Vector integration and fate in the hemophilia dog liver multiple years after AAV-FVIII gene transfer.

ABSTRACT: Gene therapy using adeno-associated virus (AAV) vectors is a promising approach for the treatment of monogenic disorders. Long-term multiyear transgene expression has been demonstrated in animal models and clinical studies. Nevertheless, uncertainties remain concerning the nature of AAV vector persistence and whether there is a potential for genotoxicity. Here, we describe the mechanisms of AAV vector persistence in the liver of a severe hemophilia A dog model (male = 4, hemizygous; and female = 4, homozygous), more than a decade after portal vein delivery. The predominant vector form was nonintegrated episomal structures with levels correlating with long-term transgene expression. Random integration was seen in all samples (median frequency, 9.3e-4 sites per cell), with small numbers of nonrandom common integration sites associated with open chromatin. No full-length integrated vectors were found, supporting predominant episomal vector-mediated long-term transgene expression. Despite integration, this was not associated with oncogene upregulation or histopathological evidence of tumorigenesis. These findings support the long-term safety of this therapeutic modality.

Improved RNA stability estimation indicates that transcriptional interference is frequent in diverse bacteria.

We used stochastic simulations and experimental data from E. coli, K. aerogenes, Synechococcus PCC 7002 and Synechocystis PCC 6803 to provide evidence that transcriptional interference via the collision mechanism is likely a prevalent mechanism for bacterial gene regulation. Rifampicin time-series data can be used to globally monitor and quantify collision between sense and antisense transcription-complexes. Our findings also highlight that transcriptional events, such as differential RNA decay, partial termination, and internal transcriptional start sites often deviate from gene annotations. Consequently, within a single gene annotation, there exist transcript segments with varying half-lives and transcriptional properties. To address these complexities, we introduce 'rifi', an R-package that analyzes transcriptomic data from rifampicin time series. 'rifi' employs a dynamic programming-based segmentation approach to identify individual transcripts, enabling accurate assessment of RNA stability and detection of diverse transcriptional events.

Intercellular communication is required for trap formation in the nematode-trapping fungus Duddingtonia flagrans.

Nematode-trapping fungi (NTF) are a large and diverse group of fungi, which may switch from a saprotrophic to a predatory lifestyle if nematodes are present. Different fungi have developed different trapping devices, ranging from adhesive cells to constricting rings. After trapping, fungal hyphae penetrate the worm, secrete lytic enzymes and form a hyphal network inside the body. We sequenced the genome of Duddingtonia flagrans, a biotechnologically important NTF used to control nematode populations in fields. The 36.64 Mb genome encodes 9,927 putative proteins, among which are more than 638 predicted secreted proteins. Most secreted proteins are lytic enzymes, but more than 200 were classified as small secreted proteins (< 300 amino acids). 117 putative effector proteins were predicted, suggesting interkingdom communication during the colonization. As a first step to analyze the function of such proteins or other phenomena at the molecular level, we developed a transformation system, established the fluorescent proteins GFP and mCherry, adapted an assay to monitor protein secretion, and established gene-deletion protocols using homologous recombination or CRISPR/Cas9. One putative virulence effector protein, PefB, was transcriptionally induced during the interaction. We show that the mature protein is able to be imported into nuclei in Caenorhabditis elegans cells. In addition, we studied trap formation and show that cell-to-cell communication is required for ring closure. The availability of the genome sequence and the establishment of many molecular tools will open new avenues to studying this biotechnologically relevant nematode-trapping fungus.

Echinocandin B biosynthesis: a biosynthetic cluster from Aspergillus nidulans NRRL 8112 and reassembly of the subclusters Ecd and Hty from Aspergillus pachycristatus NRRL 11440 reveals a single coherent gene cluster.

