Showing papers on "Replicon published in 2019"
TL;DR: A literature search for English research papers, published between 2013 and 2018, reporting on plasmid‐mediated carbapenem resistance saw a rise in both carbapanemase types and associated plasmID replicon groups was seen, with China, Canada, and the United States recording a higher increase than other countries.
Abstract: Carbapenem-resistant Enterobacteriaceae (CRE) have been listed by the WHO as high-priority pathogens owing to their high association with mortalities and morbidities. Resistance to multiple β-lactams complicates effective clinical management of CRE infections. Using plasmid typing methods, a wide distribution of plasmid replicon groups has been reported in CREs around the world, including IncF, N, X, A/C, L/M, R, P, H, I, and W. We performed a literature search for English research papers, published between 2013 and 2018, reporting on plasmid-mediated carbapenem resistance. A rise in both carbapenemase types and associated plasmid replicon groups was seen, with China, Canada, and the United States recording a higher increase than other countries. blaKPC was the most prevalent, except in Angola and the Czech Republic, where OXA-181 (n = 50, 88%) and OXA-48-like (n = 24, 44%) carbapenemases were most prevalent, respectively; blaKPC-2/3 accounted for 70% (n = 956) of all reported carbapenemases. IncF plasmids were found to be responsible for disseminating different antibiotic resistance genes worldwide, accounting for almost 40% (n = 254) of plasmid-borne carbapenemases. blaCTX-M , blaTEM , blaSHV , blaOXA-1/9 , qnr, and aac-(6')-lb were mostly detected concurrently with carbapenemases. Most reported plasmids were conjugative but not present in multiple countries or species, suggesting limited interspecies and interboundary transmission of a common plasmid. A major limitation to effective characterization of plasmid evolution was the use of PCR-based instead of whole-plasmid sequencing-based plasmid typing.
120 citations
TL;DR: It is reported that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides and dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types.
Abstract: Zinc finger antiviral protein (ZAP) is a powerful restriction factor for viruses with elevated CpG dinucleotide frequencies. We report that ZAP similarly mediates antiviral restriction against echovirus 7 (E7) mutants with elevated frequencies of UpA dinucleotides. Attenuation of both CpG- and UpA-high viruses and replicon mutants was reversed in ZAP k/o cell lines, and restored by plasmid-derived reconstitution of expression in k/o cells. In pull-down assays, ZAP bound to viral RNA transcripts with either CpG- and UpA-high sequences inserted in the R2 region. We found no evidence that attenuation of CpG- or UpA-high mutants was mediated through either translation inhibition or accelerated RNA degradation. Reversal of the attenuation of CpG-high, and UpA-high E7 viruses and replicons was also achieved through knockout of RNAseL and oligodenylate synthetase 3 (OAS3), but not OAS1. WT levels of replication of CpG- and UpA-high mutants were observed in OAS3 k/o cells despite abundant expression of ZAP, indicative of synergy or complementation of these hitherto unconnected pathways. The dependence on expression of ZAP, OAS3 and RNAseL for CpG/UpA-mediated attenuation and the variable and often low level expression of these pathway proteins in certain cell types, such as those of the central nervous system, has implications for the use of CpG-elevated mutants as attenuated live vaccines against neurotropic viruses.
67 citations
TL;DR: Horizontal transfer mediated by IncFII-like plasmids plays an important role in the pandemic expansion of blaKPC-2 among K. pneumoniae ST11 in China.
55 citations
TL;DR: Data suggest replicon delivery of Env immunogens may be a promising avenue for HIV vaccine development.
49 citations
TL;DR: It is proposed that the alphavirus unique domain (AUD) of nsP3 is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.
