Genetikk, biosyntese, virknings- og resistensmekanismer for det sirkulære bakteriosinet garvicin ML

Bacteriocins are ribosomally synthesized antimicrobial peptides, produced by many lactic acid bacteria, which show high promise as antimicrobial agents for use in both food industry and for medical applications. In this work, we have studied the bacteriocin garvicin ML (GarML), which is a head-to-tail ligated circular bacteriocin that has a broad spectrum of activity and is active against a range of pathogenic bacteria. This class of bacteriocins is furthermore attracting interest due to their favourable characteristics for potential industrial use, i.e. high pH and thermal stability in addition to resistance to many proteases. However, there are many aspects of circular bacteriocin biology that are still not known, and in this work, we have attempted to shed light on the processes which govern the biosynthesis, mode of action and resistance to this bacteriocin. Circular bacteriocins are synthesized with a leader sequence, and maturation of these peptides is thought to occur through three steps: cleavage of the leader sequence, head-to-tail circularization and export out of the cell. However, the mechanisms involved or indeed the enzymes responsible have not yet been characterized. Furthermore, the sequence of events and potential coupling of these processes is unknown. In paper I and II we have sequenced the producer strain of GarML, which allowed identification and characterization of the gene cluster involved in biosynthesis and immunity to GarML. The gene cluster was shown to share several traits, both in genetic organization and in the putative functions of the encoded proteins, with other circular bacteriocin gene clusters. Functional analysis combined with mass spectrometry of deletion mutants of the GarML operons revealed new insights into biosynthesis of GarML, which may thus apply to circular bacteriocins in general. Firstly, we have provided evidence for leader sequence cleavage occurring without subsequent circularization in two knock-out mutants (ΔgarBCDE and garX∷pCG47), which demonstrates not only that these processes are independent, but that leader sequence cleavage precedes circularization in time (paper II). Furthermore, the evidence suggests that leader sequence cleavage is not performed by any of the proteins encoded by the GarML gene cluster, i.e. garX, garBCDE or garFGH, because we still observe cleavage in their absence (paper II). Two of the operons, namely garX, garBCDE, were implicated in biosynthesis of GarML, specifically in the circularization reaction, as well as providing immunity towards GarML, while the third operon (garFGH) was demonstrated to be non-essential. For circular bacteriocins it has been and remains a controversial issue whether these peptides require a target receptor or docking molecule like the class Ia lantibiotics and IIa pediocin-like bacteriocins for antimicrobial activity, or whether the peptides interact unspecifically with the target cell membrane to create pores. A few circular bacteriocins have been demonstrated to act on liposomes and/or lipid bilayers, which may indicate that a target receptor is not required, at least at high bacteriocin concentrations. In paper III we however provide evidence for a maltose ABC transporter being implicated in sensitivity to GarML in L. lactis. The deletion of this complex led to 6-11-fold lowered sensitivity to GarML, whereas complementation restored high-level sensitivity to the bacteriocin. However, consistent with other circular bacteriocins, we observe receptor-independent killing at higher concentrations of GarML. These results therefore suggest that this class of bacteriocins may indeed require a specific interaction with a target receptor/mediator for antimicrobial activity at low concentrations. Resistance mechanisms to bacteriocins, both developed and innate, are poorly understood for many classes of bacteriocins. Gaining insight into these processes is essential in order to be able to minimize resistance, which is an important prerequisite for the potential use of bacteriocins in many applications. In this work, we have demonstrated examples of both adaptive and inherent resistance to GarML. In paper III, we have shown that L. lactis can develop resistance to GarML by loss of the maltose ABC transporter, which occurs at relatively low frequencies (from 10-7 to10-8) compared to adaptive response of class Ia lantibiotics and class IIa pediocin-like bacteriocins. However, no resistance development occurs at high bacteriocin concentrations (>250 BU mL-1), which indicates that killing is receptor-independent above this level (paper III). In paper IV, we have however provided evidence for an inherent resistance mechanism against GarML, which is conserved in a lineage of L. lactis ssp. cremoris strains. This mechanism appears to be specific for GarML, as it does not affect sensitivity towards other bacteriocins targeting lactococci, even including another circular bacteriocin (paper IV). Thus, we have evidence for a new, specific and inherent mechanism of resistance to GarML in this lineage of L. lactis ssp. cremoris strains, which contributes to the understanding of how dissemination of