocystis neurona, was further analyzed to evaluate the extent and significance of alternative poly(A) site choice in this organism. The results showed that almost 80% of S. neurona genes possess more than one poly(A) site, and that more than 780 sites showed differential usage in the two developmental stages–extracellular merozoites and intracellular schizonts–studied. These sites affected more than 450 genes, and included a disproportionate number of genes that encode membrane transporters and ribosomal proteins. Taken together, these results reveal that apicomplexan species seem to possess a poly(A) signal analogous to AAUAAA even though genes that may encode obvious counterparts of the AAUAAA-recognizing proteins are absent in these organisms. They also indicate that, as is the case in other eukaryotes, alternative polyadenylation is a widespread phenomenon in S. neurona that has the potential to impact growth and development

Giardia duodenalis – deciphering barrier break down in human, organoid-derived duodenal monolayers

Das Protozoon Giardia duodenalis ist eine der Hauptursachen für infektiöse Magen-Darm-Erkrankungen. Die zugrundeliegenden Pathomechanismen sind jedoch nach wie vor unklar. Um die Pathogenität G. duodenalis‘ untersuchen zu können, wird ein Modellsystem benötigt, dass die Komplexität des Darmepithels widerspiegelt. Diese Arbeit zeigt die Etablierung eines Zellkultursystems auf der Basis von organoid-abgeleiteten Epithelien unter Verwendung von filter-basierten Zellkultureinsätzen. Wir haben Protokolle für die Etablierung von organoid-basierten Zellkulturen (ODMs) vier verschiedener Wirte zoonotischer Protozoen unter Verwendung eines einzigen Protokolls erstellt. Die Charakterisierung zeigte, dass das Modellsystem erfolgreich die Polarisierung des Darmepithels nachahmt, aus mehreren Zelltypen besteht und eine Infektion ermöglicht. Der Schwerpunkt der Arbeit lag auf der Analyse der durch G. duodenalis induzierten Barrierestörung in ODMs auf Transkriptions-, Protein- und Funktionsebene. Die Infektion von humanen duodenalen Zellen führte zu einem Verlust der epithelialen Barrierefunktion. Mit Hilfe des transepithelialen elektrischen Widerstandes und Dextran Flux wurde eine Erhöhung der Barrieredurchlässigkeit beobachtet. Die Hemmung von zuvor in immortalisierten Zellmodellen beschriebenen Reaktionswegen konnte die Barrierefunktion nicht wiederherstellen. Stattdessen konnten Veränderungen der Ionenhomöostase sowie den Zusammenbruch der zonula occludens nachgewiesen werden. Der beobachtete Phänotyp konnte auf die Aktivierung des cAMP/PKA/CREB-Signalwegs, als einen von mehreren kausalen Faktoren, zurückgeführt werden. Hier zeigen wir die Etablierung eines aus Organoiden abgeleiteten Modells, das die Untersuchung von G. duodenalis Infektionen in vitro ermöglicht. Mit unserem Modell konnten wir eine neue Reihenfolge von Ereignissen entschlüsseln, die einen der Faktoren während symptomatischer Giardiasis darstellt.The protozoan Giardia duodenalis is a one of the major causes of gastrointestinal illness. Underlying pathomechanisms remain unclear. An in vitro model system that also mimics the complexity of intestinal epithelium is needed to allow pathogenicity studies. This thesis shows the establishment of a cell culture system based on organoid-derived epithelia using permeable cell culture inserts. We have provided guidelines on the establishment of organoid-derived monolayers (ODMs) of four different hosts of zoonotic protozoa using a single protocol. Characterization showed that the model system successfully mimics intestinal polarization, is composed of multiple cell types and allows for infection with multiple protozoan parasites. As the main focus of the thesis, analysis of G. duodenalis-induced barrier breakdown in ODMs was performed on transcriptional, protein and functional level. Infection of human duodenal, organoid-derived monolayers resulted in a time- and dose-dependent breakdown of epithelial barrier function. Barrier permeability increases were observed ranging from ions to macromolecules as measured by transepithelial electrical resistance and Dextran flux. Inhibition of previously proposed key pathogen-induced pathways observed in immortalized cell models did not rescue barrier dysfunction. We could instead show changes in ion homeostasis, and tight junctional breakdown. While none of the previously proposed effector pathways appeared to be responsible, we could pin-point the observed phenotype to activation of the cAMP/PKA/CREB signaling pathway, as one of the factors of the multifactorial barrier breakdown. The establishment of an organoid-derived infection model is shown, allowing the study of in vitro Giardia duodenalis infections. Using this model, we could decipher a new series of events that may be one of the factors causing the intestinal barrier breakdown observed in symptomatic Giardiasis

