October 23, 2019  |  

Optimized CRISPR-Cas9 genome editing for Leishmania and its use to target a multigene family, induce chromosomal translocation, and study DNA break repair mechanisms.

CRISPR-Cas9-mediated genome editing has recently been adapted for Leishmania spp. parasites, the causative agents of human leishmaniasis. We have optimized this genome-editing tool by selecting for cells with CRISPR-Cas9 activity through cotargeting the miltefosine transporter gene; mutation of this gene leads to miltefosine resistance. This cotargeting strategy integrated into a triple guide RNA (gRNA) expression vector was used to delete all 11 copies of the A2 multigene family; this was not previously possible with the traditional gene-targeting method. We found that the Leishmania donovani rRNA promoter is more efficient than the U6 promoter in driving gRNA expression, and sequential transfections of the oligonucleotide donor significantly eased the isolation of edited mutants. A gRNA and Cas9 coexpression vector was developed that was functional in all tested Leishmania species, including L. donovani, L. major, and L. mexicana. By simultaneously targeting sites from two different chromosomes, all four types of targeted chromosomal translocations were generated, regardless of the polycistronic transcription direction from the parent chromosomes. It was possible to use this CRISPR system to create a single conserved amino acid substitution (A189G) mutation for both alleles of RAD51, a DNA recombinase involved in homology-directed repair. We found that RAD51 is essential for L. donovani survival based on direct observation of the death of mutants with both RAD51 alleles disrupted, further confirming that this CRISPR system can reveal gene essentiality. Evidence is also provided that microhomology-mediated end joining (MMEJ) plays a major role in double-strand DNA break repair in L. donovani. IMPORTANCELeishmania parasites cause human leishmaniasis. To accelerate characterization of Leishmania genes for new drug and vaccine development, we optimized and simplified the CRISPR-Cas9 genome-editing tool for Leishmania. We show that co-CRISPR targeting of the miltefosine transporter gene and serial transfections of an oligonucleotide donor significantly eased isolation of edited mutants. This cotargeting strategy was efficiently used to delete all 11 members of the A2 virulence gene family. This technical advancement is valuable, since there are many gene clusters and supernumerary chromosomes in the various Leishmania species and isolates. We simplified this CRISPR system by developing a gRNA and Cas9 coexpression vector which could be used to delete genes in various Leishmania species. This CRISPR system could also be used to generate specific chromosomal translocations, which will help in the study of Leishmania gene expression and transcription control. This study also provides new information about double-strand DNA break repair mechanisms in Leishmania.


October 23, 2019  |  

Structural determination of the broadly reactive anti-IGHV1-69 anti-idiotypic antibody G6 and its idiotope.

The heavy chain IGHV1-69 germline gene exhibits a high level of polymorphism and shows biased use in protective antibody (Ab) responses to infections and vaccines. It is also highly expressed in several B cell malignancies and autoimmune diseases. G6 is an anti-idiotypic monoclonal Ab that selectively binds to IGHV1-69 heavy chain germline gene 51p1 alleles that have been implicated in these Ab responses and disease processes. Here, we determine the co-crystal structure of humanized G6 (hG6.3) in complex with anti-influenza hemagglutinin stem-directed broadly neutralizing Ab D80. The core of the hG6.3 idiotope is a continuous string of CDR-H2 residues starting with M53 and ending with N58. G6 binding studies demonstrate the remarkable breadth of binding to 51p1 IGHV1-69 Abs with diverse CDR-H3, light chain, and antigen binding specificities. These studies detail the broad expression of the G6 cross-reactive idiotype (CRI) that further define its potential role in precision medicine. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.


October 23, 2019  |  

Transmission, evolution, and endogenization: Lessons learned from recent retroviral invasions.