Abstract: Chikungunya virus (CHIKV) is a re-emerging Alphavirus causing fever, joint pain, skin rash, arthralgia, and occasionally death. Antiviral therapies and/or effective vaccines are urgently required. CHIKV biology is poorly understood, in particular the functions of the non-structural protein 3 (nsP3). Here we present the results of a mutagenic analysis of the alphavirus unique domain (AUD) of nsP3. Informed by the structure of the Sindbis virus AUD and an alignment of amino acid sequences of multiple alphaviruses, a series of mutations in the AUD were generated in a CHIKV sub-genomic replicon. This analysis revealed an essential role for the AUD in CHIKV RNA replication, with mutants exhibiting species- and cell-type specific phenotypes. To test if the AUD played a role in other stages of the virus lifecycle, the mutants were analysed in the context of infectious CHIKV. This analysis indicated that the AUD was also required for virus assembly. In particular, one mutant (P247A/V248A) exhibited a dramatic reduction in production of infectious virus. This phenotype was shown to be due to a block in transcription of the subgenomic RNA leading to reduced synthesis of the structural proteins and a concomitant reduction in virus production. This phenotype could be further explained by both a reduction in the binding of the P247A/V248A mutant nsP3 to viral genomic RNA in vivo, and the reduced affinity of the mutant AUD for the subgenomic promoter RNA in vitro. We propose that the AUD is a pleiotropic protein domain, with multiple functions during CHIKV RNA synthesis.
41 citations
TL;DR: An in vitro evolution strategy was developed and six mutations in nonstructural proteins of Venezuelan equine encephalitis replicon that promoted subgenome expression in cells were identified that may be useful for improving RNA therapeutics for vaccination, cancer immunotherapy, and gene therapy.
Abstract: Self-replicating (replicon) RNA is a promising new platform for gene therapy, but applications are still limited by short persistence of expression in most cell types and low levels of transgene expression in vivo. To address these shortcomings, we developed an in vitro evolution strategy and identified six mutations in nonstructural proteins (nsPs) of Venezuelan equine encephalitis (VEE) replicon that promoted subgenome expression in cells. Two mutations in nsP2 and nsP3 enhanced transgene expression, while three mutations in nsP3 regulated this expression. Replicons containing the most effective mutation combinations showed enhanced duration and cargo gene expression in vivo. In comparison to wildtype replicon, mutants expressing IL-2 injected into murine B16F10 melanoma showed 5.5-fold increase in intratumoral IL-2 and 2.1-fold increase in infiltrating CD8 T cells, resulting in significantly slowed tumor growth. Thus, these mutant replicons may be useful for improving RNA therapeutics for vaccination, cancer immunotherapy, and gene therapy.
40 citations
TL;DR: This work quantified genetic similarity between more than 10,000 bacterial Plasmids and reconstructed a network based on their shared k-mer content and assigned plasmids into cliques which are highly correlated with plasmid gene content, bacterial host range, GC content, as well as replicon and mobility (MOB) type classifications.
Abstract: Many bacteria can exchange genetic material through horizontal gene transfer (HGT) mediated by plasmids and plasmid-borne transposable elements. Here, we study the population structure and dynamics of over 10,000 bacterial plasmids, by quantifying their genetic similarities and reconstructing a network based on their shared k-mer content. We use a community detection algorithm to assign plasmids into cliques, which correlate with plasmid gene content, bacterial host range, GC content, and existing classifications based on replicon and mobility (MOB) types. Further analysis of plasmid population structure allows us to uncover candidates for yet undescribed replicon genes and to identify transposable elements as the main drivers of HGT at broad phylogenetic scales. Our work illustrates the potential of network-based analyses of the bacterial "mobilome" and opens up the prospect of a natural, exhaustive classification framework for bacterial plasmids.
35 citations
TL;DR: Together these studies indicate drug repurposing may be a promising pathway for development of antivirals against HEV infection.
