From parasite genomes to one healthy world: Are we having fun yet?

In 1990, the Human Genome Sequencing Project was established. This laid the ground work for an explosion of sequence data that has since followed. As a result of this effort, the first complete genome of an animal, Caenorhabditis elegans was published in 1998. The sequence of Drosophila melanogaster was made available in March, 2000 and in the following year, working drafts of the human genome were generated with the completed sequence (92%) being released in 2003. Recent advancements and next-generation technologies have made sequencing common place and have infiltrated every aspect of biological research, including parasitology. To date, sequencing of 32 apicomplexa and 24 nematode genomes are either in progress or near completion, and over 600k nematode EST and 200k apicomplexa EST submissions fill the databases. However, the winds have shifted and efforts are now refocusing on how best to store, mine and apply these data to problem solving. Herein we tend not to summarize existing X-omics datasets or present new technological advances that promise future benefits. Rather, the information to follow condenses up-to-date-applications of existing technologies to problem solving as it relates to parasite research. Advancements in non-parasite systems are also presented with the proviso that applications to parasite research are in the making

Tracing Lifestyle Adaptation in Prokaryotic Genomes

Lifestyle adaptation of microbes due to changes in their ecological niches or acquisition of new environments is a major driving force for genetic changes in their respective genomes. Moving into more specialized niches often results in the acquisition of new gene sets via horizontal gene transfer to utilize previously unavailable metabolites, while genetic ballast is shed by gene loss and/or gene inactivation. In some cases, larger genome rearrangements can be observed, such as the incorporation of whole genetic islands, providing a range of new phenotypic capabilities. Until recently these changes could not be comprehensively followed and identified due to the lack of complete microbial genome sequences. The advent of high-throughput DNA sequencing has dramatically changed the scientific landscape and today microbial genomes have become increasingly abundant. Currently, more than 2,900 genomes are published and more than 11,000 genome projects are listed in the Genomes Online Database‡. Although this wealth of information provides many new opportunities to assess microbial functionality, it also creates a new array of challenges when a comparison between multiple microbial genomes is required. Here, functional genome distribution (FGD) is introduced, analyzing the diversity between microbes based on their predicted ORFeome. FGD is therefore a comparative genomics approach, emphasizing the assessments of gene complements. To further facilitate the comparison between two or more genomes, degrees of amino-acid similarities between ORFeomes can be visualized in the Artemis comparison tool, graphically depicting small and large scale genome rearrangements, insertion and deletion events, and levels of similarity between individual open reading frames. FGD provides a new tool for comparative microbial genomics and the interpretation of differences in the genetic makeup of bacteria

Creation, evaluation, and use of PSI, a program for identifying protein-phenotype relationships and comparing protein content in groups of organisms

Recent advances in DNA sequencing technology have enabled entire genomes to be sequenced quickly and accurately, resulting in an exponential increase in the number of organisms whose genome sequences have been elucidated. While the genome sequence of a given organism represents an important starting point in understanding its physiology, the functions of the protein products of many genes are still unknown; as such, computational methods for studying protein function are becoming increasingly important. In addition, this wealth of genomic information has created an unprecedented opportunity to compare the protein content of different organisms; among other applications, this can enable us to improve taxonomic classifications, to develop more accurate diagnostic tests for identifying particular bacteria, and to better understand protein content relationships in both closely-related and distantly-related organisms. This thesis describes the design, evaluation, and use of a program called Proteome Subtraction and Intersection (PSI) that uses an idea called genome subtraction for discovering protein-phenotype relationships and for characterizing differences in protein content in groups of organisms. PSI takes as input a set of proteomes, as well as a partitioning of that set into a subset of "included" proteomes and a subset of "excluded" proteomes. Using reciprocal BLAST hits, PSI finds orthologous relationships among all the proteins in the proteomes from the original set, and then finds groups of orthologous proteins containing at least one orthologue from each of the proteomes in the "included" subset, and none from any of the proteomes in the "excluded" subset. PSI is first applied to finding protein-phenotype relationships. By identifying proteins that are present in all sequenced isolates of the genus Lactobacillus, but not in the related bacterium Pediococcus pentosaceus, proteins are discovered that are likely to be responsible for the difference in cell shape between the lactobacilli and P. pentosaceus. In addition, proteins are identified that may be responsible for resistance to the antibiotic gatifloxacin in some lactic acid bacteria. This thesis also explores the use of PSI for comparing protein content in groups of organisms. Based on the idea of genome subtraction, a novel metric is proposed for comparing the difference in protein content between two organisms. This metric is then used to create a phylogenetic tree for a large set of bacteria, which to the author's knowledge represents the largest phylogenetic tree created to date using protein content. In addition, PSI is used to find the proteomic cohesiveness of isolates of several bacterial species in order to support or refute their current taxonomic classifications. Overall, PSI is a versatile tool with many interesting applications, and should become more and more valuable as additional genomic information becomes available

