Inhibition of mitosomal alternative oxidase causes lifecycle arrest of early-stage Trachipleistophora hominis meronts during intracellular infection of mammalian cells.

Mitosomes are highly reduced forms of mitochondria which have lost two of the 'defining' features of the canonical organelle, the mitochondrial genome, and the capacity to generate energy in the form of ATP. Mitosomes are found in anaerobic protists and obligate parasites and, in most of the studied organisms, have a conserved function in the biosynthesis of iron-sulfur clusters (ISC) that are indispensable cofactors of many essential proteins. The genomes of some mitosome-bearing human pathogenic Microsporidia encode homologues of an alternative oxidase (AOX). This mitochondrial terminal respiratory oxidase is absent from the human host, and hence is a potential target for the development of new antimicrobial agents. Here we present experimental evidence for the mitosomal localization of AOX in the microsporidian Trachipleistophora hominis and demonstrate that it has an important role during the parasite's life cycle progression. Using a recently published methodology for synchronising T. hominis infection of mammalian cell lines, we demonstrated specific inhibition of T. hominis early meront growth and replication by an AOX inhibitor colletochlorin B. Treatment of T. hominis-infected host cells with the drug also inhibited re-infection by newly formed dispersive spores. Addition of the drug during the later stages of the parasite life cycle, when our methods suggest that AOX is not actively produced and T. hominis mitosomes are mainly active in Fe/S cluster biosynthesis, had no inhibitory effects on the parasites. Control experiments with the AOX-deficient microsporidian species Encephalitozoon cuniculi, further demonstrated the specificity of inhibition by the drug. Using the same methodology, we demonstrate effects of two clinically used anti-microsporidian drugs albendazole and fumagillin on the cell biology and life cycle progression of T. hominis infecting mammalian host cells. In summary, our results reveal that T. hominis mitosomes have an active role to play in the progression of the parasite life cycle as well as an important role in the biosynthesis of essential Fe/S clusters. Our work also demonstrates that T. hominis is a useful model for testing the efficacy of therapeutic agents and for studying the physiology and cell biology of microsporidian parasites growing inside infected mammalian cells.

Reshaping the Binding Pocket of the Neurotransmitter:Solute Symporter (NSS) Family Transporter SLC6A14 (ATB0,+) Selectively Reduces Access for Cationic Amino Acids and Derivatives.

SLC6A14 (ATB0,+) is unique among SLC proteins in its ability to transport 18 of the 20 proteinogenic (dipolar and cationic) amino acids and naturally occurring and synthetic analogues (including anti-viral prodrugs and nitric oxide synthase (NOS) inhibitors). SLC6A14 mediates amino acid uptake in multiple cell types where increased expression is associated with pathophysiological conditions including some cancers. Here, we investigated how a key position within the core LeuT-fold structure of SLC6A14 influences substrate specificity. Homology modelling and sequence analysis identified the transmembrane domain 3 residue V128 as equivalent to a position known to influence substrate specificity in distantly related SLC36 and SLC38 amino acid transporters. SLC6A14, with and without V128 mutations, was heterologously expressed and function determined by radiotracer solute uptake and electrophysiological measurement of transporter-associated current. Substituting the amino acid residue occupying the SLC6A14 128 position modified the binding pocket environment and selectively disrupted transport of cationic (but not dipolar) amino acids and related NOS inhibitors. By understanding the molecular basis of amino acid transporter substrate specificity we can improve knowledge of how this multi-functional transporter can be targeted and how the LeuT-fold facilitates such diversity in function among the SLC6 family and other SLC amino acid transporters.

Hundreds of Out-of-Frame Remodeled Gene Families in the Escherichia coli Pangenome.