Abstract: Globally, hepatitis E virus (HEV) causes significant morbidity and mortality each year. Despite this burden, there are no specific antivirals available to treat HEV patients, and the only licensed vaccine is not available outside China. Ribavirin and alpha interferon are used to treat chronic HEV infections; however, severe side effects and treatment failure are commonly reported. Therefore, this study aimed to identify potential antivirals for further development to combat HEV infection. We selected 16 compounds from the nucleoside and nonnucleoside antiviral classes that range in developmental status from late preclinical to FDA approved and evaluated them as potential antivirals for HEV infection, using genotype 1 replicon luminescence studies and replicon RNA quantification. Two potent inhibitors of HEV replication included NITD008 (half-maximal effective concentration [EC50], 0.03 μM; half-maximal cytotoxic concentration [CC50], >100 μM) and GPC-N114 (EC50, 1.07 μM, CC50, >100 μM), and both drugs reduced replicon RNA levels in cell culture (>50% reduction with either 10 μM GPC-N114 or 2.50 μM NITD008). Furthermore, GPC-N114 and NITD008 were synergistic in combinational treatment (combination index, 0.4) against HEV replication, allowing for dose reduction indices of 20.42 and 8.82 at 50% inhibition, respectively. Sofosbuvir has previously exhibited mixed results against HEV as an antiviral, both in vitro and in a few clinical applications; however, in this study it was effective against the HEV genotype 1 replicon (EC50, 1.97 μM; CC50, >100 μM) and reduced replicon RNA levels (47.2% reduction at 10 μM). Together these studies indicate drug repurposing may be a promising pathway for development of antivirals against HEV infection.
35 citations
TL;DR: Overall, these studies demonstrate that VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro.
Abstract: Many mRNA-based vaccines have been investigated for their specific potential to activate dendritic cells (DCs), the highly-specialized antigen-presenting cells of the immune system that play a key role in inducing effective CD4+ and CD8+ T-cell responses. In this paper we report a new vaccine/gene delivery platform that demonstrates the benefits of using a self-amplifying ("replicon") mRNA that is protected in a viral-protein capsid. Purified capsid protein from the plant virus Cowpea Chlorotic Mottle Virus (CCMV) is used to in vitro assemble monodisperse virus-like particles (VLPs) containing reporter proteins (e.g., Luciferase or eYFP) or the tandem-repeat model antigen SIINFEKL in RNA gene form, coupled to the RNA-dependent RNA polymerase from the Nodamura insect virus. Incubation of immature DCs with these VLPs results in increased activation of maturation markers - CD80, CD86 and MHC-II - and enhanced RNA replication levels, relative to incubation with unpackaged replicon mRNA. Higher RNA uptake/replication and enhanced DC activation were detected in a dose-dependent manner when the CCMV-VLPs were pre-incubated with anti-CCMV antibodies. In all experiments the expression of maturation markers correlates with the RNA levels of the DCs. Overall, these studies demonstrate that: VLP protection enhances mRNA uptake by DCs; coupling replicons to the gene of interest increases RNA and protein levels in the cell; and the presence of anti-VLP antibodies enhances mRNA levels and activation of DCs in vitro. Finally, preliminary in vivo experiments involving mouse vaccinations with SIINFEKL-replicon VLPs indicate a small but significant increase in antigen-specific T cells that are doubly positive for IFN and TFN induction.
33 citations
TL;DR: Tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.
Abstract: Hepatitis E virus (HEV) is one of the most common causes of acute hepatitis and jaundice in the world. Current understanding of the molecular virology and pathogenesis of hepatitis E is incomplete, due particularly to the limited availability of functional tools. Here, we report the development of tagged HEV genomes as a novel tool to investigate the viral life cycle. A selectable subgenomic HEV replicon was subjected to random 15-nucleotide sequence insertion using transposon-based technology. Viable insertions in the open reading frame 1 (ORF1) protein were selected in a hepatoblastoma cell line. Functional insertion sites were identified downstream of the methyltransferase domain, in the hypervariable region (HVR), and between the helicase and RNA-dependent RNA polymerase domains. HEV genomes harboring a hemagglutinin (HA) epitope tag or a small luciferase (NanoLuc) in the HVR were found to be fully functional and to allow the production of infectious virus. NanoLuc allowed quantitative monitoring of HEV infection and replication by luciferase assay. The use of HA-tagged replicons and full-length genomes allowed localization of putative sites of HEV RNA replication by the simultaneous detection of viral RNA by fluorescence in situ hybridization and of ORF1 protein by immunofluorescence. Candidate HEV replication complexes were found in cytoplasmic dot-like structures which partially overlapped ORF2 and ORF3 proteins as well as exosomal markers. Hence, tagged HEV genomes yield new insights into the viral life cycle and should allow further investigation of the structure and composition of the viral replication complex.IMPORTANCE Hepatitis E virus (HEV) infection is an important cause of acute hepatitis and may lead to chronic infection in immunocompromised patients. Knowledge of the viral life cycle is incomplete due to the limited availability of functional tools. In particular, low levels of expression of the ORF1 protein or limited sensitivity of currently available antibodies or both limit our understanding of the viral replicase. Here, we report the successful establishment of subgenomic HEV replicons and full-length genomes harboring an epitope tag or a functional reporter in the ORF1 protein. These novel tools should allow further characterization of the HEV replication complex and to improve our understanding of the viral life cycle.