Viruses of the subfamily Orthoretrovirinaeare defined by the ability to reverse transcribe an RNA genome into DNA that integrates into the host cell genome during the intracellular virus life cycle. Exogenous retroviruses (XRVs) are horizontally transmitted between host individuals, with disease outcome depending on interactions between the retrovirus and the host organism. When retroviruses infect germ line cells of the host, they may become endogenous retroviruses (ERVs), which are permanent elements in the host germ line that are subject to vertical transmission. These ERVs sometimes remain infectious and can themselves give rise to XRVs. This review integrates recent developments in the phylogenetic classification of retroviruses and the identification of retroviral receptors to elucidate the origins and evolution of XRVs and ERVs. We consider whether ERVs may recurrently pressure XRVs to shift receptor usage to sidestep ERV interference. We discuss how related retroviruses undergo alternative fates in different host lineages after endogenization, with koala retrovirus (KoRV) receiving notable interest as a recent invader of its host germ line. KoRV is heritable but also infectious, which provides insights into the early stages of germ line invasions as well as XRV generation from ERVs. The relationship of KoRV to primate and other retroviruses is placed in the context of host biogeography and the potential role of bats and rodents as vectors for interspecies viral transmission. Combining studies of extant XRVs and “fossil” endogenous retroviruses in koalas and other Australasian species has broadened our understanding of the evolution of retroviruses and host-retrovirus interactions. Copyright © 2017 American Society for Microbiology.


September 22, 2019  |  

Transcriptional adaptations during long-term persistence of Staphylococcus aureus in the airways of a cystic fibrosis patient.

The lungs of Cystic fibrosis (CF) patients are often colonized and/or infected by Staphylococcus aureus for years, mostly by one predominant clone. For long-term survival in this environment, S. aureus needs to adapt during its interactions with host factors, antibiotics, and other pathogens. Here, we study long-term transcriptional as well as genomic adaptations of an isogenic pair of S. aureus isolates from a single patient using RNA sequencing (RNA-Seq) and whole genome sequencing (WGS). Mimicking in vivo conditions, we cultivated the S. aureus isolates using artificial sputum medium before harvesting RNA for subsequent analysis. We confirmed our RNA-Seq data using quantitative real-time (qRT)-PCR and additionally investigated intermediate isolates from the same patient representing in total 13.2 years of persistence in the CF airways. Comparative RNA-Seq analysis of the first and the last (“late”) isolate revealed significant differences in the late isolate after 13.2 years of persistence. Of the 2545 genes expressed in both isolates that were cultivated aerobically, 256 genes were up- and 161 were down-regulated with a minimum 2-fold change (2f). Focusing on 25 highly (=8f) up- (n=9) or down- (n=16) regulated genes, we identified several genes encoding for virulence factors involved in immune evasion, bacterial spread or secretion (e.g. spa, sak, and esxA). Moreover, these genes displayed similar expression trends under aerobic, microaerophilic and anaerobic conditions. Further qRT-PCR-experiments of highly up- or down-regulated genes within intermediate S. aureus isolates resulted in different gene expression patterns over the years. Using sequencing analysis of the differently expressed genes and their upstream regions in the late S. aureus isolate resulted in only few genomic alterations. Comparative transcriptomic analysis revealed adaptive changes affecting mainly genes involved in host-pathogen interaction. Although the underlying mechanisms were not known, our results suggest adaptive processes beyond genomic mutations triggered by local factors rather than by activation of global regulators. Copyright © 2014 The Authors. Published by Elsevier GmbH.. All rights reserved.


September 22, 2019  |  

Advantages of genome sequencing by long-read sequencer using SMRT technology in medical area.

PacBio RS II is the first commercialized third-generation DNA sequencer able to sequence a single molecule DNA in real-time without amplification. PacBio RS II’s sequencing technology is novel and unique, enabling the direct observation of DNA synthesis by DNA polymerase. PacBio RS II confers four major advantages compared to other sequencing technologies: long read lengths, high consensus accuracy, a low degree of bias, and simultaneous capability of epigenetic characterization. These advantages surmount the obstacle of sequencing genomic regions such as high/low G+C, tandem repeat, and interspersed repeat regions. Moreover, PacBio RS II is ideal for whole genome sequencing, targeted sequencing, complex population analysis, RNA sequencing, and epigenetics characterization. With PacBio RS II, we have sequenced and analyzed the genomes of many species, from viruses to humans. Herein, we summarize and review some of our key genome sequencing projects, including full-length viral sequencing, complete bacterial genome and almost-complete plant genome assemblies, and long amplicon sequencing of a disease-associated gene region. We believe that PacBio RS II is not only an effective tool for use in the basic biological sciences but also in the medical/clinical setting.


September 22, 2019  |  

Complete genome sequence of multidrug-resistant Staphylococcus cohnii ssp. urealyticus strain SNUDS-2 isolated from farmed duck, Republic of Korea.

