Distinctive characters of Nostoc genomes in cyanolichens

Publisher's version (útgefin grein)Background Cyanobacteria of the genus Nostoc are capable of forming symbioses with a wide range of organism, including a diverse assemblage of cyanolichens. Only certain lineages of Nostoc appear to be able to form a close, stable symbiosis, raising the question whether symbiotic competence is determined by specific sets of genes and functionalities. Results We present the complete genome sequencing, annotation and analysis of two lichen Nostoc strains. Comparison with other Nostoc genomes allowed identification of genes potentially involved in symbioses with a broad range of partners including lichen mycobionts. The presence of additional genes necessary for symbiotic competence is likely reflected in larger genome sizes of symbiotic Nostoc strains. Some of the identified genes are presumably involved in the initial recognition and establishment of the symbiotic association, while others may confer advantage to cyanobionts during cohabitation with a mycobiont in the lichen symbiosis. Conclusions Our study presents the first genome sequencing and genome-scale analysis of lichen-associated Nostoc strains. These data provide insight into the molecular nature of the cyanolichen symbiosis and pinpoint candidate genes for further studies aimed at deciphering the genetic mechanisms behind the symbiotic competence of Nostoc. Since many phylogenetic studies have shown that Nostoc is a polyphyletic group that includes several lineages, this work also provides an improved molecular basis for demarcation of a Nostoc clade with symbiotic competence.This research was funded by the Icelandic Research Fund RANNIS (project number 134055-051) and by the University of Iceland. Sample collection, study design, analysis, data interpretation and manuscript writing was carried out by the authors.Peer Reviewe

Unravelling morphoea aetiopathogenesis by next-generation sequencing of paired skin biopsies

BACKGROUND: Morphoea can have a significant disease burden. Aetiopathogenesis remains poorly understood, with very limited existing genetic studies. Linear morphoea (LM) may follow Blascho's lines of epidermal development, providing potential pathogenic clues. OBJECTIVE: The first objective of this study was to identify the presence of primary somatic epidermal mosaicism in LM. The second objective was tTo explore differential gene expression in morphoea epidermis and dermis to identify potential pathogenic molecular pathways and tissue layer cross-talk. METHODOLOGY: Skin biopsies from paired affected and contralateral unaffected skin were taken from 16 patients with LM. Epidermis and dermis were isolated using a 2-step chemical-physical separation protocol. Whole Genome Sequencing (WGS; n = 4 epidermal) and RNA-seq (n = 5-epidermal, n = 5-dermal) with gene expression analysis via GSEA-MSigDBv6.3 and PANTHER-v14.1 pathway analyses, were performed. RTqPCR and immunohistochemistry were used to replicate key results. RESULTS: Sixteen participants (93.8% female, mean age 27.7 yrs disease-onset) were included. Epidermal WGS identified no single affected gene or SNV. However, many potential disease-relevant pathogenic variants were present, including ADAMTSL1 and ADAMTS16. A highly proliferative, inflammatory and profibrotic epidermis was seen, with significantly-overexpressed TNFα-via-NFkB, TGFβ, IL6/JAKSTAT and IFN-signaling, apoptosis, p53 and KRAS-responses. Upregulated IFI27 and downregulated LAMA4 potentially represent initiating epidermal 'damage' signals and enhanced epidermal-dermal communication. Morphoea dermis exhibited significant profibrotic, B-cell and IFN-signatures, and upregulated morphogenic patterning pathways such as Wnt. CONCLUSION: This study supports the absence of somatic epidermal mosaicism in LM, and identifies potential disease-driving epidermal mechanisms, epidermal-dermal interactions and disease-specific dermal differential-gene-expression in morphoea. We propose a potential molecular narrative for morphoea aetiopathogenesis which could help guide future targeted studies and therapies

Pathogenic variants in the human m(6)A reader YTHDC2 are associated with primary ovarian insufficiency