32 citations
TL;DR: It is shown that multiple replicons in coordination with maintenance and conjugation regions of various origins would maintain a broad host range and a stable replication at a steady-state plasmid copy number.
Abstract: Background IncR, IncFII, IncpA1763-KPC, and IncN1 plasmids have been increasingly found among Enterobacteriaceae species, but plasmids with hybrid structures derived from the above-mentioned incompatibility groups have not yet been described. Methods Plasmids p721005-KPC, p504051-KPC, and pA3295-KPC were fully sequenced and compared with previously sequenced related plasmids pHN84KPC (IncR), pKPHS2 (IncFIIK), pKOX_NDM1 (IncFIIY), pHN7A8 (IncFIIpHN7A8), and R46 (IncN1). Results The backbone of p721005-KPC/p504051-KPC was a hybrid of the entire 10-kb IncR-type backbone from pHN84KPC, the entire 64.3-kb IncFIIK-type maintenance, and conjugal transfer regions from pKPHS2, a 15.5-kb IncFIIY-type maintenance region from pKOX_NDM1 and a 5.6-kb IncpA1763-KPC-type backbone region from pA1763-KPC, and it contained a primary IncR replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. The backbone of pA3295-KPC was a hybrid of a 7.2-kb IncFIIpHN7A8-type backbone region from pHN7A8, the almost entire 33.3-kb IncN1-type maintenance and conjugal transfer regions highly similar to R46, a 26.2-kb IncFIIK-type maintenance regions from pKPHS2, the above 15.5-kb IncFIIY-type maintenance region, and the above 5.6-kb IncpA1763-KPC-type backbone region, and it contained a primary Inc-FIIpHN7A8 replicon and two auxiliary IncpA1763-KPC and IncN1 replicons. Each of p721005-KPC, p504051-KPC, and pA3295-KPC acquired a wealth of accessory modules, carrying a range of intact and residue mobile elements (such as insertion sequences, unit transposons, and integrons) and resistance markers (such as bla KPC, tetA, dfrA, and qnr). Conclusion In each of p721005-KPC, p504051-KPC, and pA3295-KPC, multiple replicons in coordination with maintenance and conjugation regions of various origins would maintain a broad host range and a stable replication at a steady-state plasmid copy number.
TL;DR: This study confirmed that the widely used temperature-sensitive pSG5 replicon is indeed not suitable for editing modular polyketide synthase (PKS) genes due to causing unpredicted gene recombination and developed a new CRISPR-Cas9 system pMWCas9, which was successfully used to delete/replace erythromycin PKS and other biosynthetic genes in Saccharopolyspora and Streptomyces sp.
TL;DR: It is demonstrated that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome.
Abstract: Chikungunya virus (CHIKV) is a re-emerging, pathogenic Alphavirus transmitted to humans by Aedes spp. mosquitoes. We have mapped the RNA structure of the 5′ region of the CHIKV genome using selective 2′-hydroxyl acylation analysed by primer extension (SHAPE) to investigate intramolecular base-pairing at single-nucleotide resolution. Taking a structure-led reverse genetic approach, in both infectious virus and sub-genomic replicon systems, we identified six RNA replication elements essential to efficient CHIKV genome replication - including novel elements, either not previously analysed in other alphaviruses or specific to CHIKV. Importantly, through a reverse genetic approach we demonstrate that the replication elements function within the positive-strand genomic copy of the virus genome, in predominantly structure-dependent mechanisms during efficient replication of the CHIKV genome. Comparative analysis in human and mosquito-derived cell lines reveal that a novel element within the 5′UTR is essential for efficient replication in both host systems, while those in the adjacent nsP1 encoding region are specific to either vertebrate or invertebrate host cells. In addition to furthering our knowledge of fundamental aspects of the molecular virology of this important human pathogen, we foresee that results from this study will be important for rational design of a genetically stable attenuated vaccine.