Staphylococcus cohnii has become increasingly recognized as a potential pathogen of clinically significant nosocomial and farm animal infections. This study was designed to determine the genome of a multidrug-resistant S. cohnii subsp. urealyticus strain SNUDS-2 isolated from a farmed duck in Korea.Genomic DNA was sequenced using the PacBio RS II system. The complete genome was annotated and the presence of antimicrobial resistance and virulence genes were identified.The annotated 2,625,703 bp genome contained various antimicrobial resistance genes conferring resistance to ß-lactam, aminoglycosides, fluoroquinolones, phenicols and trimethoprim. The virulence-associated three synergistic hemolysins have been identified in the strain.To the best of our knowledge, this is the first complete genome of S. cohnii, and will provide important insights into the biodiversity of CoNS and valuable information for the control of this emerging pathogen. Copyright © 2017 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.


September 22, 2019  |  

Influenza virus infection causes global RNAPII termination defects.

Viral infection perturbs host cells and can be used to uncover regulatory mechanisms controlling cellular responses and susceptibility to infections. Using cell biological, biochemical, and genetic tools, we reveal that influenza A virus (IAV) infection induces global transcriptional defects at the 3′ ends of active host genes and RNA polymerase II (RNAPII) run-through into extragenic regions. Deregulated RNAPII leads to expression of aberrant RNAs (3′ extensions and host-gene fusions) that ultimately cause global transcriptional downregulation of physiological transcripts, an effect influencing antiviral response and virulence. This phenomenon occurs with multiple strains of IAV, is dependent on influenza NS1 protein, and can be modulated by SUMOylation of an intrinsically disordered region (IDR) of NS1 expressed by the 1918 pandemic IAV strain. Our data identify a strategy used by IAV to suppress host gene expression and indicate that polymorphisms in IDRs of viral proteins can affect the outcome of an infection.


September 22, 2019  |  

Next-generation sequencing for pathogen detection and identification

Over the past decade, the field of genomics has seen such drastic improvements in sequencing chemistries that high-throughput sequencing, or next-generation sequencing (NGS), is being applied to generate data across many disciplines. NGS instruments are becoming less expensive, faster, and smaller, and therefore are being adopted in an increasing number of laboratories, including clinical laboratories. Thus far, clinical use of NGS has been mostly focused on the human genome, for purposes such as characterizing the molecular basis of cancer or for diagnosing and understanding the basis of rare genetic disorders. There are, however, an increasing number of examples whereby NGS is employed to discover novel pathogens, and these cases provide precedent for the use of NGS in microbial diagnostics. NGS has many advantages over traditional microbial diagnostic methods, such as unbiased rather than pathogen-specific protocols, ability to detect fastidious or non-culturable organisms, and ability to detect co-infections. One of the most impressive advantages of NGS is that it requires little or no prior knowledge of the pathogen, unlike many other diagnostic assays; therefore for pathogen discovery, NGS is very valuable. However, despite these advantages, there are challenges involved in implementing NGS for routine clinical microbiological diagnosis. We discuss these advantages and challenges in the context of recently described research studies.


September 22, 2019  |  

Single molecule real-time (SMRT) sequencing comes of age: applications and utilities for medical diagnostics.

Short read massive parallel sequencing has emerged as a standard diagnostic tool in the medical setting. However, short read technologies have inherent limitations such as GC bias, difficulties mapping to repetitive elements, trouble discriminating paralogous sequences, and difficulties in phasing alleles. Long read single molecule sequencers resolve these obstacles. Moreover, they offer higher consensus accuracies and can detect epigenetic modifications from native DNA. The first commercially available long read single molecule platform was the RS system based on PacBio’s single molecule real-time (SMRT) sequencing technology, which has since evolved into their RSII and Sequel systems. Here we capsulize how SMRT sequencing is revolutionizing constitutional, reproductive, cancer, microbial and viral genetic testing.© The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research.


September 22, 2019  |  

Indoleacrylic acid produced by commensal Peptostreptococcus species suppresses inflammation.