resistance factors leads to intraspecies variations in sensitivity to bacteriocins.Bakteriosiner er ribosomalt syntetiserte antimikrobielle peptider som blant annet produseres av mange melkesyrebakterier, og som har stort potensial som antimikrobielle forbindelser til bruk i matindustri og i medisinske applikasjoner. I dette arbeidet har vi studert bakteriosinet garvicin ML (GarML), som er et peptid med sirkulær peptidkjede med bredt aktivitetsspektrum og som er aktivt mot mange patogene bakterier. Denne klassen av bakteriosiner anses som interessante fordi de har egenskaper som gjør dem godt egnet til eventuelle industrielle formål, dette er blant annet høy pH- og temperaturstabilitet i tillegg til resistens mot en rekke proteaser. Det er allikevel flere aspekter ved sirkulære bakteriosiner som ikke er tilstrekkelig forstått, og i dette arbeidet har vi ønsket å undersøke nettopp de prosessene som bestemmer biosyntese, virkningsmekanisme og resistensmekanismer for dette bakteriosinet. Sirkulære bakteriosiner syntetiseres med en ledersekvens, og modning av peptidene er antatt å omfatte tre steg: kløyving av ledersekvensen, sirkulering ved ligering av N- og C-terminus, og eksport ut av cellen. Imidlertid er mekanismene involvert og de ansvarlige enzymene ikke kjent. I tillegg er rekkefølgen av disse stegene, og de mulige koblingene mellom dem, ennå ukjent. I artikkel I og II har vi sekvensert produsentstammen av GarML, som igjen tillot identifisering og karakterisering av gruppen av gener, bestående av fire operoner, som er involvert i biosyntese av og immunitet mot GarML. Denne gruppen av gener ble vist å ha mye til felles, både når det gjelder organisering og antatte funksjoner av de proteinene disse genene koder for, med tilsvarende gener for andre sirkulære bakteriosiner. Funksjonell analyse kombinert med massespektrometri ga ny innsikt i biosyntesen av GarML, som dermed kan gjelde også for sirkulære bakteriosiner generelt. Først og fremst har vi påvist at kløyving av ledersekvensen skjer uten sirkularisering i to knock-out mutanter (ΔgarBCDE and garX∷pCG47), noe som demonstrerer at disse to prosessene er uavhengige, men også at kløyving skjer forut for sirkularisering i tid (artikkel II). Videre viser resultatene at kløyving av ledersekvensen ikke utføres av noen av proteinene som er kodet for i GarML operonene, det vil si garX, garBCDE eller garFGH, fordi man observerer kløyving også uten deres tilstedeværelse (paper II). To av operonene i gruppen, garX og garBCDE, ble vist å være involvert i biosyntesen av GarML, spesifikt i sirkulariseringsreaksjonen, og samtidig gi immunitet mot GarML, mens et tredje operon (garFGH) ble vist å være ikke-essensielt. Når det gjelder sirkulære bakteriosiner, så er det kontroversielt hvorvidt disse peptidene trenger en målreseptor eller et dokking-molekyl for antimikrobiell aktivitet som klasse Ia lantibiotika og IIa pediocin-liknende bakteriosiner eller om de interagerer uspesifikt med cellemembranen for å danne porer. I noen tilfeller har det blitt vist at sirkulære bakteriosiner virker på lipid bilag og/eller liposomer, noe som kan indikere at et målmolekyl ikke er nødvendig, i hvert fall ved høye konsentrasjoner av bakteriosin. I artikkel III viser vi derimot at en maltose ABC transporter medvirker til sensitivitet mot GarML i L. lactis. Delesjon av dette komplekset gav 6-11-ganger lavere sensitivitet til GarML, mens komplementering gjenopprettet høy sensitivitet til bakteriosinet. Allikevel ble det ved svært høye konsentrasjoner av bakteriosin observert reseptor-uavhengig dreping. Disse resultatene indikerer dermed at det ved lave konsentrasjoner av bakteriosin kan være nødvendig med en spesifikk interaksjon med et målmolekyl for antimikrobiell aktivitet også for denne klassen bakteriosiner. Resistensmekanismer mot bakteriosiner, bade utviklede og iboende, er ikke godt forstått for mange klasser av bakteriosiner. Det å få innsikt i disse prosessene er essensielt for å kunne minimere nettopp resistensutvikling, noe som er en forutsetning for den potensielle utnyttelsen av bakteriosiner til ulike formål. I dette arbeidet har vi vist eksempler på både utviklet og iboende resistens til GarML. I artikkel III har vi vist at L. lactis kan utvikle resistens mot GarML ved tap av maltose ABC transporter komplekset, som skjer ved en relativt lav frekvens (fra 10-7 til10-8) sammenliknet med utviklet resistens for klasse Ia lantibiotika og klasse IIa pediocin-liknende bakteriosiner. I tillegg ble det ikke observert noen resistensutvikling ved høy konsentrasjon av bakteriosin (>250 BU mL-1), noe som indikerer at over dette nivået så er drepingen ikke reseptor-mediert. I artikkel IV har vi derimot påvist en iboende resistensmekanisme mot GarML som er konservert i en avstamming av L. lactis ssp. cremoris. Denne mekanismen ser ut til å være spesifikk for GarML, da den ikke påvirker sensitivitet mot andre bakteriosiner som virker mot laktokokker, bl.a. et annet sirkulært bakteriosin. Derav tyder resultatene på at vi har en ny, spesifikk og iboende resistensmekanisme mot GarML i denne avstammingen av L. lactis ssp. cremoris stammer, noe som bidrar til forståelsen av hvordan spredning av resistensfaktorer fører til variasjon i sensitivitet mot bakteriosiner innad i arter

