Mutations introduce diversity into genomes, leading to selective changes and driving evolution. These changes have contributed to the emergence of many of the current major health concerns of the 21st century, from the development of genetic diseases and cancers to the rise and spread of drug resistance. The experimental systematic testing of all mutations in a system of interest is impractical and not cost-effective, which has created interest in the development of computational tools to understand the molecular consequences of mutations to aid and guide rational experimentation. Areas covered: Here, the authors discuss the recent development of computational methods to understand the effects of coding mutations to protein function and interactions, particularly in the context of the 3D structure of the protein. Expert opinion: While significant progress has been made in terms of innovative tools to understand and quantify the different range of effects in which a mutation or a set of mutations can give rise to a phenotype, a great gap still exists when integrating these predictions and drawing causality conclusions linking variants. This often requires a detailed understanding of the system being perturbed. However, as part of the drug development process it can be used preemptively in a similar fashion to pharmacokinetics predictions, to guide development of therapeutics to help guide the design and analysis of clinical trials, patient treatment and public health policy strategies.

Abstract

While numerous genomes of Leishmania spp. have been sequenced and analyzed, an understanding of the evolutionary history of these organisms remains limited due to the unavailability of the sequence data for their closest known relatives, Endotrypanum and Porcisia spp., infecting sloths and porcupines. We have sequenced and analyzed genomes of three members of this clade in order to fill this gap. Their comparative analyses revealed only minute differences from Leishmaniamajor genome in terms of metabolic capacities. We also documented that the number of genes under positive selection on the Endotrypanum/Porcisia branch is rather small, with the flagellum-related group of genes being over-represented. Most significantly, the analysis of gene family evolution revealed a substantially reduced repertoire of surface proteins, such as amastins and biopterin transporters BT1 in the Endotrypanum/Porcisia species when compared to amastigote-dwelling Leishmania. This reduction was especially pronounced for δ-amastins, a subfamily of cell surface proteins crucial in the propagation of Leishmania amastigotes inside vertebrate macrophages and, apparently, dispensable for Endotrypanum/Porcisia, which do not infect such cells.

Background Telomeres are indispensable for genome stability maintenance. They are maintained by the telomere-associated protein complex, which include Ku proteins and a telomerase among others. Here, we investigated a role of Ku80 in Leishmania mexicana. Leishmania is a genus of parasitic protists of the family Trypanosomatidae causing a vector-born disease called leishmaniasis. Methodology/Principal findings We used the previously established CRISPR/Cas9 system to mediate ablation of Ku80- and Ku70-encoding genes in L. mexicana. Complete knock-outs of both genes were confirmed by Southern blotting, whole-genome Illumina sequencing, and RT-qPCR. Resulting telomeric phenotypes were subsequently investigated using Southern blotting detection of terminal restriction fragments. The genome integrity in the Ku80- deficient cells was further investigated by whole-genome sequencing. Our work revealed that telomeres in the ΔKu80 L. mexicana are elongated compared to those of the wild type. This is a surprising finding considering that in another model trypanosomatid, Trypanosoma brucei, they are shortened upon ablation of the same gene. A telomere elongation phenotype has been documented in other species and associated with a presence of telomerase-independent alternative telomere lengthening pathway. Our results also showed that Ku80 appears to be not involved in genome stability maintenance in L. mexicana. Conclusion/Significance Ablation of the Ku proteins in L. mexicana triggers telomere elongation, but does not have an adverse impact on genome integrity.

Leishmaniasis is a parasitic vector-borne disease caused by the protistan flagellates of the genus Leishmania. Leishmania (Viannia) guyanensis is one of the most common causative agents of the American tegumentary leishmaniasis. It has previously been shown that L. guyanensis strains that carry the endosymbiotic Leishmania RNA virus 1 (LRV1) cause more severe form of the disease in a mouse model than those that do not. The presence of the virus was implicated into the parasite’s replication and spreading. In this respect, studying the molecular mechanisms of cellular control of viral infection is of great medical importance. Here, we report ~30.5 Mb high-quality genome assembly of the LRV1-positive L. guyanensis M4147. This strain was turned into a model by establishing the CRISPR-Cas9 system and ablating the gene encoding phosphatidate phosphatase 2-like (PAP2L) protein. The orthologue of this gene is conspicuously absent from the genome of an unusual member of the family Trypanosomatidae, Vickermania ingenoplastis, a species with mostly bi-flagellated cells. Our analysis of the PAP2L-null L. guyanensis showed an increase in the number of cells strikingly resembling the bi-flagellated V. ingenoplastis, likely as a result of the disruption of the cell cycle, significant accumulation of phosphatidic acid, and increased virulence compared to the wild type cells.

No abstract

Most trypanosomatid flagellates do not have catalase. In the evolution of this group, the gene encoding catalase has been independently acquired at least three times from three different bacterial groups. Here, we demonstrate that the catalase of Vickermania was obtained by horizontal gene transfer from Gammaproteobacteria, extending the list of known bacterial sources of this gene. Comparative biochemical analyses revealed that the enzymes of V. ingenoplastis, Leptomonas pyrrhocoris, and Blastocrithidia sp., representing the three independent catalase-bearing trypanosomatid lineages, have similar properties, except for the unique cyanide resistance in the catalase of the latter species.

BackgroundAmyotrophic lateral sclerosis (ALS) is a progressively fatal, neurodegenerative disease associated with both motor and non-motor symptoms, including frontotemporal dementia. Approximately 10% of cases are genetically inherited (familial ALS), while the majority are sporadic. Mutations across a wide range of genes have been associated; however, the underlying molecular effects of these mutations and their relation to phenotypes remain poorly explored.MethodsWe initially curated an extensive list (n=1343) of missense mutations identified in the clinical literature, which spanned across 111 unique genes. Of these, mutations in genes SOD1, FUS and TDP43 were analysed using in silico biophysical tools, which characterised changes in protein stability, interactions, localisation and function. The effects of pathogenic and non-pathogenic mutations within these genes were statistically compared to highlight underlying molecular drivers.ResultsCompared with previous ALS-dedicated databases, we have curated the most extensive missense mutation database to date and observed a twofold increase in unique implicated genes, and almost a threefold increase in the number of mutations. Our gene-specific analysis identified distinct molecular drivers across the different proteins, where SOD1 mutations primarily reduced protein stability and dimer formation, and those in FUS and TDP-43 were present within disordered regions, suggesting different mechanisms of aggregate formation.ConclusionUsing our three genes as case studies, we identified distinct insights which can drive further research to better understand ALS. The information curated in our database can serve as a resource for similar gene-specific analyses, further improving the current understanding of disease, crucial for the development of treatment strategies.