Host factors in the intestine help select for bacteria that promote health. Certain commensals can utilize mucins as an energy source, thus promoting their colonization. However, health conditions such as inflammatory bowel disease (IBD) are associated with a reduced mucus layer, potentially leading to dysbiosis associated with this disease. We characterize the capability of commensal species to cleave and transport mucin-associated monosaccharides and identify several Clostridiales members that utilize intestinal mucins. One such mucin utilizer, Peptostreptococcus russellii, reduces susceptibility to epithelial injury in mice. Several Peptostreptococcus species contain a gene cluster enabling production of the tryptophan metabolite indoleacrylic acid (IA), which promotes intestinal epithelial barrier function and mitigates inflammatory responses. Furthermore, metagenomic analysis of human stool samples reveals that the genetic capability of microbes to utilize mucins and metabolize tryptophan is diminished in IBD patients. Our data suggest that stimulating IA production could promote anti-inflammatory responses and have therapeutic benefits. Copyright © 2017 Elsevier Inc. All rights reserved.


September 22, 2019  |  

Comparative genome and transcriptome analysis reveals distinctive surface characteristics and unique physiological potentials of Pseudomonas aeruginosa ATCC 27853.

Pseudomonas aeruginosa ATCC 27853 was isolated from a hospital blood specimen in 1971 and has been widely used as a model strain to survey antibiotics susceptibilities, biofilm development, and metabolic activities of Pseudomonas spp.. Although four draft genomes of P. aeruginosa ATCC 27853 have been sequenced, the complete genome of this strain is still lacking, hindering a comprehensive understanding of its physiology and functional genome.Here we sequenced and assembled the complete genome of P. aeruginosa ATCC 27853 using the Pacific Biosciences SMRT (PacBio) technology and Illumina sequencing platform. We found that accessory genes of ATCC 27853 including prophages and genomic islands (GIs) mainly contribute to the difference between P. aeruginosa ATCC 27853 and other P. aeruginosa strains. Seven prophages were identified within the genome of P. aeruginosa ATCC 27853. Of the predicted 25 GIs, three contain genes that encode monoxoygenases, dioxygenases and hydrolases that could be involved in the metabolism of aromatic compounds. Surveying virulence-related genes revealed that a series of genes that encode the B-band O-antigen of LPS are lacking in ATCC 27853. Distinctive SNPs in genes of cellular adhesion proteins such as type IV pili and flagella biosynthesis were also observed in this strain. Colony morphology analysis confirmed an enhanced biofilm formation capability of ATCC 27853 on solid agar surface compared to Pseudomonas aeruginosa PAO1. We then performed transcriptome analysis of ATCC 27853 and PAO1 using RNA-seq and compared the expression of orthologous genes to understand the functional genome and the genomic details underlying the distinctive colony morphogenesis. These analyses revealed an increased expression of genes involved in cellular adhesion and biofilm maturation such as type IV pili, exopolysaccharide and electron transport chain components in ATCC 27853 compared with PAO1. In addition, distinctive expression profiles of the virulence genes lecA, lasB, quorum sensing regulators LasI/R, and the type I, III and VI secretion systems were observed in the two strains.The complete genome sequence of P. aeruginosa ATCC 27853 reveals the comprehensive genetic background of the strain, and provides genetic basis for several interesting findings about the functions of surface associated proteins, prophages, and genomic islands. Comparative transcriptome analysis of P. aeruginosa ATCC 27853 and PAO1 revealed several classes of differentially expressed genes in the two strains, underlying the genetic and molecular details of several known and yet to be explored morphological and physiological potentials of P. aeruginosa ATCC 27853.


September 22, 2019  |  

The expressed portion of the barley genome

In this chapter, we refer to the expressed portion of the barley genome as the relatively small fraction of the total cellular DNA that either contains the genes that ultimately produce proteins, or that directly/indirectly controls the level, location and/or timing of when these genes are expressed and proteins are produced. We start by describing the dynamics of tissue and time-dependent gene expression and how common patterns across multiple samples can provide clues about gene networks involved in common biological processes. We then describe some of the complexities of how a single mRNA template can be differentially processed by alternative splicing to generate multiple different proteins or provide a mechanism to regulate the amount of functional gene product in a cell at a given point in time. We extend our analysis, using a number of biological examples, to address how diverse families of small non-coding microRNAs specifically regulate gene expression, and complete our appraisal by looking at the physical/molecular environment around genes that can result in either the promotion or repression of gene expression. We conclude by assessing some of the issues that remain around our ability to fully exploit the depth and power of current approaches for analysing gene expression and propose improvements that could be made using new but available sequencing and bioinformatics technologies.


