Computational genomics of lactobacilli

Lactobacilli are generally harmless gram-positive lactic acid bacteria and well known for their broad spectrum of beneficial effects on human health and usage in food production. However, relatively little is known at the molecular level about the relationships between lactobacilli and humans and about their food processing abilities. The aim of this thesis was to establish bioinformatics approaches for classifying proteins involved in the health effects and food production abilities of lactobacilli and to elucidate the functional potential of two biomedically important Lactobacillus species using whole-genome sequencing. To facilitate the genome-based analysis of lactobacilli, two new bioinformatics approaches were developed for the systematic analysis of protein function. The first approach, called LOCP, fulfilled the need for accurate genome-wide annotation of putative pilus operons in gram-positive bacteria, whereas the second approach, BLANNOTATOR, represented an improved homology-based solution for general function annotation of bacterial proteins. Importantly, both approaches showed superior accuracy in evaluation tests and proved to be useful in finding information ignored by other homology-search methods, illustrating their added value to the current repertoire of function classification systems. Their application also led to the discovery of several putative pilus operons and new potential effector molecules in lactobacilli, including many of the key findings of this thesis work. Lactobacillus rhamnosus GG is one of the clinically best-studied Lactobacillus strains and has a long history of safe use in the food industry. The whole-genome sequencing of the strain GG and a closely related dairy strain L. rhamnosus LC705 revealed two almost identical genomes, despite the physiological differences between the strains. Nevertheless of the extensive genomic similarity, present only in GG was a genomic region containing genes for three pilin subunits and a pilin-dedicated sortase. The presence of these pili on the cell surface of L. rhamnosus GG was also confirmed, and one of the GG-specific pilins was demonstrated to be central for the mucus interaction of strain GG. These discoveries established the presence of gram-positive pilus structures also in non-pathogenic bacteria and provided a long-awaited explanation for the highly efficient adhesion of the strain GG to the intestinal mucosa. The other Lactobacillus species investigated in this thesis was Lactobacillus crispatus. To gain insights into its physiology and to identify components by which this important constituent of the healthy human vagina may promote urogenital health, the genome of a representative L. crispatus strain was sequenced and compared to those of nine others. These analyses provided an accurate account of features associated with vaginal health and revealed a set of 1,224 gene families that were universally conserved across all the ten strains, and, most likely, also across the entire L. crispatus species. Importantly, this set of genes was shown to contain adhesion genes involved in the displacement of the bacterial vaginosis-associated Gardnerella vaginalis from vaginal cells and provided a molecular explanation for the inverse association between L. crispatus and G. vaginalis colonisation in the vagina. Taken together, the present study demonstrates the power of whole-genome sequencing and computer-assisted genome annotation in identifying genes that are involved in host-interactions and have industrial value. The discovery of gram-positive pili in L. rhamnosus GG and the mechanism by which L. crispatus excludes G. vaginalis from vaginal cells are both major steps forward in understanding the interaction between lactobacilli and host. We envisage that these findings together with the developed bioinformatics methods will aid the improvement of probiotic products and human health in the future.Laktobasillit ovat enimmäkseen harmittomia gram-positiivisia maitohappobakteereja. Vaikka näitä terveysvaikutteisiakin hyötybakteereja on hyödynnetty elintarvikkeiden valmistuksessa jo vuosisatoja, tietämyksemme