TL;DR: The authors provide the structure of the HBx BH3-like domain and Bcl-xL, which shows an unusual mode of interaction, and identify a short peptide that inhibits HBV replication in cultured human hepatic cells.
Abstract: Hepatitis B virus (HBV) X protein, HBx, interacts with anti-apoptotic Bcl-2 and Bcl-xL proteins through its BH3-like motif to promote HBV replication and cytotoxicity. Here we report the crystal structure of HBx BH3-like motif in complex with Bcl-xL where the BH3-like motif adopts a short α-helix to snuggle into a hydrophobic pocket in Bcl-xL via its noncanonical Trp120 residue and conserved Leu123 residue. This binding pocket is ~2 A away from the canonical BH3-only binding pocket in structures of Bcl-xL with proapoptotic BH3-only proteins. Mutations altering Trp120 and Leu123 in HBx impair its binding to Bcl-xL in vitro and HBV replication in vivo, confirming the importance of this motif to HBV. A HBx BH3-like peptide, HBx-aa113-135, restores HBV replication from a HBx-null HBV replicon, while a shorter peptide, HBx-aa118-127, inhibits HBV replication. These results provide crucial structural and functional insights into drug designs for inhibiting HBV replication and treating HBV patients. Hepatitis B virus X protein (HBx) binds anti-apoptotic Bcl-xL through its BH3-like motif to promote viral replication. Here, the authors provide the structure of the HBx BH3-like domain and Bcl-xL, which shows an unusual mode of interaction, and identify a short peptide that inhibits HBV replication in cultured human hepatic cells.
TL;DR: It is suggested that PLE DNA replication is one of multiple mechanisms contributing to ICP1 restriction, and expression of the PLE initiation factor was not sufficient for PLE replication in the absence of phage.
Abstract: Phage-inducible chromosomal island-like elements (PLEs) are bacteriophage satellites found in Vibrio cholerae. PLEs parasitize the lytic phage ICP1, excising from the bacterial chromosome, replicating, and mobilizing to new host cells following cell lysis. PLEs protect their host cell populations by completely restricting the production of ICP1 progeny. Previously, it was found that ICP1 replication was reduced during PLE(+) infection. Despite robust replication of the PLE genome, relatively few transducing units are produced. We investigated if PLE DNA replication itself is antagonistic to ICP1 replication. Here we identify key constituents of PLE replication and assess their role in interference of ICP1. PLE encodes a RepA_N initiation factor that is sufficient to drive replication from the PLE origin of replication during ICP1 infection. In contrast to previously characterized bacteriophage satellites, expression of the PLE initiation factor was not sufficient for PLE replication in the absence of phage. Replication of PLE was necessary for interference of ICP1 DNA replication, but replication of a minimalized PLE replicon was not sufficient for ICP1 DNA replication interference. Despite restoration of ICP1 DNA replication, non-replicating PLE remained broadly inhibitory against ICP1. These results suggest that PLE DNA replication is one of multiple mechanisms contributing to ICP1 restriction.
TL;DR: These findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster the understanding of EBOv and NNSVs.
Abstract: Ebola virus (EBOV) is a non-segmented, negative-sense RNA virus (NNSV) in the family Filoviridae, and is recognized as one of the most lethal pathogens in the planet. For RNA viruses, cellular or virus-encoded RNA helicases play pivotal roles in viral life cycles by remodelling viral RNA structures and/or unwinding viral dsRNA produced during replication. However, no helicase or helicase-like activity has ever been found to associate with any NNSV-encoded proteins, and it is unknown whether the replication of NNSVs requires the participation of any viral or cellular helicase. Here, we show that despite of containing no conserved NTPase/helicase motifs, EBOV VP35 possesses the NTPase and helicase-like activities that can hydrolyse all types of NTPs and unwind RNA helices in an NTP-dependent manner, respectively. Moreover, guanidine hydrochloride, an FDA-approved compound and inhibitor of certain viral helicases, inhibited the NTPase and helicase-like activities of VP35 as well as the replication/transcription of an EBOV minigenome replicon in cells, highlighting the importance of VP35 helicase-like activity during EBOV life cycle. Together, our findings provide the first demonstration of the NTPase/helicase-like activity encoded by EBOV, and would foster our understanding of EBOV and NNSVs.