September 22, 2019  |  

PacBio sequencing and its applications.

Single-molecule, real-time sequencing developed by Pacific BioSciences offers longer read lengths than the second-generation sequencing (SGS) technologies, making it well-suited for unsolved problems in genome, transcriptome, and epigenetics research. The highly-contiguous de novo assemblies using PacBio sequencing can close gaps in current reference assemblies and characterize structural variation (SV) in personal genomes. With longer reads, we can sequence through extended repetitive regions and detect mutations, many of which are associated with diseases. Moreover, PacBio transcriptome sequencing is advantageous for the identification of gene isoforms and facilitates reliable discoveries of novel genes and novel isoforms of annotated genes, due to its ability to sequence full-length transcripts or fragments with significant lengths. Additionally, PacBio’s sequencing technique provides information that is useful for the direct detection of base modifications, such as methylation. In addition to using PacBio sequencing alone, many hybrid sequencing strategies have been developed to make use of more accurate short reads in conjunction with PacBio long reads. In general, hybrid sequencing strategies are more affordable and scalable especially for small-size laboratories than using PacBio Sequencing alone. The advent of PacBio sequencing has made available much information that could not be obtained via SGS alone. Copyright © 2015 The Authors. Production and hosting by Elsevier Ltd.. All rights reserved.


September 22, 2019  |  

Complete genome sequence of Enterobacter cloacae R11 reveals multiple genes potentially associated with high-level polymyxin E resistance.

Enterobacter cloacae strain R11 is a multidrug-resistant bacterium isolated from sewage water near a swine feedlot in China. Strain R11 can survive in medium containing up to 192 µg/mL polymyxin E, indicating a tolerance for this antibiotic that is significantly higher than that reported for other gram-negative bacteria. In this study, conjugation experiments showed that partial polymyxin E resistance could be transferred from strain R11 to Escherichia coli strain 25922, revealing that some genes related to polymyxin E resistance are plasmid-based. The complete genome sequence of this strain was determined, yielding a total of 4?993?008 bp (G+C content, 53.15%) and 4908 genes for the circular chromosome and 4 circular plasmids. Genome analysis revealed a total of 73 putative antibiotic resistance genes, including several polymyxin E resistance genes and genes potentially involved in multidrug resistance. These data provide insights into the genetic basis of the polymyxin E resistance and multidrug resistance of E. cloacae.


September 22, 2019  |  

Extensively drug-resistant Escherichia coli sequence type 1642 carrying an IncX3 plasmid containing the blaKPC-2 gene associated with transposon Tn4401a.

Extensively drug-resistant (XDR) Enterobacteriaceae carrying the bla(KPC) gene have emerged as a major global therapeutic concern. The purpose of this study was to analyze the complete sequences of plasmids from KPC-2 carbapenemase-producing XDR Escherichia coli sequence type (ST) 1642 isolates.We performed antimicrobial susceptibility testing, PCR, multilocus sequence typing (MLST), and whole-genome sequencing to characterize the plasmid-mediated KPC-2-producing E. coli clinical isolates.The isolates were resistant to most available antibiotics, including meropenem, ampicillin, ceftriaxone, gentamicin, and ciprofloxacin, but susceptible to tigecycline and colistin. The isolates were identified as the rare ST1642 by MLST. The isolates carried four plasmids: the first 69-kb conjugative IncX3 plasmid harbors bla(KPC-2) within a truncated Tn4401a transposon and bla(SHV-11) with duplicated conjugative elements. The second 142-kb plasmid with a multireplicon consisting of IncQ, IncFIA, and IncIB carries bla(TEM-1b) and two class 1 integrons. This plasmid also harbors a wide variety of additional antimicrobial resistance genes including aadA5, dfrA17, mph(A), sul1, tet(B), aac(3′)-IId, strA, strB, and sul2.The complete sequence analysis of plasmids from an XDR E. coli strain related to persistent infection showed the coexistence of a bla(KPC-2)-carrying IncX3-type plasmid and a class 1 integron-harboring multireplicon, suggesting its potential to cause outbreaks. Of additional clinical significance, the rare ST1642, identified in a cat, could constitute the source of human infection.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.