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September 22, 2019

The genome of tapeworm Taenia multiceps sheds light on understanding parasitic mechanism and control of coenurosis disease.

Coenurosis, caused by the larval coenurus of the tapeworm Taenia multiceps, is a fatal central nervous system disease in both sheep and humans. Though treatment and prevention options are available, the control of coenurosis still faces presents great challenges. Here, we present a high-quality genome sequence of T. multiceps in which 240 Mb (96%) of the genome has been successfully assembled using Pacbio single-molecule real-time (SMRT) and Hi-C data with a N50 length of 44.8 Mb. In total, 49.5 Mb (20.6%) repeat sequences and 13, 013 gene models were identified. We found that Taenia spp. have an expansion of transposable elements and recent small-scale gene duplications following the divergence of Taenia from Echinococcus, but not in Echinococcus genomes, and the genes underlying environmental adaptability and dosage effect tend to be over-retained in the T. multiceps genome. Moreover, we identified several genes encoding proteins involved in proglottid formation and interactions with the host central nervous system, which may contribute to the adaption of T. multiceps to its parasitic life style. Our study not only provides insights into the biology and evolution of T. multiceps, but also identifies a set of species-specific gene targets for developing novel treatment and control tools for coenurosis.

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September 22, 2019

Cloning of the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase family.

Yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is a devastating fungal disease threatening much of global wheat production. Race-specific resistance (R)-genes are used to control rust diseases, but the rapid emergence of virulent Pst races has prompted the search for a more durable resistance. Here, we report the cloning of Yr15, a broad-spectrum R-gene derived from wild emmer wheat, which encodes a putative kinase-pseudokinase protein, designated as wheat tandem kinase 1, comprising a unique R-gene structure in wheat. The existence of a similar gene architecture in 92 putative proteins across the plant kingdom, including the barley RPG1 and a candidate for Ug8, suggests that they are members of a distinct family of plant proteins, termed here tandem kinase-pseudokinases (TKPs). The presence of kinase-pseudokinase structure in both plant TKPs and the animal Janus kinases sheds light on the molecular evolution of immune responses across these two kingdoms.

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September 22, 2019

Antiviral adaptive immunity and tolerance in the mosquito Aedes aegyti

Mosquitoes spread pathogenic arboviruses while themselves tolerate infection. We here characterize an immunity pathway providing long-term antiviral protection and define how this pathway discriminates between self and non-self. Mosquitoes use viral RNAs to create viral derived cDNAs (vDNAs) central to the antiviral response. vDNA molecules are acquired through a process of reverse-transcription and recombination directed by endogenous retrotransposons. These vDNAs are thought to integrate in the host genome as endogenous viral elements (EVEs). Sequencing of pre-integrated vDNA revealed that the acquisition process exquisitely distinguishes viral from host RNA, providing one layer of self-nonself discrimination. Importantly, we show EVE-derived piRNAs have antiviral activity and are loaded onto Piwi4 to inhibit virus replication. In a second layer of self-non-self discrimination, Piwi4 preferentially loads EVE-derived piRNAs, discriminating against transposon-targeting piRNAs. Our findings define a fundamental virus-specific immunity pathway in mosquitoes that uses EVEs as a potent and specific antiviral transgenerational mechanism.

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September 22, 2019

Comparative genomics of Czech vaccine strains of Bordetella pertussis.

Bordetella pertussis is a strictly human pathogen causing the respiratory infectious disease called whooping cough or pertussis. B. pertussis adaptation to acellular pertussis vaccine pressure has been repeatedly highlighted, but recent data indicate that adaptation of circulating strains started already in the era of the whole cell pertussis vaccine (wP) use. We sequenced the genomes of five B. pertussis wP vaccine strains isolated in the former Czechoslovakia in the pre-wP (1954-1957) and early wP (1958-1965) eras, when only limited population travel into and out of the country was possible. Four isolates exhibit a similar genome organization and form a distinct phylogenetic cluster with a geographic signature. The fifth strain is rather distinct, both in genome organization and SNP-based phylogeny. Surprisingly, despite isolation of this strain before 1966, its closest sequenced relative appears to be a recent isolate from the US. On the genome content level, the five vaccine strains contained both new and already described regions of difference. One of the new regions contains duplicated genes potentially associated with transport across the membrane. The prevalence of this region in recent isolates indicates that its spread might be associated with selective advantage leading to increased strain fitness.

