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September 22, 2019

Genomic characterization of Lactobacillus delbrueckii TUA4408L and evaluation of the antiviral activities of its extracellular polysaccharides in porcine intestinal epithelial cells.

In lactic acid bacteria, the synthesis of exopolysaccharides (EPS) has been associated with some favorable technological properties as well as health-promoting benefits. Research works have shown the potential of EPS produced by lactobacilli to differentially modulate immune responses. However, most studies were performed in immune cells and few works have concentrated in the immunomodulatory activities of EPS in non-immune cells such as intestinal epithelial cells. In addition, the cellular and molecular mechanisms involved in the immunoregulatory effects of EPS have not been studied in detail. In this work, we have performed a genomic characterization of Lactobacillus delbrueckii subsp. delbrueckii TUA4408L and evaluated the immunomodulatory and antiviral properties of its acidic (APS) and neutral (NPS) EPS in porcine intestinal epithelial (PIE) cells. Whole genome sequencing allowed the analysis of the general features of L. delbrueckii TUA4408L genome as well as the characterization of its EPS genes. A typical EPS gene cluster was found in the TUA4408L genome consisting in five highly conserved genes epsA-E, and a variable region, which includes the genes for the polymerase wzy, the flippase wzx, and seven glycosyltransferases. In addition, we demonstrated here for the first time that L. delbrueckii TUA4408L and its EPS are able to improve the resistance of PIE cells against rotavirus infection by reducing viral replication and regulating inflammatory response. Moreover, studies in PIE cells demonstrated that the TUA4408L strain and its EPS differentially modulate the antiviral innate immune response triggered by the activation of Toll-like receptor 3 (TLR3). L. delbrueckii TUA4408L and its EPS are capable of increasing the activation of interferon regulatory factor (IRF)-3 and nuclear factor ?B (NF-?B) signaling pathways leading to an improved expression of the antiviral factors interferon (IFN)-ß, Myxovirus resistance gene A (MxA) and RNaseL.

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September 22, 2019

Genomic characterization and probiotic potency of Bacillus sp. DU-106, a highly effective producer of L-lactic acid isolated from fermented yogurt.

Bacillus sp. DU-106, a newly isolated member of Bacillus cereus group, exhibits the predominant ability to produce L-lactic acid. The probiotic potency of test strain revealed its survivability at acidic pH, bile salts and viability in simulated gastric juice in vitro. The acute oral toxicity test indicated its no toxicity to laboratory mice in vivo. We further determined the complete genome of strain DU-106 to understand genetic basis as a potential probiotic. It has a circular chromosome and three plasmids for a total genome 5,758,208 bp in size with a G + C content of 35.10%. Genes associated with lactate synthesis were found in the DU-106 genome. We also annotated various stress-related, bile salt resistance, and adhesion-related domains in this strain, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival under gastrointestinal tract. Moreover, strain DU-106 genome lacks the virulence genes encodes cereulide synthetase, enterotoxin FM, and cytotoxin K. These phenotypic and genomic probiotic potencies facilitate its potential candidate as probiotic starter in food industry.

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September 22, 2019

Methylation of the reelin gene promoter in peripheral blood and its relationship with the cognitive function of schizophrenia patients.

There is a decrease in the expression of the reelin gene (RELN) in the brain of schizophrenia patients, which can underlie observed cognitive abnormalities. It is suggested that this decrease is caused by the hypermethylation of the RELN promoter. The aim of the study was to investigate methylation of the RELN promoter in the peripheral blood of schizophrenia patients and its association with their cognitive deficits. A modified SMRT-BS (single-molecule real-time bisulfite sequencing) was used. We determined the methylation rate of 170 CpG sites within a 1465 bp DNA region containing the entire CpG island in the RELN promoter in 51 schizophrenia patients and 52 healthy controls. All subjects completed a battery of neuropsychological tests. There were no DNA methylation changes associated with schizophrenia. Most CpGs sites were unmethylated in both groups. At the same time, there was a variability in the methylation level of different regions within the promoter. The methylation level in the area from -258 to -151 bp relative to RELN transcription start site was a significant predictor of the index of patients’ cognitive functioning if sex, age, smoking, education, and polymorphism rsl858815 had been considered. The positive correlation between the methylation rate in this region and cognitive index suggests that the hypomethylation of the RELN promoter could contribute to the development of cognitive deficits in schizophrenia.