A Zn-Dependent Metallopeptidase Is Responsible for Sensitivity to LsbB, a Class II Leaderless Bacteriocin of Lactococcus lactis subsp lactis BGMN1-5

Lactococcus lactis subsp. lactis BGMN1-5 produces a leaderless class II bacteriocin called LsbB. To identify the receptor for LsbB, a cosmid library of the LsbB-sensitive strain BGMN1-596 was constructed. About 150 cosmid clones were individually isolated and transferred to LsbB-resistant mutants of BGMN1-596. Cosmid pAZILcos/MN2, carrying a 40-kb insert, was found to restore LsbB sensitivity in LsbB-resistant mutants. Further subcloning revealed that a 1.9-kb fragment, containing only one open reading frame, was sufficient to restore sensitivity. The fragment contains the gene yvjB coding for a Zn-dependent membrane-bound metallopeptidase, suggesting that this gene may serve as the receptor for LsbB. Further support for this notion derives from several independent experiments: (i) whole-genome sequencing confirmed that all LsbB-resistant mutants contain mutations in yvjB; (ii) disruption of yvjB by direct gene knockout rendered sensitive strains BGMN1-596 and IL1403 resistant to LsbB; and (iii) most compellingly, heterologous expression of yvjB in naturally resistant strains of other species, such as Lactobacillus paracasei and Enterococcus faecalis, also rendered them sensitive to the bacteriocin. To our knowledge, this is the first time a membrane-bound peptidase gene has been shown to be involved in bacteriocin sensitivity in target cells. We also demonstrated a novel successful approach for identifying bacteriocin receptors

Livestock-Associated MRSA CC1 in Norway; Introduction to Pig Farms, Zoonotic Transmission, and Eradication