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September 22, 2019

Whole genome sequencing for investigations of meningococcal outbreaks in the United States: a retrospective analysis.

Although rare in the U.S., outbreaks due to Neisseria meningitidis do occur. Rapid, early outbreak detection is important for timely public health response. In this study, we characterized U.S. meningococcal isolates (N?=?201) from 15 epidemiologically defined outbreaks (2009-2015) along with temporally and geographically matched sporadic isolates using multilocus sequence typing, pulsed-field gel electrophoresis (PFGE), and six whole genome sequencing (WGS) based methods. Recombination-corrected maximum likelihood (ML) and Bayesian phylogenies were reconstructed to identify genetically related outbreak isolates. All WGS analysis methods showed high degree of agreement and distinguished isolates with similar or indistinguishable PFGE patterns, or the same strain genotype. Ten outbreaks were caused by a single strain; 5 were due to multiple strains. Five sporadic isolates were phylogenetically related to 2 outbreaks. Analysis of 9 outbreaks using timed phylogenies identified the possible origin and estimated the approximate time that the most recent common ancestor emerged for outbreaks analyzed. U.S. meningococcal outbreaks were caused by single- or multiple-strain introduction, with organizational outbreaks mainly caused by a clonal strain and community outbreaks by divergent strains. WGS can infer linkage of meningococcal cases when epidemiological links are uncertain. Accurate identification of outbreak-associated cases requires both WGS typing and epidemiological data.

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September 22, 2019

First draft genome sequence of the rock bream in the family Oplegnathidae.

The rock bream (Oplegnathus fasciatus) is one of the most economically valuable marine fish in East Asia, and due to various environmental factors, there is substantial revenue loss in the production sector. Therefore, knowledge of its genome is required to uncover the genetic factors and the solutions to these problems. In this study, we constructed the first draft genome of O. fasciatus as a reference for the family Oplegnathidae. The genome size is estimated to be 749?Mb, and it was assembled into 766?Mb by combining Illumina and PacBio sequences. A total of 24,053 transcripts (23,338 genes) are predicted, and among those transcripts, 23,362 (97%), are annotated with functional terms. Finally, the completeness of the genome assembly was assessed by CEGMA, which resulted in the complete mapping of 220 (88.7%) core genes in the genome. To the best of our knowledge, this is the first draft genome for the family Oplegnathidae.

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September 22, 2019

Loss of bacitracin resistance due to a large genomic deletion among Bacillus anthracis strains.

Bacillus anthracis is a Gram-positive endospore-forming bacterial species that causes anthrax in both humans and animals. In Zambia, anthrax cases are frequently reported in both livestock and wildlife, with occasional transmission to humans, causing serious public health problems in the country. To understand the genetic diversity of B. anthracis strains in Zambia, we sequenced and compared the genomic DNA of B. anthracis strains isolated across the country. Single nucleotide polymorphisms clustered these strains into three groups. Genome sequence comparisons revealed a large deletion in strains belonging to one of the groups, possibly due to unequal crossing over between a pair of rRNA operons. The deleted genomic region included genes conferring resistance to bacitracin, and the strains with the deletion were confirmed with loss of bacitracin resistance. Similar deletions between rRNA operons were also observed in a few B. anthracis strains phylogenetically distant from Zambian strains. The structure of bacitracin resistance genes flanked by rRNA operons was conserved only in members of the Bacillus cereus group. The diversity and genomic characteristics of B. anthracis strains determined in this study would help in the development of genetic markers and treatment of anthrax in Zambia. IMPORTANCE Anthrax is caused by Bacillus anthracis, an endospore-forming soil bacterium. The genetic diversity of B. anthracis is known to be low compared with that of Bacillus species. In this study, we performed whole-genome sequencing of Zambian isolates of B. anthracis to understand the genetic diversity between closely related strains. Comparison of genomic sequences revealed that closely related strains were separated into three groups based on single nucleotide polymorphisms distributed throughout the genome. A large genomic deletion was detected in the region containing a bacitracin resistance gene cluster flanked by rRNA operons, resulting in the loss of bacitracin resistance. The structure of the deleted region, which was also conserved among species of the Bacillus cereus group, has the potential for both deletion and amplification and thus might be enabling the species to flexibly control the level of bacitracin resistance for adaptive evolution.