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September 22, 2019

Genomic surveillance of Neisseria gonorrhoeae to investigate the distribution and evolution of antimicrobial-resistance determinants and lineages.

The first extensively drug resistant (XDR) Neisseria gonorrhoeae strain with high resistance to the extended-spectrum cephalosporin ceftriaxone was identified in 2009 in Japan, but no other strain with this antimicrobial-resistance profile has been reported since. However, surveillance to date has been based on phenotypic methods and sequence typing, not genome sequencing. Therefore, little is known about the local population structure at the genomic level, and how resistance determinants and lineages are distributed and evolve. We analysed the whole-genome sequence data and the antimicrobial- susceptibility testing results of 204 strains sampled in a region where the first XDR ceftriaxone-resistant N. gonorrhoeae was isolated, complemented with 67 additional genomes from other time frames and locations within Japan. Strains resistant to ceftriaxone were not found, but we discovered a sequence type (ST)7363 sub-lineage susceptible to ceftriaxone and cefixime in which the mosaic penA allele responsible for reduced susceptibility had reverted to a susceptible allele by recombination. Approximately 85% of isolates showed resistance to fluoroquinolones (ciprofloxacin) explained by linked amino acid substitutions at positions 91 and 95 of GyrA with 99% sensitivity and 100% specificity. Approximately 10% showed resistance to macrolides (azithromycin), for which genetic determinants are less clear. Furthermore, we revealed different evolutionary paths of the two major lineages: single acquisition of penA X in the ST7363-associated lineage, followed by multiple independent acquisitions of the penA X and XXXIV in the ST1901-associated lineage. Our study provides a detailed picture of the distribution of resistance determinants and disentangles the evolution of the two major lineages spreading worldwide.

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September 22, 2019

Spread of carbapenem resistance by transposition and conjugation among Pseudomonas aeruginosa.

The emergence of carbapenem-resistant Pseudomonas aeruginosa represents a worldwide problem. To understand the carbapenem-resistance mechanisms and their spreading among P. aeruginosa strains, whole genome sequences were determined of two extensively drug-resistant strains that are endemic in Dutch hospitals. Strain Carb01 63 is of O-antigen serotype O12 and of sequence type ST111, whilst S04 90 is a serotype O11 strain of ST446. Both strains carry a gene for metallo-ß-lactamase VIM-2 flanked by two aacA29 genes encoding aminoglycoside acetyltransferases on a class 1 integron. The integron is located on the chromosome in strain Carb01 63 and on a plasmid in strain S04 90. The backbone of the 159-kb plasmid, designated pS04 90, is similar to a previously described plasmid, pND6-2, from Pseudomonas putida. Analysis of the context of the integron showed that it is present in both strains on a ~30-kb mosaic DNA segment composed of four different transposons that can presumably act together as a novel, active, composite transposon. Apart from the presence of a 1237-bp insertion sequence element in the composite transposon on pS04 90, these transposons show > 99% sequence identity indicating that transposition between plasmid and chromosome could have occurred only very recently. The pS04 90 plasmid could be transferred by conjugation to a susceptible P. aeruginosa strain. A second class 1 integron containing a gene for a CARB-2 ß-lactamase flanked by an aacA4′-8 and an aadA2 gene, encoding an aminoglycoside acetyltransferase and adenylyltransferase, respectively, was present only in strain Carb01 63. This integron is located also on a composite transposon that is inserted in an integrative and conjugative element on the chromosome. Additionally, this strain contains a frameshift mutation in the oprD gene encoding a porin involved in the transport of carbapenems across the outer membrane. Together, the results demonstrate that integron-encoded carbapenem and carbapenicillin resistance can easily be disseminated by transposition and conjugation among Pseudomonas aeruginosa strains.

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September 22, 2019

Ma orthologous genes in Prunus spp. shed light on a noteworthy NBS-LRR cluster conferring differential resistance to root-knot nematodes.