Homoplasy in genome-wide analysis of rare amino acid replacements: the molecular-evolutionary basis for Vavilov's law of homologous series

<p>Abstract</p> <p>Background</p> <p>Rare genomic changes (RGCs) that are thought to comprise derived shared characters of individual clades are becoming an increasingly important class of markers in genome-wide phylogenetic studies. Recently, we proposed a new type of RGCs designated RGC_CAMs (after Conserved Amino acids-Multiple substitutions) that were inferred using genome-wide identification of amino acid replacements that were: i) located in unambiguously aligned regions of orthologous genes, ii) shared by two or more taxa in positions that contain a different, conserved amino acid in a much broader range of taxa, and iii) require two or three nucleotide substitutions. When applied to animal phylogeny, the RGC_CAM approach supported the coelomate clade that unites deuterostomes with arthropods as opposed to the ecdysozoan (molting animals) clade. However, a non-negligible level of homoplasy was detected.</p> <p>Results</p> <p>We provide a direct estimate of the level of homoplasy caused by parallel changes and reversals among the RGC_CAMs using 462 alignments of orthologous genes from 19 eukaryotic species. It is shown that the impact of parallel changes and reversals on the results of phylogenetic inference using RGC_CAMs cannot explain the observed support for the Coelomata clade. In contrast, the evidence in support of the Ecdysozoa clade, in large part, can be attributed to parallel changes. It is demonstrated that parallel changes are significantly more common in internal branches of different subtrees that are separated from the respective common ancestor by relatively short times than in terminal branches separated by longer time intervals. A similar but much weaker trend was detected for reversals. The observed evolutionary trend of parallel changes is explained in terms of the covarion model of molecular evolution. As the overlap between the covarion sets in orthologous genes from different lineages decreases with time after divergence, the likelihood of parallel changes decreases as well.</p> <p>Conclusion</p> <p>The level of homoplasy observed here appears to be low enough to justify the utility of RGC_CAMs and other types of RGCs for resolution of hard problems in phylogeny. Parallel changes, one of the major classes of events leading to homoplasy, occur much more often in relatively recently diverged lineages than in those separated from their last common ancestor by longer time intervals of time. This pattern seems to provide the molecular-evolutionary underpinning of Vavilov's law of homologous series and is readily interpreted within the framework of the covarion model of molecular evolution.</p> <p>Reviewers</p> <p>This article was reviewed by Alex Kondrashov, Nicolas Galtier, and Maximilian Telford and Robert Lanfear (nominated by Laurence Hurst).</p

The Antibiotic Resistance Growth Plate (ARGP) as an experimental evolution tool to explore the phenotypic and genotypic mutational pathways underlying the emergence of antimicrobial resistance in 'Escherichia coli'