BACKGROUND: Echinocandins are nonribosomal lipopeptides produced by ascommycete fungi. Due to their strong inhibitory effect on fungal cell wall biosynthesis and lack of human toxicity, they have been developed to an important class of antifungal drugs. Since 2012, the biosynthetic gene clusters of most of the main echinocandin variants have been characterized. Especially the comparison of the clusters allows a deeper insight for the biosynthesis of these complex structures. RESULTS: In the genome of the echinocandin B producer Aspergillus nidulans NRRL 8112 we have identified a gene cluster (Ani) that encodes echinocandin biosynthesis. Sequence analyses showed that Ani is clearly delimited from the genomic context and forms a monophyletic lineage with the other echinocandin gene clusters. Importantly, we found that the disjunct genomic location of the echinocandin B gene cluster in A. pachycristatus NRRL 11440 on two separate subclusters, Ecd and Hty, at two loci was likely an artifact of genome misassembly in the absence of a reference sequence. We show that both sequences can be aligned resulting a single cluster with a gene arrangement collinear compared to other clusters of Aspergillus section Nidulantes. The reassembled gene cluster (Ecd/Hty) is identical to a putative gene cluster (AE) that was previously deposited at the NCBI as a sequence from A. delacroxii NRRL 3860. PCR amplification of a part of the gene cluster resulted a sequence that was very similar (97 % identity), but not identical to that of AE. CONCLUSIONS: The Echinocandin B biosynthetic cluster from A. nidulans NRRL 8112 (Ani) is particularly similar to that of A. pachycristatus NRRL 11440 (Ecd/Hty). Ecd/Hty was originally reported as two disjunct sub-clusters Ecd and Hty, but is in fact a continuous sequence with the same gene order as in Ani. According to sequences of PCR products amplified from genomic DNA, the echinocandin B producer A. delacroxii NRRL 3860 is closely related to A. pachycristatus NRRL 11440. A PCR-product from the gene cluster was very similar, but clearly distinct from the sequence published for A. delacroxii NRRL 3860 at the NCBI (No. AB720074). As the NCBI entry is virtually identical with the re-assembled Ecd/Hty cluster, it is likely that it originates from A. pachycristatus NRRL 11440 rather than A. delacroxii NRRL 3860.

Influence of a non-hospital medical care facility on antimicrobial resistance in wastewater.

The global widespread use of antimicrobials and accompanying increase in resistant bacterial strains is of major public health concern. Wastewater systems and wastewater treatment plants are considered a niche for antibiotic resistance genes (ARGs), with diverse microbial communities facilitating ARG transfer via mobile genetic element (MGE). In contrast to hospital sewage, wastewater from other health care facilities is still poorly investigated. At the instance of a nursing home located in south-west Germany, in the present study, shotgun metagenomics was used to investigate the impact on wastewater of samples collected up- and down-stream in different seasons. Microbial composition, ARGs and MGEs were analyzed using different annotation approaches with various databases, including Antibiotic Resistance Ontologies (ARO), integrons and plasmids. Our analysis identified seasonal differences in microbial communities and abundance of ARG and MGE between samples from different seasons. However, no obvious differences were detected between up- and downstream samples. The results suggest that, in contrast to hospitals, sewage from the nursing home does not have a major impact on ARG or MGE in wastewater, presumably due to much less intense antimicrobial usage. Possible limitations of metagenomic studies using high-throughput sequencing for detection of genes that seemingly confer antibiotic resistance are discussed.

Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase.

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far.

Characterization and phylogenetic analysis of the mitochondrial genome of Glarea lozoyensis indicates high diversity within the order Helotiales.