Root-knot nematodes (RKNs) are considerable polyphagous pests that severely challenge plants worldwide and especially perennials. The specific genetic resistance of plants mainly relies on the NBS-LRR genes that are pivotal factors for pathogens control. In Prunus spp., the Ma plum and RMja almond genes possess different spectra for resistance to RKNs. While previous works based on the Ma gene allowed to clone it and to decipher its peculiar TIR-NBS-LRR (TNL) structure, we only knew that the RMja gene mapped on the same chromosome as Ma. We carried out a high-resolution mapping using an almond segregating F2 progeny of 1448 seedlings from resistant (R) and susceptible (S) parental accessions, to locate precisely RMja on the peach genome, the reference sequence for Prunus species. We showed that the RMja gene maps in the Ma resistance cluster and that the Ma ortholog is the best candidate for RMja. This co-localization is a crucial step that opens the way to unravel the molecular determinants involved in the resistance to RKNs. Then we sequenced both almond parental NGS genomes and aligned them onto the RKN susceptible reference peach genome. We produced a BAC library of the R parental accession and, from two overlapping BAC clones, we obtained a 336-kb sequence encompassing the RMja candidate region. Thus, we could benefit from three Ma orthologous regions to investigate their sequence polymorphism, respectively, within plum (complete R spectrum), almond (incomplete R spectrum) and peach (null R spectrum). We showed that the Ma TNL cluster has evolved orthologs with a unique conserved structure comprised of five repeated post-LRR (PL) domains, which contain most polymorphism. In addition to support the Ma and RMja orthologous relationship, our results suggest that the polymorphism contained in the PL sequences might underlie differential resistance interactions with RKNs and an original immune mechanism in woody perennials. Besides, our study illustrates how PL exon duplications and losses shape TNL structure and give rise to atypical PL domain repeats of yet unknown role.

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September 22, 2019

Structural variants exhibit allelic heterogeneity and shape variation in complex traits

Despite extensive effort to reveal the genetic basis of complex phenotypic variation, studies typically explain only a fraction of trait heritability. It has been hypothesized that individually rare hidden structural variants (SVs) could account for a significant fraction of variation in complex traits. To investigate this hypothesis, we assembled 14 Drosophila melanogaster genomes and systematically identified more than 20,000 euchromatic SVs, of which ~40% are invisible to high specificity short read genotyping approaches. SVs are common in Drosophila genes, with almost one third of diploid individuals harboring an SV in genes larger than 5kb, and nearly a quarter harboring multiple SVs in genes larger than 10kb. We show that SV alleles are rarer than amino acid polymorphisms, implying that they are more strongly deleterious. A number of functionally important genes harbor previously hidden structural variants that likely affect complex phenotypes (e.g., Cyp6g1, Drsl5, Cyp28d1&2, InR, and Gss1&2). Furthermore, SVs are overrepresented in quantitative trait locus candidate genes from eight Drosophila Synthetic Population Resource (DSPR) mapping experiments. We conclude that SVs are pervasive in genomes, are frequently present as heterogeneous allelic series, and can act as rare alleles of large effect.

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September 22, 2019

De novo assembly, delivery and expression of a 101 kb human gene in mouse cells

Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger, mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here we describe a pipeline for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kb. The DNA assembly step is supported by an integrated robotic workcell. We assemble the 101 kb human HPRT1 gene in yeast, deliver it to mouse cells, and show expression of the human protein from its full-length gene. This pipeline provides a framework for producing systematic, designer variants of any mammalian gene locus for functional evaluation in cells.

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September 22, 2019

Parliament2: Fast structural variant calling using optimized combinations of callers

Here we present Parliament2: a structural variant caller which combines multiple best-in-class structural variant callers to create a highly accurate callset. This captures more events than the individual callers achieve independently. Parliament2 uses a call-overlap-genotype approach that is highly extensible to new methods and presents users the choice to run some or all of Breakdancer, Breakseq, CNVnator, Delly, Lumpy, and Manta to run. Parliament2 applies an additional parallelization framework to speed certain callers and executes these in parallel, taking advantage of the different resource requirements to complete structural variant calling much faster than running the programs individually. Parliament2 is available as a Docker container, which pre-installs all required dependencies. This allows users to run any caller with easy installation and execution. This Docker container can easily be deployed in cloud or local environments and is available as an app on DNAnexus.