All genomes include gene families with very limited taxonomic distributions that potentially represent new genes and innovations in protein-coding sequence, raising questions on the origins of such genes. Some of these genes are hypothesized to have formed de novo, from noncoding sequences, and recent work has begun to elucidate the processes by which de novo gene formation can occur. A special case of de novo gene formation, overprinting, describes the origin of new genes from noncoding alternative reading frames of existing open reading frames (ORFs). We argue that additionally, out-of-frame gene fission/fusion events of alternative reading frames of ORFs and out-of-frame lateral gene transfers could contribute to the origin of new gene families. To demonstrate this, we developed an original pattern-search in sequence similarity networks, enhancing the use of these graphs, commonly used to detect in-frame remodeled genes. We applied this approach to gene families in 524 complete genomes of Escherichia coli. We identified 767 gene families whose evolutionary history likely included at least one out-of-frame remodeling event. These genes with out-of-frame components represent ∼2.5% of all genes in the E. coli pangenome, suggesting that alternative reading frames of existing ORFs can contribute to a significant proportion of de novo genes in bacteria.

Phylogenomic fingerprinting of tempo and functions of horizontal gene transfer within ochrophytes.

Horizontal gene transfer (HGT) is an important source of novelty in eukaryotic genomes. This is particularly true for the ochrophytes, a diverse and important group of algae. Previous studies have shown that ochrophytes possess a mosaic of genes derived from bacteria and eukaryotic algae, acquired through chloroplast endosymbiosis and from HGTs, although understanding of the time points and mechanisms underpinning these transfers has been restricted by the depth of taxonomic sampling possible. We harness an expanded set of ochrophyte sequence libraries, alongside automated and manual phylogenetic annotation, in silico modeling, and experimental techniques, to assess the frequency and functions of HGT across this lineage. Through manual annotation of thousands of single-gene trees, we identify continuous bacterial HGT as the predominant source of recently arrived genes in the model diatom Phaeodactylum tricornutum Using a large-scale automated dataset, a multigene ochrophyte reference tree, and mathematical reconciliation of gene trees, we note a probable elevation of bacterial HGTs at foundational points in diatom evolution, following their divergence from other ochrophytes. Finally, we demonstrate that throughout ochrophyte evolutionary history, bacterial HGTs have been enriched in genes encoding secreted proteins. Our study provides insights into the sources and frequency of HGTs, and functional contributions that HGT has made to algal evolution.

A new family of cell surface located purine transporters in Microsporidia and related fungal endoparasites.

Plasma membrane-located transport proteins are key adaptations for obligate intracellular Microsporidia parasites, because they can use them to steal host metabolites the parasites need to grow and replicate. However, despite their importance, the functions and substrate specificities of most Microsporidia transporters are unknown. Here, we provide functional data for a family of transporters conserved in all microsporidian genomes and also in the genomes of related endoparasites. The universal retention among otherwise highly reduced genomes indicates an important role for these transporters for intracellular parasites. Using Trachipleistophora hominis, a Microsporidia isolated from an HIV/AIDS patient, as our experimental model, we show that the proteins are ATP and GTP transporters located on the surface of parasites during their intracellular growth and replication. Our work identifies a new route for the acquisition of essential energy and nucleotides for a major group of intracellular parasites that infect most animal species including humans.

The Methodology Behind Network Thinking: Graphs to Analyze Microbial Complexity and Evolution.

In the post genomic era, large and complex molecular datasets from genome and metagenome sequencing projects expand the limits of what is possible for bioinformatic analyses. Network-based methods are increasingly used to complement phylogenetic analysis in studies in molecular evolution, including comparative genomics, classification, and ecological studies. Using network methods, the vertical and horizontal relationships between all genes or genomes, whether they are from cellular chromosomes or mobile genetic elements, can be explored in a single expandable graph. In recent years, development of new methods for the construction and analysis of networks has helped to broaden the availability of these approaches from programmers to a diversity of users. This chapter introduces the different kinds of networks based on sequence similarity that are already available to tackle a wide range of biological questions, including sequence similarity networks, gene-sharing networks and bipartite graphs, and a guide for their construction and analyses.

MultiTwin: A Software Suite to Analyze Evolution at Multiple Levels of Organization Using Multipartite Graphs.