BACKGROUND: Glarea lozoyensis is a filamentous fungus used for the industrial production of non-ribosomal peptide pneumocandin B0. In the scope of a whole genome sequencing the complete mitochondrial genome of the fungus has been assembled and annotated. It is the first one of the large polyphyletic Helotiaceae family. A phylogenetic analysis was performed based on conserved proteins of the oxidative phosphorylation system in mitochondrial genomes. RESULTS: The total size of the mitochondrial genome is 45,038 bp. It contains the expected 14 genes coding for proteins related to oxidative phosphorylation,two rRNA genes, six hypothetical proteins, three intronic genes of which two are homing endonucleases and a ribosomal protein rps3. Additionally there is a set of 33 tRNA genes. All genes are located on the same strand. Phylogenetic analyses based on concatenated mitochondrial protein sequences confirmed that G. lozoyensis belongs to the order of Helotiales and that it is most closely related to Phialocephala subalpina. However, a comparison with the three other mitochondrial genomes known from Helotialean species revealed remarkable differences in size, gene content and sequence. Moreover, it was found that the gene order found in P. subalpina and Sclerotinia sclerotiorum is not conserved in G. lozoyensis. CONCLUSION: The arrangement of genes and other differences found between the mitochondrial genome of G. lozoyensis and those of other Helotiales indicates a broad genetic diversity within this large order. Further mitochondrial genomes are required in order to determine whether there is a continuous transition between the different forms of mitochondrial genomes or G. lozoyensis belongs to a distinct subgroup within Helotiales.

Genome sequence of the fungus Glarea lozoyensis: the first genome sequence of a species from the Helotiaceae family.

The anamorphic fungus Glarea lozoyensis mutant strain 74030 is an overproducer of pneumocandin B(0), which is chemically converted into Cancidas, a potent antibiotic against clinically important fungal pathogens. Pneumocandins are acylated, cyclic hexapeptides with unusual hydroxylated amino acids. With the Glarea lozoyensis genome, the first species from the large polyphyletic family Helotiaceae has been sequenced.

The ylo-1 gene encodes an aldehyde dehydrogenase responsible for the last reaction in the Neurospora carotenoid pathway.

The accumulation of the apocarotenoid neurosporaxanthin and its carotene precursors explains the orange pigmentation of the Neurospora surface cultures. Neurosporaxanthin biosynthesis requires the activity of the albino gene products (AL-1, AL-2 and AL-3), which yield the precursor torulene. Recently, we identified the carotenoid oxygenase CAO-2, which cleaves torulene to produce the aldehyde beta-apo-4'-carotenal. This revealed a last missing step in Neurospora carotenogenesis, namely the oxidation of the CAO-2 product to the corresponding acid neurosporaxanthin. The mutant ylo-1, which exhibits a yellow colour, lacks neurosporaxanthin and accumulates several carotenes, but its biochemical basis is unknown. Based on available genetic data, we identified ylo-1 in the Neurospora genome, which encodes an enzyme representing a novel subfamily of aldehyde dehydrogenases, and demonstrated that it is responsible for the yellow phenotype, by sequencing and complementation of mutant alleles. In contrast to the precedent structural genes in the carotenoid pathway, light does not induce the synthesis of ylo-1 mRNA. In vitro incubation of purified YLO-1 protein with beta-apo-4'-carotenal produced neurosporaxanthin through the oxidation of the terminal aldehyde into a carboxyl group. We conclude that YLO-1 completes the set of enzymes needed for the synthesis of this major Neurospora pigment.

The biochemical characterization of a high-stearic acid sunflower mutant reveals the coordinated regulation of stearoyl-acyl carrier protein desaturases.

In this study, we have biochemically characterized the high-stearic sunflower (Helianthus annuus L.) mutant CAS-14. This mutant displays an abnormal fatty acid composition along the length of the seed when grown at high temperatures. Thus, at the expense of oleate and linoleate, CAS-14 seeds present an increasing axial gradient of stearic acid from the embryo to the distal extreme of the seed. The accumulation of oil in this mutant was initially characterized by analysing the incorporation of radiolabelled acetate, a fatty acid synthetic precursor, into the developing seed tissues in vivo. These experiments indicated that the mutant phenotype was associated with a decrease in the soluble stearoyl-acyl carrier protein desaturase (SAD) activity, as later confirmed when assessing this activity in cell-free extracts from developing sunflower kernels. Furthermore, SAD enzyme gene transcription was also examined in this tissue, identifying the coordinated decrease in the transcription of the sad17 and sad6 genes as underlying the decrease in enzyme activity. On the basis of these results and those previously obtained on the inheritance of the CAS-14 trait, we discuss the possible regulatory mechanisms acting on plant soluble desaturases.

Identification of the gene responsible for torulene cleavage in the Neurospora carotenoid pathway.