Primary ovarian insufficiency (POI) affects 1% of women and carries significant medical and psychosocial sequelae. Approximately 10% of POI has a defined genetic cause, with most implicated genes relating to biological processes involved in early fetal ovary development and function. Recently, Ythdc2, an RNA helicase and N6-methyladenosine reader, has emerged as a regulator of meiosis in mice. Here, we describe homozygous pathogenic variants in YTHDC2 in 3 women with early-onset POI from 2 families: C. 2567C>G, p.P856R in the helicase-associated (HA2) domain and c.1129G>T, p.E377*. We demonstrated that YTHDC2 is expressed in the developing human fetal ovary and is upregulated in meiotic germ cells, together with related meiosisassociated factors. The p.P856R variant resulted in a less flexible protein that likely disrupted downstream conformational kinetics of the HA2 domain, whereas the p.E377*variant truncated the helicase core. Taken together, our results reveal that YTHDC2 is a key regulator of meiosis in humans and pathogenic variants within this gene are associated with POI

ZSWIM7 Is associated with human female meiosis and familial primary ovarian insufficiency

Background Primary ovarian insufficiency (POI) affects 1% of women and is associated with significant medical consequences. A genetic cause for POI can be found in up to 30% of women, elucidating key roles for these genes in human ovary development. Objective We aimed to identify the genetic mechanism underlying early-onset POI in 2 sisters from a consanguineous pedigree. Methods Genome sequencing and variant filtering using an autosomal recessive model was performed in the 2 affected sisters and their unaffected family members. Quantitative reverse transcriptase PCR (qRT-PCR) and RNA sequencing were used to study the expression of key genes at critical stages of human fetal gonad development (Carnegie Stage 22/23, 9 weeks post conception (wpc), 11 wpc, 15/16 wpc, 19/20 wpc) and in adult tissue. Results Only 1 homozygous variant cosegregating with the POI phenotype was found: a single nucleotide substitution in zinc finger SWIM-type containing 7 (ZSWIM7), NM_001042697.2: c.173C > G; resulting in predicted loss-of-function p.(Ser58*). qRT-PCR demonstrated higher expression of ZSWIM7 in the 15/16 wpc ovary compared with testis, corresponding to peak meiosis in the fetal ovary. RNA sequencing of fetal gonad samples showed that ZSWIM7 has a similar temporal expression profile in the developing ovary to other homologous recombination genes. Main conclusions Disruption of ZSWIM7 is associated with POI in humans. ZSWIM7 is likely to be important for human homologous recombination; these findings expand the range of genes associated with POI in women

Neuroinflammation, autoinflammation, splenomegaly and anemia caused by bi-allelic mutations in IRAK4

We describe a novel, severe autoinflammatory syndrome characterized by neuroinflammation, systemic autoinflammation, splenomegaly, and anemia (NASA) caused by bi-allelic mutations in IRAK4. IRAK-4 is a serine/threonine kinase with a pivotal role in innate immune signaling from toll-like receptors and production of pro-inflammatory cytokines. In humans, bi-allelic mutations in IRAK4 result in IRAK-4 deficiency and increased susceptibility to pyogenic bacterial infections, but autoinflammation has never been described. We describe 5 affected patients from 2 unrelated families with compound heterozygous mutations in IRAK4 (c.C877T (p.Q293*)/c.G958T (p.D320Y); and c.A86C (p.Q29P)/c.161 + 1G>A) resulting in severe systemic autoinflammation, massive splenomegaly and severe transfusion dependent anemia and, in 3/5 cases, severe neuroinflammation and seizures. IRAK-4 protein expression was reduced in peripheral blood mononuclear cells (PBMC) in affected patients. Immunological analysis demonstrated elevated serum tumor necrosis factor (TNF), interleukin (IL) 1 beta (IL-1β), IL-6, IL-8, interferon α2a (IFN-α2a), and interferon β (IFN-β); and elevated cerebrospinal fluid (CSF) IL-6 without elevation of CSF IFN-α despite perturbed interferon gene signature. Mutations were located within the death domain (DD; p.Q29P and splice site mutation c.161 + 1G>A) and kinase domain (p.Q293*/p.D320Y) of IRAK-4. Structure-based modeling of the DD mutation p.Q29P showed alteration in the alignment of a loop within the DD with loss of contact distance and hydrogen bond interactions with IRAK-1/2 within the myddosome complex. The kinase domain mutation p.D320Y was predicted to stabilize interactions within the kinase active site. While precise mechanisms of autoinflammation in NASA remain uncertain, we speculate that loss of negative regulation of IRAK-4 and IRAK-1; dysregulation of myddosome assembly and disassembly; or kinase active site instability may drive dysregulated IL-6 and TNF production. Blockade of IL-6 resulted in immediate and complete amelioration of systemic autoinflammation and anemia in all 5 patients treated; however, neuroinflammation has, so far proven recalcitrant to IL-6 blockade and the janus kinase (JAK) inhibitor baricitinib, likely due to lack of central nervous system penetration of both drugs. We therefore highlight that bi-allelic mutation in IRAK4 may be associated with a severe and complex autoinflammatory and neuroinflammatory phenotype that we have called NASA (neuroinflammation, autoinflammation, splenomegaly and anemia), in addition to immunodeficiency in humans