TL;DR: A novel mcr-1 gene variant, carried by an IncX4 plasmid, which strongly suggests that the presence of PMCR-encoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally.
Abstract: We studied a commensal colistin-resistant Escherichia coli isolated from a swine cecum sample collected at a slaughter, in Portugal. Antimicrobial susceptibility phenotype of E. coli LV23529 showed resistance to colistin at a minimum inhibitory concentration of 4 mg/L. Whole genome of E. coli LV23529 was sequenced using a MiSeq system and the assembled contigs were analyzed for the presence of antibiotic resistance and plasmid replicon types using bioinformatics tools. We report a novel mcr-1 gene variant (mcr-1.9), carried by an IncX4 plasmid, where one-point mutation at nucleotide T1238C leads to Val413Ala substitution. The mcr-1.9 genetic context was characterized by an IS26 element upstream of the mcr-pap2 element and by the absence of ISApl1. Bioinformatic analysis also revealed genes conferring resistance to β-lactams, sulphamethoxazole, trimethoprim, chloramphenicol and colistin, corresponding to the phenotype noticed. Moreover, we highlight the presence of mcr-1.9 plus blaCTX-M-8, a blaESBL gene rarely detected in Europe in isolates of animal origin; these two genes were located on different plasmids with 33,303 and 89,458 bp, respectively. MCR-1.9-harboring plasmid showed high identity to other X4-type mcr-1-harboring plasmids characterized worldwide, which strongly suggests that the presence of PMCR-encoding genes in food-producing animals, such as MCR-1.9, represent a potential threat to humans, as it is located in mobile genetic elements that have the potential to spread horizontally.
TL;DR: Haloarchaea are excellent models to study many aspects of genome biology in prokaryotes, exhibiting properties that have not been found in bacteria.
Abstract: All analyzed haloarachea are polyploid. In addition, haloarchaea contain more than one type of chromosome, and thus the gene dosage can be regulated independently on different replicons. Haloarchaea and several additional archaea have more than one replication origin on their major chromosome, in stark contrast with bacteria, which have a single replication origin. Two of these replication origins of Haloferax volcanii have been studied in detail and turned out to have very different properties. The chromosome copy number appears to be regulated in response to growth phases and environmental factors. Archaea typically contain about two Origin Recognition Complex (ORC) proteins, which are homologous to eukaryotic ORC proteins. However, haloarchaea are the only archaeal group that contains a multitude of ORC proteins. All 16 ORC protein paralogs from H. volcanii are involved in chromosome copy number regulation. Polyploidy has many evolutionary advantages for haloarchaea, e.g. a high resistance to desiccation, survival over geological times, and the relaxation of cell cycle-specific replication control. A further advantage is the ability to grow in the absence of external phosphate while using the many genome copies as internal phosphate storage polymers. Very efficient gene conversion operates in haloarchaea and results in the unification of genome copies. Taken together, haloarchaea are excellent models to study many aspects of genome biology in prokaryotes, exhibiting properties that have not been found in bacteria.
TL;DR: A reporter virus expressing NanoLuc luciferase was generated, which was stable after 10 rounds of continuous propagation without reporter gene deletion, and two types of replicons driven by the T7 promoter or CMV promoter were generated, both of which worked well in BHK21 cells.
TL;DR: The surprising co-existence of three copies of blaKPC-2 on an IncR/IncF plasmid due to the action of IS26 is reported and is a key factor generating genetic plasticity and could mediate the multiplication of resistance genes.