Torulene, a C(40) carotene, is the precursor of the end product of the Neurospora carotenoid pathway, the C(35) xanthophyll neurosporaxanthin. Torulene is synthesized by the enzymes AL-2 and AL-1 from the precursor geranylgeranyl diphosphate and then cleaved by an unknown enzyme into the C(35) apocarotenoid. In general, carotenoid cleavage reactions are catalyzed by carotenoid oxygenases. Using protein data bases, we identified two putative carotenoid oxygenases in Neurospora, named here CAO-1 and CAO-2. A search for novel mutants of the carotenoid pathway in this fungus allowed the identification of two torulene-accumulating strains, lacking neurosporaxanthin. Sequencing of the cao-2 gene in these strains revealed severe mutations, pointing to a role of CAO-2 in torulene cleavage. This was further supported by the identical phenotype found upon targeted disruption of cao-2. The biological function was confirmed by in vitro assays using the purified enzyme, which cleaved torulene to produce beta-apo-4'-carotenal, the corresponding aldehyde of neurosporaxanthin. The specificity of CAO-2 was shown by the lack of gamma-carotene-cleaving activity in vitro. As predicted for a structural gene of the carotenoid pathway, cao-2 mRNA was induced by light in a WC-1 and WC-2 dependent manner. Our data demonstrate that CAO-2 is the enzyme responsible for the oxidative cleavage of torulene in the neurosporaxanthin biosynthetic pathway.

Light-dependent regulation of the gene cut-1 of Neurospora, involved in the osmotic stress response.

The gene cut-1 from Neurospora encodes a protein of the haloacid dehalogenase (HAD) family, a group of enzymes usually associated to phosphatase or phosphotransferase activities. Loss of cut-1 function results in an osmosensitive phenotype and its transcription is induced by high osmotic pressure or heat-shock, but not by other stressing conditions tested. Unexpectedly, cut-1 transcript levels markedly decrease following illumination in the wild type and in some mutants tested, such as bd and vvd. The reduction was less pronounced in wc-1 and wc-2 mutants, indicating that this photoresponse is principally mediated by the WC system, a conclusion further supported by the lack of effect of blue light in a wc-1 mutant. Additionally, cut-1 mRNA levels are much higher in the dark in wc, vvd, bd, and cut mutants, showing the involvement of these genes in a cut-1 down-regulation mechanism. In the cases investigated, the mRNA levels of these strains increased further under high osmotic conditions, indicating an independent derepression effect. The environmental or genetic factors influencing cut-1 expression have no significant effect on the mRNA levels of two additional Neurospora genes encoding HAD proteins.

The Neurospora crassa gene responsible for the cut and ovc phenotypes encodes a protein of the haloacid dehalogenase family.

Light stimulation of carotenogenesis in Neurospora crassa, mediated by the White Collar proteins, is enhanced in some regulatory mutants, such as vivid and ovc. The gene responsible for the vivid mutation has been identified, but not the one responsible for the ovc phenotype. The ovc mutant is sensitive to high osmotic conditions and allelic with another mutant, cut, also osmosensitive but not affected in carotenogenesis. A phenotypic characterization of both strains is presented. Light induction of mRNA levels of the carotenoid genes al-1 and al-2, the regulatory gene wc-1 or the conidiation-specific gene con-10 is not significantly changed in the ovc mutant when compared with the wild type. We have identified the gene affected in the ovc mutant by complementation of osmosensitivity with a cosmid library. This gene, which we call cut-1, codes for an enzyme of the haloacid dehalogenase family, which includes different classes of phosphatases. cut-1 is able to restore the wild-type phenotype of the ovc and cut strains, confirming that they are affected in the same gene. DNA sequence analysis identified a point mutation in the cut mutant, leading to a truncated protein. The ovc mutant represents a deletion encompassing the entire gene and surrounding sequences. The cut-1 promoter contains putative regulatory elements involved in osmotic or thermal stress. We show that cut-1 transcription is low in illuminated or dark-grown cultures, and is induced by high osmotic conditions or by heat shock.