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September 22, 2019

Comparative genomic and methylome analysis of non-virulent D74 and virulent Nagasaki Haemophilus parasuis isolates.

Haemophilus parasuis is a respiratory pathogen of swine and the etiological agent of Glässer’s disease. H. parasuis isolates can exhibit different virulence capabilities ranging from lethal systemic disease to subclinical carriage. To identify genomic differences between phenotypically distinct strains, we obtained the closed whole-genome sequence annotation and genome-wide methylation patterns for the highly virulent Nagasaki strain and for the non-virulent D74 strain. Evaluation of the virulence-associated genes contained within the genomes of D74 and Nagasaki led to the discovery of a large number of toxin-antitoxin (TA) systems within both genomes. Five predicted hemolysins were identified as unique to Nagasaki and seven putative contact-dependent growth inhibition toxin proteins were identified only in strain D74. Assessment of all potential vtaA genes revealed thirteen present in the Nagasaki genome and three in the D74 genome. Subsequent evaluation of the predicted protein structure revealed that none of the D74 VtaA proteins contain a collagen triple helix repeat domain. Additionally, the predicted protein sequence for two D74 VtaA proteins is substantially longer than any predicted Nagasaki VtaA proteins. Fifteen methylation sequence motifs were identified in D74 and fourteen methylation sequence motifs were identified in Nagasaki using SMRT sequencing analysis. Only one of the methylation sequence motifs was observed in both strains indicative of the diversity between D74 and Nagasaki. Subsequent analysis also revealed diversity in the restriction-modification systems harbored by D74 and Nagasaki. The collective information reported in this study will aid in the development of vaccines and intervention strategies to decrease the prevalence and disease burden caused by H. parasuis.

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September 22, 2019

A complete Leishmania donovani reference genome identifies novel genetic variations associated with virulence.

Leishmania donovani is responsible for visceral leishmaniasis, a neglected and lethal parasitic disease with limited treatment options and no vaccine. The study of L. donovani has been hindered by the lack of a high-quality reference genome and this can impact experimental outcomes including the identification of virulence genes, drug targets and vaccine development. We therefore generated a complete genome assembly by deep sequencing using a combination of second generation (Illumina) and third generation (PacBio) sequencing technologies. Compared to the current L. donovani assembly, the genome assembly reported within resulted in the closure over 2,000 gaps, the extension of several chromosomes up to telomeric repeats and the re-annotation of close to 15% of protein coding genes and the annotation of hundreds of non-coding RNA genes. It was possible to correctly assemble the highly repetitive A2 and Amastin virulence gene clusters. A comparative sequence analysis using the improved reference genome confirmed 70 published and identified 15 novel genomic differences between closely related visceral and atypical cutaneous disease-causing L. donovani strains providing a more complete map of genes associated with virulence and visceral organ tropism. Bioinformatic tools including protein variation effect analyzer and basic local alignment search tool were used to prioritize a list of potential virulence genes based on mutation severity, gene conservation and function. This complete genome assembly and novel information on virulence factors will support the identification of new drug targets and the development of a vaccine for L. donovani.

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September 22, 2019

Characterization of Streptococcus pluranimalium from a cattle with mastitis by whole genome sequencing and functional validation.