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September 22, 2019

Extraordinary genome instability and widespread chromosome rearrangements during vegetative growth

The haploid genome of the pathogenic fungus Zymoseptoria tritici is contained on “core” and “accessory” chromosomes. While 13 core chromosomes are found in all strains, as many as eight accessory chromosomes show presence/absence variation and rearrangements among field isolates. The factors influencing these presence/absence polymorphisms are so far unknown. We investigated chromosome stability using experimental evolution, karyotyping, and genome sequencing. We report extremely high and variable rates of accessory chromosome loss during mitotic propagation in vitro and in planta Spontaneous chromosome loss was observed in 2 to >50% of cells during 4 weeks of incubation. Similar rates of chromosome loss in the closely related Zymoseptoria ardabiliae suggest that this extreme chromosome dynamic is a conserved phenomenon in the genus. Elevating the incubation temperature greatly increases instability of accessory and even core chromosomes, causing severe rearrangements involving telomere fusion and chromosome breakage. Chromosome losses do not affect the fitness of Zymoseptoria tritici in vitro, but some lead to increased virulence, suggesting an adaptive role of this extraordinary chromosome instability. Copyright © 2018 by the Genetics Society of America.

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September 22, 2019

4.5 years within-patient evolution of a colistin resistant KPC-producing Klebsiella pneumoniae ST258.

Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-Kp) has emerged globally over the last decade as a major nosocomial pathogen that threatens patient care. These highly resistant bacteria are mostly associated with a single Kp clonal group, CG258, but the reasons for its host and hospital adaptation remain largely unknown.We analyzed the in vivo evolution of a colistin-resistant KPC-Kp CG258 strain that contaminated a patient following an endoscopy and was responsible for a fatal bacteremia 4.5 years later. Whole-genome sequencing was performed on 17 KPC-Kp isolates from this patient; single-nucleotide polymorphisms were analyzed and their implication in antimicrobial resistance and bacterial host adaptation investigated.The patient KPC-Kp strain diversified over 4.5 years at a rate of 7.5 substitutions per genome per year, resulting in broad phenotypic modifications. After 2 years of carriage, all isolates restored susceptibility to colistin. Higher expression of the fimbriae conferred the ability to produce more biofilm, and the isolate responsible for a bacteremia grew in human serum. The convergent mutations occurring in specific pathways, such as the respiratory chain and the cell envelope, revealed a complex long-term adaptation of KPC-Kp.Broad genomic and phenotypic diversification and the parallel selection of pathoadaptive mutations might contribute to long-term carriage and virulence of KPC-Kp CG258 strains and to the dissemination of this clone.

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September 22, 2019

Dissemination and persistence of extended-spectrum cephalosporin-resistance encoding IncI1-blaCTXM-1 plasmid among Escherichia coli in pigs.

This study investigated the ecology, epidemiology and plasmid characteristics of extended-spectrum cephalosporin (ESC)-resistant E. coli in healthy pigs over a period of 4 years (2013-2016) following the withdrawal of ESCs. High carriage rates of ESC-resistant E. coli were demonstrated in 2013 (86.6%) and 2014 (83.3%), compared to 2015 (22%) and 2016 (8.5%). ESC resistance identified among E. coli isolates was attributed to the carriage of an IncI1 ST-3 plasmid (pCTXM1-MU2) encoding blaCTXM-1. Genomic characterisation of selected E. coli isolates (n?=?61) identified plasmid movement into multiple commensal E. coli (n?=?22 STs). Major STs included ST10, ST5440, ST453, ST2514 and ST23. A subset of the isolates belong to the atypical enteropathogenic E. coli (aEPEC) pathotype that harboured multiple LEE pathogenic islands. pCTXM1-MU2 was similar (99% nt identity) to IncI1-ST3 plasmids reported from Europe, encoded resistance to aminoglycosides, sulphonamides and trimethoprim, and carried colicin Ib. pCTXM1-MU2 appears to be highly stable and readily transferable. This study demonstrates that ESC resistance may persist for a protracted period following removal of direct selection pressure, resulting in the emergence of ESC-resistance in both commensal E. coli and aEPEC isolates of potential significance to human and animal health.

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September 22, 2019

Generic accelerated sequence alignment in SeqAn using vectorization and multi-threading.