laktobasillien molekyylibiologisista perusteista on varsin rajallinen. Tämän väitöskirjatyön tavoitteena oli kehittää uusia laskennallisia työkaluja laktobasillien tuottamien biomolekyylien karakterisointiin sekä selvittää kahden biolääketieteellisestikin merkittävän laktobasillilajin toimintaan perimän luentaa hyödyntäen. Väitöskirjatutkimuksessa esitellään kaksi laskennallisen biologian menetelmää laktobasillien ilmentämien ominaisuuksien ennustamiseen perimätiedosta sekä hyödynnetään näitä laktobasillien toiminnan tulkinnassa. Menetelmistä ensimmäinen, LOCP, on luotu seulomaan perimätiedosta pili-tartuntaelimien tuottamiseen tarvittavia geeniryhmiä, kun taas menetelmistä jälkimmäinen, BLANNOTATOR, on sekvenssivertailuihin ja lähisukuisista biomolekyyleistä lainattuun tietoon perustuva uusi proteiinisekvenssien luokitintyökalu. Osatöissä tehdyissä selvityksissä molemmat kehitetyistä menetelmistä osoittautuivat ennennäkemättömän tarkoiksi ja kykeneviksi löytämään muiden tehtäviin soveltuvien menetelmien erheellisesti sivuttamaa tietoa. Ohjelmien avulla pystyttiin myös löytämään uusia pili-tartuntaelimien tuottamiseen tarvittavia geeniryhmiä sekä muita mahdollisesti biolääketieteellisesti merkittäviä ominaisuuksia laktobasilleista, mukaan lukien useimmat tässäkin väitöskirjatyössä esitetyt havainnot. Ensimmäinen väitöskirjatyössä tarkasteltu bakteeri oli Lactobacillus rhamnosus GG, joka on eräs tunnetuimmista ja tutkituimmista probiooteista, eli terveysvaikutteisista bakteereista. Tämän teollisestikin merkittävän laktobasillin perimän luenta ja perimän vertailu toisen lähisukulaisen laktobasillin, L. rhamnosus LC705, perimään paljasti yllätyksellisen vähän perinnöllisiä eroja näiden kahden biologisesti erilaisen bakteerin välillä. Perimien vastaavuudesta huolimatta tutkimuksessa onnistuttiin laskennallisia menetelmiä hyödyntämällä kuitenkin myös tunnistamaan yhteensä viisi L. rhamnosus GG -bakteerille ominaista perimäjaksoa, joista merkittävimmän havaittiin sisältävän pili-tartuntaelimien biosynteesissä tarvittavan geeniryhmän. Työssä myös todistettiin pili-tartuntaelimen ilmentyminen bakteerisolun pinnalle ja tartuntaelimen erään osakomponentin merkitys L. rhamnosus GG -bakteerin sitoutumiselle ihmisen ruuansulatusjärjestelmää peittävään limaan. Yhdessä nämä löydökset todistivat kiistatta ensimmäistä kertaa pili-tartuntaelimen ilmentymisen hyötybakteerissa ja tarjosivat uraauurtavan näkökulman L. rhamnosus GG -bakteerin terveysvaikutuksille sekä kyvylle sitoutua ruuansulatusjärjestelmän eri osiin L. rhamnosus LC705 -bakteeria paremmin. Lisäksi väitöskirjatyössä selvitettiin ihmisen emättimessä runsaastikin läsnä olevan ja emätinterveydelle tärkeän Lactobacillus crispatus -bakteerin perinnöllistä perustaa. Työssä kartoitettiin L. crispatus -lajia hyvin edustavan kannan perimä. Vertaamalla kannan perimää yhdeksän muun saman lajin kannan perimiin, luotiin kattava kuvaus lajin ominaisuuksista ja tunnistettiin yhteensä 1224 geeniperhettä, joiden voidaan olettaa vastaavan bakteerin lajityypillisistä piirteistä. Nämä lajityypilliset geeniperheet muodostavat merkittävän osan kunkin L. crispatus -kannan perimästä, ja niiden joukosta onnistuttiin tunnistamaan lajin tarttumiskyvystä mahdollisesti vastaavia geenejä. Erään tällaisen tarttumisgeenin tuotteen osoitettiin myös kykenevän estämään Gardnerella vaginalis -haittabakteerin kiinnittymistä emättimen epiteelin. Tämä löydös selittää osaltaan L. crispatus -bakteerin roolia terveen emättimen valtalajina. Loppupäätelmänä voidaan esittää, että bakteerien perimän luenta ja bakteeriperäisten proteiinisekvenssien luokitusennustukset ovat äärimmäisen hyödyllinen tapa tulkita laktobasillien ilmentämiä ominaisuuksia ja löytää terveysvaikutteisia biomolekyylejä. Pili-tartuntaelimien ja G. vaginalis -haittabakteerin kiinnittymistä estävän proteiinin löytyminen ovat tärkeä edistysaskel kohti kokonaisvaltaista laktobasillien ja ihmisten vuorovaikutuksien ymmärtämistä ja voivat avata yhdessä kehitettyjen laskennallisten biologisten menetelmien kanssa täysin uudenlaisia lähestymistapoja tuottaa entistä parempia terveyttä edistäviä elintarvikkeita ja parantaa ihmisterveyttä