The inclusion of introgressive processes in evolutionary studies induces a less constrained view of evolution. Network-based methods (like large-scale similarity networks) allow to include in comparative genomics all extrachromosomic carriers (like viruses, the most abundant biological entities on the planet) with their cellular hosts. The integration of several levels of biological organization (genes, genomes, communities, environments) enables more comprehensive analyses of gene sharing and improved sequence-based classifications. However, the algorithmic tools for the analysis of such networks are usually restricted to people with high programming skills. We present an integrated suite of software tools named MultiTwin, aimed at the construction, structuring, and analysis of multipartite graphs for evolutionary biology. Typically, this kind of graph is useful for the comparative analysis of the gene content of genomes in microbial communities from the environment and for exploring patterns of gene sharing, for example between distantly related cellular genomes, pangenomes, or between cellular genomes and their mobile genetic elements. We illustrate the use of this tool with an application of the bipartite approach (using gene family-genome graphs) for the analysis of pathogenicity traits in prokaryotes.

Hundreds of novel composite genes and chimeric genes with bacterial origins contributed to haloarchaeal evolution.

BACKGROUND: Haloarchaea, a major group of archaea, are able to metabolize sugars and to live in oxygenated salty environments. Their physiology and lifestyle strongly contrast with that of their archaeal ancestors. Amino acid optimizations, which lowered the isoelectric point of haloarchaeal proteins, and abundant lateral gene transfers from bacteria have been invoked to explain this deep evolutionary transition. We use network analyses to show that the evolution of novel genes exclusive to Haloarchaea also contributed to the evolution of this group. RESULTS: We report the creation of 320 novel composite genes, both early in the evolution of Haloarchaea during haloarchaeal genesis and later in diverged haloarchaeal groups. One hundred and twenty-six of these novel composite genes derived from genetic material from bacterial genomes. These latter genes, largely involved in metabolic functions but also in oxygenic lifestyle, constitute a different gene pool from the laterally acquired bacterial genes formerly identified. These novel composite genes were likely advantageous for their hosts, since they show significant residence times in haloarchaeal genomes-consistent with a long phylogenetic history involving vertical descent and lateral gene transfer-and encode proteins with optimized isoelectric points. CONCLUSIONS: Overall, our work encourages a systematic search for composite genes across all archaeal major groups, in order to better understand the origins of novel prokaryotic genes, and in order to test to what extent archaea might have adjusted their lifestyles by incorporating and recycling laterally acquired bacterial genetic fragments into new archaeal genes.

Bipartite Network Analysis of Gene Sharings in the Microbial World.

Extensive microbial gene flows affect how we understand virology, microbiology, medical sciences, genetic modification, and evolutionary biology. Phylogenies only provide a narrow view of these gene flows: plasmids and viruses, lacking core genes, cannot be attached to cellular life on phylogenetic trees. Yet viruses and plasmids have a major impact on cellular evolution, affecting both the gene content and the dynamics of microbial communities. Using bipartite graphs that connect up to 149,000 clusters of homologous genes with 8,217 related and unrelated genomes, we can in particular show patterns of gene sharing that do not map neatly with the organismal phylogeny. Homologous genes are recycled by lateral gene transfer, and multiple copies of homologous genes are carried by otherwise completely unrelated (and possibly nested) genomes, that is, viruses, plasmids and prokaryotes. When a homologous gene is present on at least one plasmid or virus and at least one chromosome, a process of "gene externalization," affected by a postprocessed selected functional bias, takes place, especially in Bacteria. Bipartite graphs give us a view of vertical and horizontal gene flow beyond classic taxonomy on a single very large, analytically tractable, graph that goes beyond the cellular Web of Life.

Resculpting the binding pocket of APC superfamily LeuT-fold amino acid transporters.