Farm animals have been identified as an emerging reservoir for transmission of livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) to humans. The low incidence of MRSA in humans and farm animals in Norway has led to the implementation of a national strategy of surveillance and control of LA-MRSA aiming to prevent livestock becoming a domestic source of MRSA to humans. In 2015, MRSA clonal complex 1 spa-type t177 was identified in nine Norwegian pig herds in two neighboring counties. An outbreak investigation was undertaken, and measures of control through eradication were imposed. We performed a register-based cohort study including pig herds and MRSA-positive persons in Norway between 2008 and 2016 to investigate the livestock-association of MRSA CC1, the transmission of the outbreak strain to humans before and after control measures, and the effect of control measures imposed. Data from the Norwegian Surveillance System of Communicable Diseases were merged with data collected through outbreak investigations for LA-MRSA, the National Registry and the Norwegian Register for Health Personnel. Whole-genome sequencing was performed on isolates from livestock and humans identified through contact tracing, in addition to t177 and t127 isolates diagnosed in persons in the same counties. It is likely that a farm worker introduced MRSA CC1 to a sow farm, and further transmission to eight fattening pig farms through trade of live pigs confirmed the potential for livestock association of this MRSA type. The outbreak strain formed a distinct phylogenetic cluster which in addition to the pig farms included one sheep herd and five exposed persons. None of the investigated isolates from possible cases without direct contact to the MRSA positive farms were phylogenetically related to the outbreak strain. Moreover, isolates of t177 or t127 from healthcare and community-acquired cases were not closely related to the outbreak cluster. Eradication measures imposed were effective in eliminating MRSA t177 from the positive pig holdings, and the outbreak strain was not detected in the national pig population or in persons from these counties after control measures

Is paternal age associated with transfer day, developmental stage, morphology, and initial hCG-rise of the competent blastocyst leading to live birth?:A multicenter cohort study

In this study we investigated whether age of men undergoing assisted reproductive technology (ART) treatment was associated with day of transfer, stage, morphology, and initial hCG-rise of the competent blastocyst leading to a live birth? The design was a multicenter historical cohort study based on exposure (age) and outcome data (blastocyst stage and morphology and initial hCG-rise) from men whose partner underwent single blastocyst transfer resulting in singleton pregnancy/birth. The ART treatments were carried out at sixteen private and university-based public fertility clinics. We included 7246 men and women, who between 2014 and 2018 underwent controlled ovarian stimulation (COS) or Frozen-thawed Embryo Transfer (FET) with a single blastocyst transfer resulting in singleton pregnancy were identified. 4842 men with a partner giving birth were included, by linking data to the Danish Medical Birth Registry. We showed that the adjusted association between paternal age and transfer day in COS treatments was OR 1.06, 95% CI (1.00;1.13). Meaning that for every increase of one year, men had a 6% increased probability that the competent blastocyst was transferred on day 6 compared to day 5. Further we showed that the mean difference in hCG values when comparing paternal age group 30–34, 35–39 and 40–45 with the age group 25–29 in those receiving COS treatment, all showed significantly lower adjusted values for older men. In conclusion we hypothesize that the later transfer (day 6) in female partners of older men may be due to longer time spent by the oocyte to repair fragmented DNA of the sperm cells, which should be a focus of future research in men

Cluster Headache Genomewide Association Study and Meta-Analysis Identifies Eight Loci and Implicates Smoking as Causal Risk Factor

Objective: The objective of this study was to aggregate data for the first genomewide association study meta-analysis of cluster headache, to identify genetic risk variants, and gain biological insights. Methods: A total of 4,777 cases (3,348 men and 1,429 women) with clinically diagnosed cluster headache were recruited from 10 European and 1 East Asian cohorts. We first performed an inverse-variance genomewide association meta-analysis of 4,043 cases and 21,729 controls of European ancestry. In a secondary trans-ancestry meta-analysis, we included 734 cases and 9,846 controls of East Asian ancestry. Candidate causal genes were prioritized by 5 complementary methods: expression quantitative trait loci, transcriptome-wide association, fine-mapping of causal gene sets, genetically driven DNA methylation, and effects on protein structure. Gene set and tissue enrichment analyses, genetic correlation, genetic risk score analysis, and Mendelian randomization were part of the downstream analyses. Results: The estimated single nucleotide polymorphism (SNP)-based heritability of cluster headache was 14.5%. We identified 9 independent signals in 7 genomewide significant loci in the primary meta-analysis, and one additional locus in the trans-ethnic meta-analysis. Five of the loci were previously known. The 20 genes prioritized as potentially causal for cluster headache showed enrichment to artery and brain tissue. Cluster headache was genetically correlated with cigarette smoking, risk-taking behavior, attention deficit hyperactivity disorder (ADHD), depression, and musculoskeletal pain. Mendelian randomization analysis indicated a causal effect of cigarette smoking intensity on cluster headache. Three of the identified loci were shared with migraine. Interpretation: This first genomewide association study meta-analysis gives clues to the biological basis of cluster headache and indicates that smoking is a causal risk factor