Streptococcus pluranimalium is a new member of the Streptococcus genus isolated from multiple different animal hosts. It has been identified as a pathogen associated with subclinical mastitis, valvular endocarditis and septicaemia in animals. Moreover, this bacterium has emerged as a new pathogen for human infective endocarditis and brain abscess. However, the patho-biological properties of S. pluranimalium remain virtually unknown. The aim of this study was to determine the complete genome sequence of S. pluranimalium strain TH11417 isolated from a cattle with mastitis, and to characterize its antimicrobial resistance, virulence, and carbon catabolism.The genome of S. pluranimalium TH11417, determined by single-molecule real-time (SMRT) sequencing, consists of 2,065,522 base pair (bp) with a G?+?C content of 38.65%, 2,007 predicted coding sequence (CDS), 58 transfer RNA (tRNA) genes and five ribosome RNA (rRNA) operons. It contains a novel ISSpl1 element (a memeber of the IS3 family) and a ?11417.1 prophage that carries the mef(A), msr(D) and lnu(C) genes. Consistently, our antimicrobial susceptibility test confirmed that S. pluranimalium TH11417 was resistant to erythromycin and lincomycin. However, this strain did not show virulence in murine pneumonia (intranasal inoculation, 107 colony forming unit – CFU) and sepsis (intraperitoneal inoculation, 107 CFU) models. Additionally, this strain is able to grow with glucose, lactose or galactose as the sole carbon source, and possesses a lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS).We reported the first whole genome sequence of S. pluranimalium isolated from a cattle with mastitis. It harbors a prophage carrying the mef(A), msr(D) and lnu(C) genes, and is avirulent in the murine infection model.

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September 22, 2019

Streptococcus suis contains multiple phase-variable methyltransferases that show a discrete lineage distribution.

Streptococcus suis is a major pathogen of swine, responsible for a number of chronic and acute infections, and is also emerging as a major zoonotic pathogen, particularly in South-East Asia. Our study of a diverse population of S. suis shows that this organism contains both Type I and Type III phase-variable methyltransferases. In all previous examples, phase-variation of methyltransferases results in genome wide methylation differences, and results in differential regulation of multiple genes, a system known as the phasevarion (phase-variable regulon). We hypothesized that each variant in the Type I and Type III systems encoded a methyltransferase with a unique specificity, and could therefore control a distinct phasevarion, either by recombination-driven shuffling between different specificities (Type I) or by biphasic on-off switching via simple sequence repeats (Type III). Here, we present the identification of the target specificities for each Type III allelic variant from S. suis using single-molecule, real-time methylome analysis. We demonstrate phase-variation is occurring in both Type I and Type III methyltransferases, and show a distinct association between methyltransferase type and presence, and population clades. In addition, we show that the phase-variable Type I methyltransferase was likely acquired at the origin of a highly virulent zoonotic sub-population.

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September 22, 2019

Excision-reintegration at a pneumococcal phase-variable restriction-modification locus drives within- and between-strain epigenetic differentiation and inhibits gene acquisition.

Phase-variation of Type I restriction-modification systems can rapidly alter the sequence motifs they target, diversifying both the epigenetic patterns and endonuclease activity within clonally descended populations. Here, we characterize the Streptococcus pneumoniae SpnIV phase-variable Type I RMS, encoded by the translocating variable restriction (tvr) locus, to identify its target motifs, mechanism and regulation of phase variation, and effects on exchange of sequence through transformation. The specificity-determining hsdS genes were shuffled through a recombinase-mediated excision-reintegration mechanism involving circular intermediate molecules, guided by two types of direct repeat. The rate of rearrangements was limited by an attenuator and toxin-antitoxin system homologs that inhibited recombinase gene transcription. Target motifs for both the SpnIV, and multiple Type II, MTases were identified through methylation-sensitive sequencing of a panel of recombinase-null mutants. This demonstrated the species-wide diversity observed at the tvr locus can likely specify nine different methylation patterns. This will reduce sequence exchange in this diverse species, as the native form of the SpnIV RMS was demonstrated to inhibit the acquisition of genomic islands by transformation. Hence the tvr locus can drive variation in genome methylation both within and between strains, and limits the genomic plasticity of S. pneumoniae.