Sérkenni Nostoc erfðamengja í fléttum

Cyanobacteria of the genus Nostoc are capable of forming symbiosis with a wide range of living organisms, cyanolichens being the largest and most diverse group of cyanobacterial symbioses. Recently, it has become apparent that symbiotic competence is a complex trait determined by particular genes and their collectives. Biosynthetic genes specific for symbionts may lead to the production of symbiotically associated metabolites. Despite the increasing evidence for involvement of primary and secondary metabolites in symbiotic interactions, the chemical ecology of symbiosis is a relatively unexplored field. Using bioinformatic prediction, symbiont cultivation, isotopic enrichment, and advanced analytics we characterized a novel unique polyketide, nosperin, from a Nostoc sp. strain associated with the lichen Peltigera membranacea. The biosynthetic gene cluster and the structure of nosperin are related to those of the pederin group previously known only from nonphotosynthetic bacteria associated with beetles and marine sponges. The nosperin gene cluster is the first trans-AT PKS system reported for Cyanobacteria. Furthermore, the nosperin producer, Nostoc sp. N6, is the first cultured producer of a pederin family natural product providing new opportunities to study the biochemistry and physiology of the biosynthetic pathway. The unexpected discovery of nosperin in cyanolichens expands the remarkable range of symbioses associated with the pederin family of natural products and suggests that its role is associated with symbiosis. The complete genome sequencing and gene annotation of Nostoc sp. N6 and the Nostoc cyanobiont of the lichen Lobaria pulmonaria as well as draft genome sequences of three more lichen-associated Nostoc strains now allow identification of genes potentially involved in symbioses with a broad range of partners as well as genes specific to the symbiosis with lichen mycobionts. The presence of additional genes necessary for symbiotic competence is likely reflected in the larger genome sizes of symbiotic Nostoc strains. Some of the identified genes are presumably involved in the initial recognition and in establishment of the symbiotic association, while others may confer advantage to cyanobionts during cohabitation with a mycobiont in the lichen symbiosis. Since many phylogenetic studies have shown that Nostoc is a polyphyletic group that includes several lineages, this work also provides an improved molecular basis for taxonomic revision of the genus Nostoc.Blágrænbakteríur af ættkvíslinni Nostoc geta myndað sambýli við margs konar lífverur, en fléttusambýli með asksveppum eru hvað algengust og fjölbreyttust. Á undanförnum árum hefur komið í ljós að hæfni til að mynda sambýli er margþættur eiginleiki ákvarðaður af einstökum genum og samsetningum þeirra. Nýmyndunargen sértæk fyrir sambýlunga geta ákvarðað efnagerðir sem fylgja gjarnan sambýli. Enda þótt sífellt fleira bendi til þátttöku bæði miðlægra og hliðsettra efnasambanda í starfsemi sambýlislífvera, þá er efnavistfræði sambýlis fremur lítt kannað svið. Með samnýtingu lífupplýsingafræði, ræktun sambýlungs með þungri samsætu kolefnis, og háþróaðri efnagreiningu, gátum við leitt í ljós myndun og byggingu sérstaks fjölketíðefnis, nosperins, í Nostoc stofni sem ræktaður var úr himnuskóf (Peltigera membranacea). Genaþyrpingin sem ákvarðar nýmyndun nosperins, svo og bygging nosperins, eru skyld pederin efnum, en þau voru áður einungis kunn úr bakteríum án ljóstillífunar í samlífi annars vegar við bjöllur, hins vegar við svampa í sjó. Nosperin genaþyrpingin er hin fyrsta af svokallaðri trans-AT gerð sem fundist hefur í blágrænbakteríum. Ennfremur er Nostoc stofn N6 fyrsta ræktaða bakterían sem framleiðir pederin efni, og veitir þannig ný tækifæri til ýmiss konar rannsókna á nýmyndun og eiginleikum þessa efnaflokks. Hin óvænta uppgötvun nosperins í fléttum eykur þannig þekkingu okkar á útbreiðslu og gerð pederin efna og undirstrikar fylgni þeirra við sambýlislífverur. Vönduð raðgreining og genaskilgreining á erfðamengi Nostoc N6 svo og á erfðamengi Nostoc stofns úr lungnafléttu (Lobaria pulmonaria), ásamt grófraðgreiningu þriggja annarra Nostoc stofna úr fléttum, veitir nú upplýsingar um gen sem líklega eru mikilvæg í sambýli almennt, og sérstaklega í fléttusambýli. Viðbótargen vegna sambýliseiginleika kunna að valda því að erfðamengi slíkra Nostoc stofna eru stærri en annarra Nostoc stofna. Sum slíka gen kunna að eiga þátt í að bera kennsl á vænlegan sambýlung og í að tryggja fyrstu skref til myndunar sambýlis, önnur geta tengst aukinni hæfni blágrænbakteríunnar til langvarandi sambýlis við fléttusveppi. Ítarleg raðgreining og genaskilgreining er einnig mikilvæg til að skilja betur þróun og skyldleika innan ættkvíslarinnar Nostoc, sem nú er í margþættri endurskoðun