Inhibición parcial de dos genes que codifican para proteínas del empalmosoma en Giardia intestinalis

Introduction. Giardia intestinalis is an early divergent organism that was recently shown to have introns. The machinery responsible for the removal of introns in higher eukaryotes is the spliceosome, which consists of five ribonucleoproteins. Each of these ribonucleoproteins has a small nuclear RNA, a set of seven Sm proteins (B, D1, D2, D3, E, F and G) and several specific proteins. Some genes that encode spliceosome proteins have been bioinformatically identified in the parasite genome. Although it is assumed that the spliceosome is responsible for splicing in this parasite, biochemical characterization is lacking. Objective. To inhibit two G. intestinalis spliceosome protein genes in order to determine whether this inhibition affects parasite growth or encystation. Materials and methods. Antisense sequences of the genes encoding the spliceosomal parasite proteins SmB and SmD3 were cloned into a specific G. intestinalis vector. G. intestinalis individuals were subsequently transfected with the recombinant vectors and those parasites that incorporated the vector were selected. A decrease in mRNA levels by real-time PCR was confirmed and the growth and encystation in wild and transfected parasites was assessed. Results. A decrease of 40% and 70% of SmB and SmD3 mRNA levels, respectively, was observed. Growth and encystation in these parasites were not affected. Conclusion. Decrease of SmB and SmD3 mRNA levels does not affect the parasite, indicating that the spliceosome remains functional or that splicing is not essential for parasite viability.Introducción. Giardia intestinalis es un organismo tempranamente divergente en el que recientemente se demostró la presencia de intrones. La maquinaria responsable de la remoción de intrones en organismos eucariotas superiores es el empalmosoma, el cual está conformado por cinco ribonucleoproteínas, cada una de las cuales tiene un ARN pequeño nuclear, un set de siete proteínas Sm (B, D1, D2, D3, E, F y G) y varias proteínas específicas. En G. intestinalis se han identificado los genes de algunas proteínas del empalmosoma por bioinformática. Aunque se asume que este es el responsable del empalme en el parásito, su caracterización bioquímica no se ha hecho.Objetivo. Inhibir dos genes que codifican para proteínas del empalmosoma de G. intestinalis con el fin de determinar si esta inhibición afecta el crecimiento o el enquistamiento del parásito.Materiales y métodos. En un vector específico para G. intestinalis se clonaron secuencias antisentido de los genes que codifican para las proteínas SmB y SmD3 del empalmosoma del parásito. Posteriormente, se transfectó G. intestinalis con los vectores recombinantes y se seleccionaron aquellos parásitos que lo incorporaron. Se confirmó la disminución del mensajero mediante reacción en cadena de la polimerasa (PCR) en tiempo real, y se evaluaron el crecimiento y el enquistamiento en parásitos silvestres y transfectados.Resultados. Se observó una disminución de 40 y 70 % en el ARNm de SmB y SmD3, respectivamente. El crecimiento y el enquistamiento no se vieron afectados en estos parásitos.Conclusión. La disminución de SmB y SmD3 no afectó al parásito, lo que indica que el empalmosoma sigue siendo funcional, o que el empalme no es una función vital del parásito