Amino acid transporters are essential components of prokaryote and eukaryote cells, possess distinct physiological functions, and differ markedly in substrate specificity. Amino acid transporters can be both drug targets and drug transporters (bioavailability, targeting) with many monogenic disorders resulting from dysfunctional membrane transport. The largest collection of amino acid transporters (including the mammalian SLC6, SLC7, SLC32, SLC36, and SLC38 families), across all kingdoms of life, is within the Amino acid-Polyamine-organoCation (APC) superfamily. The LeuT-fold is a paradigm structure for APC superfamily amino acid transporters and carriers of sugars, neurotransmitters, electrolytes, osmolytes, vitamins, micronutrients, signalling molecules, and organic and fatty acids. Each transporter is specific for a unique sub-set of solutes, specificity being determined by how well a substrate fits into each binding pocket. However, the molecular basis of substrate selectivity remains, by and large, elusive. Using an integrated computational and experimental approach, we demonstrate that a single position within the LeuT-fold can play a crucial role in determining substrate specificity in mammalian and arthropod amino acid transporters within the APC superfamily. Systematic mutation of the amino acid residue occupying the equivalent position to LeuT V104 titrates binding pocket space resulting in dramatic changes in substrate selectivity in exemplar APC amino acid transporters including PAT2 (SLC36A2) and SNAT5 (SLC38A5). Our work demonstrates how a single residue/site within an archetypal structural motif can alter substrate affinity and selectivity within this important superfamily of diverse membrane transporters.

Transcriptomic profiling of host-parasite interactions in the microsporidian Trachipleistophora hominis.

BACKGROUND: Trachipleistophora hominis was isolated from an HIV/AIDS patient and is a member of a highly successful group of obligate intracellular parasites. METHODS: Here we have investigated the evolution of the parasite and the interplay between host and parasite gene expression using transcriptomics of T. hominis-infected rabbit kidney cells. RESULTS: T. hominis has about 30% more genes than small-genome microsporidians. Highly expressed genes include those involved in growth, replication, defence against oxidative stress, and a large fraction of uncharacterised genes. Chaperones are also highly expressed and may buffer the deleterious effects of the large number of non-synonymous mutations observed in essential T. hominis genes. Host expression suggests a general cellular shutdown upon infection, but ATP, amino sugar and nucleotide sugar production appear enhanced, potentially providing the parasite with substrates it cannot make itself. Expression divergence of duplicated genes, including transporters used to acquire host metabolites, demonstrates ongoing functional diversification during microsporidian evolution. We identified overlapping transcription at more than 100 loci in the sparse T. hominis genome, demonstrating that this feature is not caused by genome compaction. The detection of additional transposons of insect origin strongly suggests that the natural host for T. hominis is an insect. CONCLUSIONS: Our results reveal that the evolution of contemporary microsporidian genomes is highly dynamic and innovative. Moreover, highly expressed T. hominis genes of unknown function include a cohort that are shared among all microsporidians, indicating that some strongly conserved features of the biology of these enormously successful parasites remain uncharacterised.

Reduction and expansion in microsporidian genome evolution: new insights from comparative genomics.

Microsporidia are an abundant group of obligate intracellular parasites of other eukaryotes, including immunocompromised humans, but the molecular basis of their intracellular lifestyle and pathobiology are poorly understood. New genomes from a taxonomically broad range of microsporidians, complemented by published expression data, provide an opportunity for comparative analyses to identify conserved and lineage-specific patterns of microsporidian genome evolution that have underpinned this success. In this study, we infer that a dramatic bottleneck in the last common microsporidian ancestor (LCMA) left a small conserved core of genes that was subsequently embellished by gene family expansion driven by gene acquisition in different lineages. Novel expressed protein families represent a substantial fraction of sequenced microsporidian genomes and are significantly enriched for signals consistent with secretion or membrane location. Further evidence of selection is inferred from the gain and reciprocal loss of functional domains between paralogous genes, for example, affecting transport proteins. Gene expansions among transporter families preferentially affect those that are located on the plasma membrane of model organisms, consistent with recruitment to plug conserved gaps in microsporidian biosynthesis and metabolism. Core microsporidian genes shared with other eukaryotes are enriched in orthologs that, in yeast, are highly expressed, highly connected, and often essential, consistent with strong negative selection against further reduction of the conserved gene set since the LCMA. Our study reveals that microsporidian genome evolution is a highly dynamic process that has balanced constraint, reductive evolution, and genome expansion during adaptation to an extraordinarily successful obligate intracellular lifestyle.