Pairwise sequence alignment is undoubtedly a central tool in many bioinformatics analyses. In this paper, we present a generically accelerated module for pairwise sequence alignments applicable for a broad range of applications. In our module, we unified the standard dynamic programming kernel used for pairwise sequence alignments and extended it with a generalized inter-sequence vectorization layout, such that many alignments can be computed simultaneously by exploiting SIMD (single instruction multiple data) instructions of modern processors. We then extended the module by adding two layers of thread-level parallelization, where we (a) distribute many independent alignments on multiple threads and (b) inherently parallelize a single alignment computation using a work stealing approach producing a dynamic wavefront progressing along the minor diagonal.We evaluated our alignment vectorization and parallelization on different processors, including the newest Intel® Xeon® (Skylake) and Intel® Xeon PhiTM (KNL) processors, and use cases. The instruction set AVX512-BW (Byte and Word), available on Skylake processors, can genuinely improve the performance of vectorized alignments. We could run single alignments 1600 times faster on the Xeon PhiTM and 1400 times faster on the Xeon® than executing them with our previous sequential alignment module.The module is programmed in C++?using the SeqAn (Reinert et al., 2017) library and distributed with version 2.4 under the BSD license. We support SSE4, AVX2, AVX512 instructions and included UME: SIMD, a SIMD-instruction wrapper library, to extend our module for further instruction sets. We thoroughly test all alignment components with all major C++?compilers on various platforms.Supplementary data are available at Bioinformatics online.

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September 22, 2019

Development and validation of 58K SNP-array and high-density linkage map in Nile tilapia (O. niloticus).

Despite being the second most important aquaculture species in the world accounting for 7.4% of global production in 2015, tilapia aquaculture has lacked genomic tools like SNP-arrays and high-density linkage maps to improve selection accuracy and accelerate genetic progress. In this paper, we describe the development of a genotyping array containing more than 58,000 SNPs for Nile tilapia (Oreochromis niloticus). SNPs were identified from whole genome resequencing of 32 individuals from the commercial population of the Genomar strain, and were selected for the SNP-array based on polymorphic information content and physical distribution across the genome using the Orenil1.1 genome assembly as reference sequence. SNP-performance was evaluated by genotyping 4991 individuals, including 689 offspring belonging to 41 full-sib families, which revealed high-quality genotype data for 43,588 SNPs. A preliminary genetic linkage map was constructed using Lepmap2 which in turn was integrated with information from the O_niloticus_UMD1 genome assembly to produce an integrated physical and genetic linkage map comprising 40,186 SNPs distributed across 22 linkage groups (LGs). Around one-third of the LGs showed a different recombination rate between sexes, with the female being greater than the male map by a factor of 1.2 (1632.9 to 1359.6 cM, respectively), with most LGs displaying a sigmoid recombination profile. Finally, the sex-determining locus was mapped to position 40.53 cM on LG23, in the vicinity of the anti-Müllerian hormone (amh) gene. These new resources has the potential to greatly influence and improve the genetic gain when applying genomic selection and surpass the difficulties of efficient selection for invasively measured traits in Nile tilapia.

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September 22, 2019

Amycomicin is a potent and specific antibiotic discovered with a targeted interaction screen.

The rapid emergence of antibiotic-resistant pathogenic bacteria has accelerated the search for new antibiotics. Many clinically used antibacterials were discovered through culturing a single microbial species under nutrient-rich conditions, but in the environment, bacteria constantly encounter poor nutrient conditions and interact with neighboring microbial species. In an effort to recapitulate this environment, we generated a nine-strain actinomycete community and used 16S rDNA sequencing to deconvolute the stochastic production of antimicrobial activity that was not observed from any of the axenic cultures. We subsequently simplified the community to just two strains and identified Amycolatopsis sp. AA4 as the producing strain and Streptomyces coelicolor M145 as an inducing strain. Bioassay-guided isolation identified amycomicin (AMY), a highly modified fatty acid containing an epoxide isonitrile warhead as a potent and specific inhibitor of Staphylococcus aureus Amycomicin targets an essential enzyme (FabH) in fatty acid biosynthesis and reduces S. aureus infection in a mouse skin-infection model. The discovery of AMY demonstrates the utility of screening complex communities against specific targets to discover small-molecule antibiotics.

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