Abstract: Objectives Here we report the finding of three copies of the blaKPC-2 gene on a plasmid in ST11 Klebsiella pneumoniae. Methods Carbapenem-resistant K. pneumoniae clinical strain WCHKP2 was subjected to whole-genome sequencing (WGS) using both a short-read Illumina HiSeq X10 platform and long-read MinION sequencer. Hybrid assembly was performed using Unicycler, and contigs were corrected using Pilon. Based on WGS, the sequence type (ST), capsular type, plasmid replicon type and plasmid multilocus sequence type were determined and virulence and antimicrobial resistance genes were identified. Mating was performed to identify a self-transmissible plasmid mediating carbapenem resistance. Results Strain WCHKP2 was resistant to imipenem [minimum inhibitory concentration (MIC) = 64 μg/mL] and meropenem (MIC = 128 μg/mL). Strain WCHKP2 had a 5 477 148-bp circular chromosome, two small ColRNAI-like plasmids (5596 bp and 10 060 bp), and one large plasmid (177 516 bp, designated pKPC2_020002) containing an IncR and an IncFII replicon. Surprisingly, there were three copies of the blaKPC-2 carbapenemase gene on pKPC2_020002, which was not self-transmissible. Each of the blaKPC-2 genes was located in the same genetic context with insertion sequence ISKpn27 upstream and ISKpn6 downstream and bracketed by IS26. The three copies of the IS26–ISKpn27–blaKPC-2–ISKpn6–IS26 unit were present in tandem. Conclusion Here we report the surprising co-existence of three copies of blaKPC-2 on an IncR/IncF plasmid due to the action of IS26. Multiple copies of IS26 are a key factor generating genetic plasticity and could mediate the multiplication of resistance genes.
TL;DR: This new genome editing tool called Inducible Plasmid Self-Destruction (IPSD) was successfully used to perform gene knock-out and knock-in in lactobacilli and bifidobacteria and may provide a general approach for genetic engineering of various bacterial species.
Abstract: Genome engineering is essential for application of synthetic biology in probiotics including lactobacilli and bifidobacteria. Several homologous recombination system-based mutagenesis tools have been developed for these bacteria, but still have many limitations in different species or strains. Here we developed a genome engineering method based on an inducible self-destruction plasmid delivering homologous DNA into bacteria. Excision of the replicon by induced recombinase facilitates selection of homologous recombination events. This new genome editing tool called inducible plasmid self-destruction (IPSD) was successfully used to perform gene knockout and knock-in in lactobacilli and bifidobacteria. Due to its simplicity and universality, the IPSD strategy may provide a general approach for genetic engineering of various bacterial species.
TL;DR: Molecular surveillance of mcr gene co-occurrence warrants further investigation to evaluate the public health risk and suggest that evolution of mCR genes among various plasmids was being driven by mobile elements.
Abstract: Objectives To characterize the genome of an Escherichia coli harbouring both mcr-1 and mcr-3.19 on a hybrid plasmid and the underlying transmission mechanisms. Methods Broth microdilution was used to perform antimicrobial susceptibility testing. Conjugation assays and S1-PFGE were used to assess the transferability of mcr genes. Resistance genotypes and genetic contexts were investigated, based on WGS data from the Illumina and MinION platforms. Inverse PCR was performed to test the mcr-3.19-bearing circular intermediate. Bioinformatic tools were used to further characterize the hybrid plasmid. Results E. coli CP53 was identified as harbouring both mcr-1 and mcr-3.19 on a 231 859 bp hybrid plasmid pCP53-mcr1_3 containing IncFIA, IncHI1A, IncHI1B and IncN replicons. The genetic structures of mcr-1 and mcr-3.19 were similar to those reported in other mcr-1 and mcr-3.19-bearing plasmids, which suggested that recombination between mcr-bearing plasmids had been mediated by ISs. However, the MDR plasmid pCP53-mcr1_3 cannot transfer via conjugation. Furthermore, another three plasmids were identified in the isolate, two of which encoded resistance genes. In640 duplication between two MDR plasmids was observed. An MDR-region recombination existed in E. coli CP53. A core structure consisting of mcr-3-dgkA existed in mcr-3-bearing plasmids reported, to date. Circular intermediates were observed for mcr-1 and mcr-3.19 regions. Conclusions A novel mcr-3.19 was identified along with mcr-1 contained in a hybrid plasmid. This finding suggested that evolution of mcr genes among various plasmids was being driven by mobile elements. Molecular surveillance of mcr gene co-occurrence warrants further investigation to evaluate the public health risk.