The increasing threat of an antimicrobial resistance crisis is a significant global concern. Antimicrobial treatment failures are worsened by the rapid evolution of resistance amongst bacterial pathogens. Therefore, in addition to developing novel antimicrobial agents, there is growing interest in exploring the underlying genotypic factors of resistance evolution. Traditionally, such studies have focused heavily on well-established mechanisms of acquired resistance involving horizontal gene transfer, yet the evolution of resistance through the acquisitions of mutations is yet to be fully elucidated. Adaptive laboratory evolution studies have provided insights into the genetic basis of adaptation through the direct observation of the evolutionary process. Experimental evolution has advanced from serial passage in well mixed systems to the incorporation of spatiotemporal antibiotic concentration gradients. During this study the Antibiotic Resistance Growth Plate (ARGP) was developed as a simple experimental tool to explore the ability of bacteria to evolve resistance across an antibiotic landscape. The device (90mm × 15mm) facilitates the direct observation of the evolutionary trajectories of mutational lineages within a circular format supporting the radial growth and the exploration of phenotypic space within bacterial populations. Whole genome sequencing of the evolved resistant strains, identified key mutations in the 16s rRNA genes and the fusA gene encoding elongation factor-G, specific to antimicrobial agent gentamicin. Additional gene sequencing revealed parallel gentamicin resistant bacterial populations, evolved identical mutations within the fusA gene. Combined bioinformatic, phylogenetic and molecular docking analysis uncovered the biological significance of the fusA gene in the mutational pathways of gentamicin resistance in E. coli MG1655 in vitro. In contrast, the observed biological fitness costs associated with the acquisition of resistance conferring mutations, emphasised why such costly resistant genotypes were unidentified in natural and clinical settings. This study has established an experimental evolution model to explore the mutational pathways underlying antimicrobial resistance development in vitro. The ARGP offers a platform for the continued research into the acquisition of antimicrobial resistance through mutations, for more complex bacterial pathogens selected against a range of antimicrobial agents. As a tool, the ARGP can be utilised to inform therapeutic decisions based on the evolutionary risk management, provide new opportunities within the field of drug development and holds scope for its application within an educational setting

Kinetoplastid Phylogenomics and Evolution

This Special Issue, Kinetoplastid Phylogenomics and Evolution, unites a series of research and review papers related to kinetoplastid parasites. The diverse topics represented in this collection display a variety of scientific questions and methodological approaches currently used to study these fascinating organisms

Mutatsioonisagedust mõjutavate tegurite otsinguil: tRNA modifikatsiooniensüümid TruA ja RluA mutatsiooniprotsessides

Väitekirja elektrooniline versioon ei sisalda publikatsiooneBakterid suudavad elada kõikjal, kuid karmide ja muutlike keskkonnatingimustega kohanemiseks on aga vaja geneetilist varieeruvust. Bakterites on selle põhiliseks allikaks mutatsioonid. Evolutsiooni mõistmiseks on vaja selgitada molekulaarseid mehhanisme, mis mõjutavad mutatsioonide tekkesagedust. Käesolevas töös kirjeldasin ja analüüsisin uut testsüsteemi, mis võimaldab tuvastada mutatsioonisagedust mõjutavaid faktoreid bakteriperekonnas Pseudomonas. Kirjeldatud testsüsteemi abil õnnestus mullabakteris Pseudomonas putida tuvastada nii varem kirjeldatud kui ka uusi mutatsioonisagedust mõjutavaid geene. Üllatavaim leid oli tRNA modifikatsiooniensüümide TruA ja RluA mõju mutatsioonisagedusele. tRNAd on väikesed molekulid, mis valgusünteesil kannavad valkude ehituskive ribosoomi. Selleks, et paremini oma funktsiooni täita, on paljud nukleotiidid tRNAdes modifitseeritud. Modifikatsioonidel võib olla palju ülesandeid, näiteks aitavad modifikatsioonid tRNAdel saavutada õiget struktuuri või suurendavad translatsiooni täpsust. TruA ja RluA modifitseerivad U nukleotiidi pseudouridiiniks, tehes seda erineval poolel tRNA antikoodonist. Näitasime, et TruA ja RluA tehtud modifikatsioonide puudumisel suureneb P. putidas mutatsioonisagedus. Mõistmaks paremini nende ensüümide olulisust, analüüsisime translatsiooni täpsust, stressi taluvust, proteoomi ja üldist elulemust P. putida TruA ja RluA defektsetes tüvedes. Lisaks sellele selgitasime võrdlevalt TruA ja RluA rolle ka Pseudomonas aeruginosa ja Escherichia coli rakkudes. Saadud tulemustest on näha, kuidas konserveerunud funktsiooniga valgud võivad põhjustada erinevates bakterites erinevaid fenotüüpe. Samuti ilmestab käesolev töö, kuivõrd mitmekesised ja ootamatud tegurid võivad mõjutada DNAs mutatsioonide teket.Bacteria can live everywhere. To cope with harsh and everchanging environmental conditions there is a constant need for genetic versatility. Mutations are the main source of genetic versatility in bacteria. To understand the evolution and adaptive abilities of bacteria, it is vital to investigate the processes affecting the mutation frequency. We have created, verified, and applied a new test system for detecting factors affecting the mutation frequency in bacteria from the genus Pseudomonas. By exploiting the new assay, we identified several genes affecting the mutation frequency in the soil bacterium Pseudomonas putida, many of which were not previously associated with the mutation frequency. Most surprising finding was that the tRNA modification enzymes TruA and RluA affect mutagenesis. tRNAs are small adaptor molecules that carry the building blocks of enzymes to the ribosome and thus are essential for translation. To improve their performance, tRNAs are extensively modified. Among other roles, modifications help tRNA’s to achieve the correct structure and improve the translation fidelity. TruA and RluA modify U nucleotide into pseudouridines in the close vicinity of tRNA anticodon. We demonstrated that the lack of TruA- and RluA-catalyzed modifications remarkably increases the mutation frequency in P. putida. To further analyze the importance of the modification enzymes TruA and RluA, we measured the stress tolerance, translation fidelity, protein expression and general fitness of P. putida strains lacking TruA or RluA. For comparison we analyzed the phenotypes caused by TruA and RluA deficiency in Pseudomonas aeruginosa and Escherichia coli cells. Our research demonstrates how an enzyme with conserved function can cause diverse phenotypes in different bacteria. Also, the thesis illustrates how the complex world of DNA mutations can be affected by many nonobvious factors.