Mutational landscape of chronic myeloid leukemia : more than a single oncogene leukemia

The BCR-ABL1 fusion gene, which causes aberrant kinase activity and uncontrolled cell proliferation, is the hallmark of chronic myeloid leukemia (CML). The development of tyrosine kinase inhibitors (TKI) that target the BCR-ABL oncoprotein has led to dramatic improvement in CML management. However, some challenges remain to be addressed in the TKI era, including patient stratification and the selection of frontline TKIs and CML progression. Additionally, with the emerging goal of treatment-free remission (TFR) in CML management, biomarkers that predict the outcomes of stopping TKI remain to be identified. Notably, recent reports have revealed the power of genome screening in understanding the role of genome aberrations other than BCR-ABL1 in CML pathogenesis. These studies have discovered the presence of disease-phase specific mutations and linked certain mutations to inferior responses to TKI treatment and CML progression. A personalized approach that incorporates genetic data in tailoring treatment strategies has been successfully implemented in acute leukemia, and it represents a promising approach for the management of high-risk CML patients. In this article, we will review current knowledge about the mutational profile in different phases of CML as well as patterns of mutational dynamics in patients having different outcomes. We highlight the effects of somatic mutations involving certain genes (e.g. epigenetic modifiers) on the outcomes of TKI treatment. We also discuss the potential value of incorporating genetic data in treatment decisions and the routine care of CML patients as a future direction for optimizing CML management.Peer reviewe

Neutralizing natural anti-IL-17F autoantibodies protect Autoimmune Polyendocrine Syndrome Type 1 (APS-1) patients from asthma

Non peer reviewe

BLANNOTATOR: enhanced homology-based function prediction of bacterial proteins

Peer reviewe

Exome Sequencing Identifies Susceptibility Loci for Sarcoidosis Prognosis

Many sarcoidosis-associating immunological genes have been shown to be shared between other immune-mediated diseases. In Finnish sarcoidosis patients, good prognosis subjects more commonly have HLA-DRB1*03:01 and/or HLA-DRB1*04:01-DPB1*04:01 haplotype, but no marker for persistent disease have been found. The objective was to further pinpoint genetic differences between prognosis subgroups in relation to the HLA markers. Whole-exome sequencing was conducted for 72 patients selected based on disease activity (resolved disease, n = 36; persistent disease, n = 36). Both groups were further divided by the HLA markers (one/both markers, n = 18; neither of the markers, n = 18). The Finnish exome data from the Genome Aggregation Database was used as a control population in the WES sample. Statistical analyses included single-variant analysis for common variants and gene level analysis for rare variants. We attempted to replicate associated variants in 181 Finnish sarcoidosis patients and 150 controls. An association was found in chromosome 1p36.21 (AADACL3 and C1orf158), which has recently been associated with sarcoidosis in another WES study. In our study, variations in these genes were associated with resolved disease (AADACL3, p = 0.0001 and p = 0.0003; C1orf158, p = 7.03E-05). Another interesting chromosomal region also peaked, Leucocyte Receptor Complex in 19q13.42, but the association diminished in the replication sample. In conclusion, this WES study supports the previously found association in the region 1p36.21. Furthermore, a novel to sarcoidosis region was found, but additional studies are warranted to verify this association.Peer reviewe

A novel partial de novo duplication of JARID2 gene causing a neurodevelopmental phenotype

Publisher Copyright: © 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.Background: Deletions covering the entire or partial JARID2 gene as well as pathogenic single nucleotide variants leading to haploinsufficiency of JARID2 have recently been shown to cause a clinically distinct neurodevelopmental syndrome. Here, we present a previously undescribed partial de novo duplication of the JARID2 gene in a patient displaying features similar to those of patients with JARID2 loss-of-function variants. Case report: The index patient presents with abnormalities in gross motor skills and speech development as well as neuropsychiatric disorders. The patient has markedly dark infraorbital circles and slightly prominent supraorbital ridges.Whole-genome sequencing and array comparative genomic hybridization revealed a novel disease-causing variant type, a partial tandem duplication of JARID2, covering the exons 1–7. Furthermore, RNA sequencing validated the increased expression of these exons. Expression alterations were also detected in target genes of the PRC2 complex, in which JARID2 acts as an essential member. Conclusion: Our data add to the variety of different pathogenic variants associated with JARID2 neurodevelopmental syndrome.Peer reviewe