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September 22, 2019

Genomic insights into virulence mechanisms of Leishmania donovani: evidence from an atypical strain.

Leishmaniasis is a neglected tropical disease with diverse clinical phenotypes, determined by parasite, host and vector interactions. Despite the advances in molecular biology and the availability of more Leishmania genome references in recent years, the association between parasite species and distinct clinical phenotypes remains poorly understood. We present a genomic comparison of an atypical variant of Leishmania donovani from a South Asian focus, where it mostly causes cutaneous form of leishmaniasis.Clinical isolates from six cutaneous leishmaniasis patients (CL-SL); 2 of whom were poor responders to antimony (CL-PR), and two visceral leishmaniasis patients (VL-SL) were sequenced on an Illumina MiSeq platform. Chromosome aneuploidy was observed in both groups but was more frequent in CL-SL. 248 genes differed by 2 fold or more in copy number among the two groups. Genes involved in amino acid use (LdBPK_271940) and energy metabolism (LdBPK_271950), predominated the VL-SL group with the same distribution pattern reflected in gene tandem arrays. Genes encoding amastins were present in higher copy numbers in VL-SL and CL-PR as well as being among predicted pseudogenes in CL-SL. Both chromosome and SNP profiles showed CL-SL and VL-SL to form two distinct groups. While expected heterozygosity was much higher in VL-SL, SNP allele frequency patterns did not suggest potential recent recombination breakpoints. The SNP/indel profile obtained using the more recently generated PacBio sequence did not vary markedly from that based on the standard LdBPK282A1 reference. Several genes previously associated with resistance to antimonials were observed in higher copy numbers in the analysis of CL-PR. H-locus amplification was seen in one cutaneous isolate which however did not belong to the CL-PR group.The data presented suggests that intra species variations at chromosome and gene level are more likely to influence differences in tropism as well as response to treatment, and contributes to greater understanding of parasite molecular mechanisms underpinning these differences. These findings should be substantiated with a larger sample number and expression/functional studies.

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September 22, 2019

An improved genome assembly for Larimichthys crocea reveals hepcidin gene expansion with diversified regulation and function.

Larimichthys crocea (large yellow croaker) is a type of perciform fish well known for its peculiar physiological properties and economic value. Here, we constructed an improved version of the L. crocea genome assembly, which contained 26,100 protein-coding genes. Twenty-four pseudo-chromosomes of L. crocea were also reconstructed, comprising 90% of the genome assembly. This improved assembly revealed several expansions in gene families associated with olfactory detection, detoxification, and innate immunity. Specifically, six hepcidin genes (LcHamps) were identified in L. crocea, possibly resulting from lineage-specific gene duplication. All LcHamps possessed similar genomic structures and functional domains, but varied substantially with respect to expression pattern, transcriptional regulation, and biological function. LcHamp1 was associated specifically with iron metabolism, while LcHamp2s were functionally diverse, involving in antibacterial activity, antiviral activity, and regulation of intracellular iron metabolism. This functional diversity among gene copies may have allowed L. crocea to adapt to diverse environmental conditions.

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September 22, 2019

Emergence of an extensively drug-resistant (XDR) Streptococcus pneumoniae serotype 15A by capsular switching.

Recently, we have identified an extensively drug-resistant (XDR) Streptococcus pneumoniae serotype 15A isolate from a patient with bacterial meningitis. It belonged to sequence type 8279 (ST8279), a clone identified as XDR serotype 11A isolated in South Korea. We obtained and compared the genome sequences of an XDR 15A and an XDR 11A isolate. The genomes of two XDR isolates were highly identical, except for the capsular polysaccharide (cps) locus and another small region. Capsular switching from 11A to 15A may have occurred via recombination of the cps locus. The emergence of a new XDR clone via capsular switching would be a great concern for public health and in clinical settings. Copyright © 2018 Elsevier GmbH. All rights reserved.

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