Heterologous expression of a lichen polyketide synthase in filamentous fungi

Polyketides are a group of secondary metabolites produced by a wide range of living organisms – bacteria, fungi, plants and animals. These compounds exhibit remarkable structural diversity and possess an extremely broad spectrum of biological activity. Polyketides are of great commercial interest for drug discovery since many of these compounds have desirable pharmaceutical properties and they are a source of novel antibiotics, anti-tumor and anti-cancer agents, as well as cholesterol-lowering drugs. The biosynthesis of polyketides is catalyzed by large multifunctional enzymes called polyketide synthases (PKSs) that assemble core polyketide molecules from simple starter carboxylic acid precursors and several malonyl-CoA units in a manner similar to fatty acid synthesis. Although polyketide synthesis is widespread in filamentous fungi and lichen mycobionts, relatively few PKS genes have been isolated from filamentous fungi and no PKS gene from lichens can be found in the GenBank database except the one from the lichen Solorina crocea recently submitted. Although lichens produce various pharmaceutically relevant polyketides and have been appreciated in traditional medicines, their value has largely been ignored by the modern pharmaceutical industry because slow growth and difficulties in establishing pure cultures do not allow their routine use in most conventional screening processes. Recently, molecular genetic techniques using PCR, genomic library construction and heterologous expression in surrogate hosts have provided an alternative approach to begin accessing, exploring and harnessing the diversity of polyketide biosynthetic pathways in lichens. The aim of this work was the surrogate expression of a PKS gene from the lichen Solorina crocea in the filamentous fungus Aspergillus oryzae, analysis of the product(s) of its expression and determination of the gene structure: exon–intron boundaries, 5′- and 3′-prime ends, the native promoter region and the domain organization of the deduced protein sequence

Additional file 5 of Distinctive characters of Nostoc genomes in cyanolichens

Comparison of the cyanobacterial core and shell gene set to Nostoc sp. N6 and Nostoc sp. ‘Lobaria pulmonaria cyanobiont’. (XLS 172 kb

Additional file 3 of Distinctive characters of Nostoc genomes in cyanolichens

Proteins encoded in 10 most prominent locally collinear blocks in Nostoc punctiforme PCC 73102, Nostoc sp. N6 and Nostoc sp. ‘Lobaria pulmonaria cyanobiont’, identified by Mauve. (XLS 84 kb

Additional file 2 of Distinctive characters of Nostoc genomes in cyanolichens

Supporting Information (SI) Appendix. (PDF 8156 kb