POXO: a web-enabled tool series to discover transcription factor binding sites

We present POXO, a comprehensive tool series to discover transcription factor binding sites from co-expressed genes (). POXO manages tasks such as functional evaluation and grouping of genes, sequence retrieval, pattern discovery and pattern verification. It also allows users to tailor analytical pipelines from these tools, with single mouse clicks. One typical pipeline of POXO begins by examining the biological functions that a set of co-expressed genes are involved in. In this examination, the functional coherence of the gene set is evaluated and representative functions are associated with the gene set. This examination can also be used to group genes into functionally similar subsets, if several biological processes are affected in the experiment. The next step in the pipeline is then to discover over-represented nucleotide patterns from the upstream sequences of the selected gene sets. This enables to investigate the possibility that the genes are co-regulated by common cis-elements. If over-represented patterns are found, similar ones can then be clustered together and be verified. The performance of POXO is demonstrated by analysing expression data from pathogen treated Arabidopsis thaliana. In this example, POXO detected activated gene sets and suggested transcription factors responsible for their regulation

Characterizing the Key Metabolic Pathways of the Neonatal Mouse Heart Using a Quantitative Combinatorial Omics Approach

The heart of a newborn mouse has an exceptional capacity to regenerate from myocardial injury that is lost within the first week of its life. In order to elucidate the molecular mechanisms taking place in the mouse heart during this critical period we applied an untargeted combinatory multiomics approach using large-scale mass spectrometry-based quantitative proteomics, metabolomics and mRNA sequencing on hearts from 1-day-old and 7-day-old mice. As a result, we quantified 1.937 proteins (366 differentially expressed), 612 metabolites (263 differentially regulated) and revealed 2.586 differentially expressed gene loci (2.175 annotated genes). The analyses pinpointed the fructose-induced glycolysis-pathway to be markedly active in 1-day-old neonatal mice. Integrated analysis of the data convincingly demonstrated cardiac metabolic reprogramming from glycolysis to oxidative phosphorylation in 7-days old mice, with increases of key enzymes and metabolites in fatty acid transport (acylcarnitines) and beta-oxidation. An upsurge in the formation of reactive oxygen species and an increase in oxidative stress markers, e.g., lipid peroxidation, altered sphingolipid and plasmalogen metabolism were also evident in 7-days mice. In vitro maintenance of physiological fetal hypoxic conditions retained the proliferative capacity of cardiomyocytes isolated from newborn mice hearts. In summary, we provide here a holistic, multiomics view toward early postnatal changes associated with loss of a tissue regenerative capacity in the neonatal mouse heart. These results may provide insight into mechanisms of human cardiac diseases associated with tissue regenerative incapacity at the molecular level, and offer a prospect to discovery of novel therapeutic targets.Peer reviewe

Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae

<p>Abstract</p> <p>Background</p> <p>Considerable interest in the bioconversion of lignocellulosic biomass into ethanol has led to metabolic engineering of <it>Saccharomyces cerevisiae </it>for fermentation of xylose. In the present study, the transcriptome and proteome of recombinant, xylose-utilising <it>S. cerevisiae </it>grown in aerobic batch cultures on xylose were compared with those of glucose-grown cells both in glucose repressed and derepressed states. The aim was to study at the genome-wide level how signalling and carbon catabolite repression differ in cells grown on either glucose or xylose. The more detailed knowledge whether xylose is sensed as a fermentable carbon source, capable of catabolite repression like glucose, or is rather recognised as a non-fermentable carbon source is important for further engineering this yeast for more efficient anaerobic fermentation of xylose.</p> <p>Results</p> <p>Genes encoding respiratory proteins, proteins of the tricarboxylic acid and glyoxylate cycles, and gluconeogenesis were only partially repressed by xylose, similar to the genes encoding their transcriptional regulators <it>HAP4</it>, <it>CAT8 </it>and <it>SIP1-2 </it>and <it>4</it>. Several genes that are repressed via the Snf1p/Mig1p-pathway during growth on glucose had higher expression in the cells grown on xylose than in the glucose repressed cells but lower than in the glucose derepressed cells. The observed expression profiles of the transcription repressor <it>RGT1 </it>and its target genes <it>HXT2-3</it>, encoding hexose transporters suggested that extracellular xylose was sensed by the glucose sensors Rgt2p and Snf3p. Proteome analyses revealed distinct patterns in phosphorylation of hexokinase 2, glucokinase and enolase isoenzymes in the xylose- and glucose-grown cells.</p> <p>Conclusion</p> <p>The results indicate that the metabolism of yeast growing on xylose corresponds neither to that of fully glucose repressed cells nor that of derepressed cells. This may be one of the major reasons for the suboptimal fermentation of xylose by recombinant <it>S. cerevisiae </it>strains. Phosphorylation of different isoforms of glycolytic enzymes suggests that regulation of glycolysis also occurred at a post-translational level, supporting prior findings.</p