Large expert-curated database for benchmarking document similarity detection in biomedical literature search

Document recommendation systems for locating relevant literature have mostly relied on methods developed a decade ago. This is largely due to the lack of a large offline gold-standard benchmark of relevant documents that cover a variety of research fields such that newly developed literature search techniques can be compared, improved and translated into practice. To overcome this bottleneck, we have established the RElevant LIterature SearcH consortium consisting of more than 1500 scientists from 84 countries, who have collectively annotated the relevance of over 180 000 PubMed-listed articles with regard to their respective seed (input) article/s. The majority of annotations were contributed by highly experienced, original authors of the seed articles. The collected data cover 76% of all unique PubMed Medical Subject Headings descriptors. No systematic biases were observed across different experience levels, research fields or time spent on annotations. More importantly, annotations of the same document pairs contributed by different scientists were highly concordant. We further show that the three representative baseline methods used to generate recommended articles for evaluation (Okapi Best Matching 25, Term Frequency-Inverse Document Frequency and PubMed Related Articles) had similar overall performances. Additionally, we found that these methods each tend to produce distinct collections of recommended articles, suggesting that a hybrid method may be required to completely capture all relevant articles. The established database server located at https://relishdb.ict.griffith.edu.au is freely available for the downloading of annotation data and the blind testing of new methods. We expect that this benchmark will be useful for stimulating the development of new powerful techniques for title and title/abstract-based search engines for relevant articles in biomedical research.Peer reviewe

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke — the second leading cause of death worldwide — were conducted predominantly in populations of European ancestry1,2. Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis3, and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach4, we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry5. Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries

Stroke genetics informs drug discovery and risk prediction across ancestries

Previous genome-wide association studies (GWASs) of stroke - the second leading cause of death worldwide - were conducted predominantly in populations of European ancestry(1,2). Here, in cross-ancestry GWAS meta-analyses of 110,182 patients who have had a stroke (five ancestries, 33% non-European) and 1,503,898 control individuals, we identify association signals for stroke and its subtypes at 89 (61 new) independent loci: 60 in primary inverse-variance-weighted analyses and 29 in secondary meta-regression and multitrait analyses. On the basis of internal cross-ancestry validation and an independent follow-up in 89,084 additional cases of stroke (30% non-European) and 1,013,843 control individuals, 87% of the primary stroke risk loci and 60% of the secondary stroke risk loci were replicated (P < 0.05). Effect sizes were highly correlated across ancestries. Cross-ancestry fine-mapping, in silico mutagenesis analysis(3), and transcriptome-wide and proteome-wide association analyses revealed putative causal genes (such as SH3PXD2A and FURIN) and variants (such as at GRK5 and NOS3). Using a three-pronged approach(4), we provide genetic evidence for putative drug effects, highlighting F11, KLKB1, PROC, GP1BA, LAMC2 and VCAM1 as possible targets, with drugs already under investigation for stroke for F11 and PROC. A polygenic score integrating cross-ancestry and ancestry-specific stroke GWASs with vascular-risk factor GWASs (integrative polygenic scores) strongly predicted ischaemic stroke in populations of European, East Asian and African ancestry(5). Stroke genetic risk scores were predictive of ischaemic stroke independent of clinical risk factors in 52,600 clinical-trial participants with cardiometabolic disease. Our results provide insights to inform biology, reveal potential drug targets and derive genetic risk prediction tools across ancestries.</p