Identification and characterisation of rumen bacteria with prominent roles in the ruminal metabolism of forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Microbiology and Genetics) at Massey University, Palmerston North, New Zealand

Foigures 1.2, 1.4, 1.5 & 1.6 are re-used with permission.This thesis documents the characterisation of two groups of rumen bacteria that are both prominent in forage-fed ruminants, with the aim to better understand their roles in ruminal metabolism. The first group, referred to as the R-7 group, has in recent years been shown to be one of the most abundant rumen bacterial groups, though the few isolated representative strains available were uncharacterised. Two strains of the group included in the Hungate1000 culture collection, R-7 and WTE2008, were selected for characterisation. To facilitate phylogenetic analyses of this group, the complete genomes of an additional three previously isolated R-7 group strains were sequenced. Genomic, phylogenetic and phenotypic characterisation of R-7 and WTE2008 demonstrated that despite their 16S rRNA gene sequences sharing 98.6-99.0% nucleotide identity, their genome-wide average nucleotide identity of 84% assigned them as separate species of a novel genus and family of the proposed order ‘Christensenellales’ using the Genome Taxonomy Database. Phenotypic characterisation showed that the strains were identical in morphology, and both possessed the ability to degrade plant cell wall polysaccharides xylan and pectin, but not cellulose. Acetate, ethanol, hydrogen and lactate were produced by both strains, though R-7 produced greater amounts of hydrogen than WTE2008, which instead produced more lactate. Based on these analyses, it is proposed that R-7 and WTE2008 belong to separate species (Aristaeella gen. nov. hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively) of a newly proposed family (Aristaeellaceae fam. nov.). The second bacterial group of interest, due to their dominant role in ruminal propionate production, was the Prevotella 1 group, following analyses of metatranscriptome datasets of rumen microbial communities of lucerne-fed sheep for dominant community members that express propionate pathway genes from succinate. Screening of 14 strains spanning the diversity of Prevotella 1 found that all except one P. brevis strain produced propionate in a cobalamin (vitamin B12)-dependent manner. To better understand the pathway and regulation of propionate production from succinate, a comparative multi-omics approach was used to test the hypothesis that propionate production is regulated by a cobalamin-binding riboswitch. Scanning of a completed genome assembly of Prevotella ruminicola KHP1 identified four ‘cobalamin’ family riboswitches. However, the riboswitches were not in close proximity to genes putatively involved in converting succinate to propionate, nor were these genes arranged in a single operon. Comparative genomics of the 14 screened strains found that all strains possessed all homologues of candidate propionate pathway genes identified in the KHP1 genome. However, the 13 propionate-producing strains possessed a putative transporter and three subunits encoding a putative methylmalonyl-CoA decarboxylase upstream but antisense to two genes encoding methylmalonyl-CoA mutase subunits, whereas the non-producing strain did not. Comparative transcriptomics and proteomics of KHP1 cultures in the presence and absence of cobalamin demonstrated that some gene candidates were upregulated by cobalamin at the transcriptome level, including co-located genes annotated as phosphate butyryltransferase and butyrate kinase, despite the strain not producing butyrate, suggesting that propionate production may occur via propionyl phosphate. However, only both subunits of methylmalonyl-CoA mutase showed greater transcript and protein abundances in the presence of cobalamin. These results show that while some propionate pathway candidate genes were differentially expressed between cobalamin treatments, they did not appear to be under direct control of a cobalamin-binding riboswitch. This study has contributed to our understanding of the roles of both Aristaeellaceae fam. nov. and Prevotella 1 in ruminal metabolism

An integrative systems biology study to understand immune aging in people living with HIV

Antiretroviral therapy (ART) reduces viral replication, restores T helper cells and improves the survival of people living with HIV (PWH), transforming a life-threatening disease into a manageable chronic infection. Nevertheless, PWH under ART shows aging-related diseases such as bone abnormalities, non-HIV-associated cancers, and cardiovascular and neurocognitive diseases. The complex immune metabolic dysregulation leading to these comorbidities is called immune aging. The main question raised by my thesis was, what are the complex mechanisms responsible for immune aging in HIV? Using advanced system biology and machine learning tools, I used multi-omics-based patient stratification to identify biologic perturbations associated with immune aging in PWH. First, we investigated PWH with Metabolic Syndrome (MetS), a relatively common agingrelated disease in HIV-1. In paper I, we identified the dysregulation of glutamate metabolism in PWH with MetS using plasma metabolomics and measure of cell transporters by flow cytrometry. Then, we investigated the mechanisms of differing PWH on long-term successful ART from HIV-negative controls (HC). In paper II, we identified the dysregulation of amino acids and, more specifically, glutaminolysis (i.e., lysis of glutamine to glutamate) in PWH compared to HC using metabolomics in two independent cohorts to avoid the potential cohort biases. We identified five neurosteroids to be lower in PWH and potentially create neurological impairments in PWH. The glutaminolysis inhibition in chronically infected HIV-1 promonocytic (U1) cells induced apoptosis and latency reversal which could clear HIV reservoirs. The first two papers universally clarified our knowledge about dysregulated metabolic traits following a prolonged ART in PWH. However, we observed heterogeneity among the clinically defined PWH. Therefore, we focused more on the multi-omics data-driven approaches to stratify the at-risk group who were either dysregulated metabolically atrisk PWH (paper III) or immunometabolic at-risk group (paper IV) and clarified the biological aging process by measuring transcriptomics age (paper V). In paper III, we found three groups of PWH based on multi-omics integration of lipidomics, metabolomics, and microbiome. The severe at-risk metabolic complications showed increased weight-related comorbidities and di- and triglycerides compared to the other clusters. At-risk and HC-like groups displayed similar metabolic profiles but were different from HC. An increase in Prevotella was linked to the overrepresentation of men having sex with men (MSM) in the at-risk group. The microbiome-associated metabolites (MAM) appeared dysregulated in all HIV groups compared to controls. We improved this clustering by adding transcriptomics and proteomics data for a refined immunometabolic at-risk-related clustering in PWH. In paper IV, immune-driven HC-like and at-risk groups were clustered based on metabolomics, transcriptomics, and proteomics. Several biomarkers from central carbon metabolism (CCM) and senescence-associated proteins were linked to the at-risk phenotype based on random forest, structural causal modeling, and co-expression networks. Senescent protein changes were associated with a deficiency in macrophage function based on single-cell data, cell profiling, flow cytometry, and proteomics from macrophage data and in vitro validation. We also developed personalized and group-level genome-scale metabolic models (GSMM) and confirmed the implication of metabolites from CCM and polyamides in at-risk phenotypes. Finally, we investigated the accelerated aging process (AAP) in PWH. In paper V, we calculated the biological age of PWH using transcriptomics data and grouped patients into aging groups; The decelerated aging process (DAP) group was linked with higher age, European origin, and a higher proportion of tenofovir disoproxil fumarate /alafenamide (TDF/TAF). AAP had a downregulation of metabolic pathways and an upregulation of inflammatory pathways. In conclusion, my thesis identifies underlying mechanisms of immune aging using system biology tools in three independent cohorts of PWH for mechanistic studies and to improve their care and achieve healthy aging