TL;DR: A replicon-based scheme was proposed for typing IncI-complex plasmids, which shared conserved gene organization in the replication and conjugal transfer regions, but displaying considerable sequence diversity among different subgroups.
Abstract: IncI-complex plasmids can be divided into seven subgroups IncI1, IncI2, IncIγ, IncB/O, IncK1, IncK2, and IncZ. In this study, a replicon-based scheme was proposed for typing IncI-complex plasmids into four separately clustering subgroups IncI2, IncI1/B/O, IncIγ/K1 and IncK2/Z, the last three of which were combined from IncI1 and IncB/O, IncIγ and IncK1, and IncK2 and IncZ, respectively. Four IncIγ/K1 plasmids p205880-NR2, p14E509-CTXM, p11011-CTXM and p61806-CTXM were fully sequenced and compared with IncIγ/K1 reference pCT, IncI2 reference R721, IncI1/B/O reference R64 and IncK2/Z reference pO26-CRL-125. These plasmids shared conserved gene organization in the replication and conjugal transfer regions, but displaying considerable sequence diversity among different subgroups. Remarkable modular differences were observed in the maintenance and transfer leading regions. p205880-NR2 contained no resistance genes or accessory modules, while the other seven plasmids acquired one or more accessory modules, which harbored mobile elements [including unit transposons, insertion sequence (IS)-based transposition units and individual IS elements] and associated resistance markers (especially including those involved in resistance to β-lactams, aminoglycosides, tetracyclins, phenicols, streptomycins, trimethoprims, sulphonamides, tunicamycins and erythromycins). Data presented here provided a deeper insight into diversification and evolution of IncI-complex plasmids.
TL;DR: Evaluating the antiviral potential of ARB against another picornavirus, foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus and an important veterinary pathogen demonstrates that ARB can inhibit some non-enveloped picornviruses.
Abstract: Arbidol (ARB, also known as umifenovir) is used clinically in several countries as an anti-influenza virus drug. ARB inhibits multiple enveloped viruses in vitro and the primary mode of action is inhibition of virus entry and/or fusion of viral membranes with intracellular endosomal membranes. ARB is also an effective inhibitor of non-enveloped poliovirus types 1 and 3. In the current report, we evaluate the antiviral potential of ARB against another picornavirus, foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus and an important veterinary pathogen. ARB inhibits the replication of FMDV RNA sub-genomic replicons. ARB inhibition of FMDV RNA replication is not a result of generalized inhibition of cellular uptake of cargo, such as transfected DNA, and ARB can be added to cells up to 3 h post-transfection of FMDV RNA replicons and still inhibit FMDV replication. ARB prevents the recovery of FMDV replication upon withdrawal of the replication inhibitor guanidine hydrochloride (GuHCl). Although restoration of FMDV replication is known to require de novo protein synthesis upon GuHCl removal, ARB does not suppress cellular translation or FMDV internal ribosome entry site (IRES)-driven translation. ARB also inhibits infection with the related Aphthovirus, equine rhinitis A virus (ERAV). Collectively, the data demonstrate that ARB can inhibit some non-enveloped picornaviruses. The data are consistent with inhibition of picornavirus genome replication, possibly via the disruption of intracellular membranes on which replication complexes are located.
TL;DR: An in vitro potato virus X translation/replication system is developed using vacuole- and nucleus-free lysates from tobacco protoplasts, and it is revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp).
Abstract: Understanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication.IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits de novo potexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.
TL;DR: The presence of the RNA replicon genes responsible for extensive RNA replication in the RNA/DNA layered alphavirus vectors provides superior transgene expression in comparison to conventional plasmid DNA-based expression.
TL;DR: The ultrastability of an mcr-1-encoding element in a bacterial plasmid and chromosome renders it unlikely to be eradicated quickly by the reduced use of colistin, and factors leading to the frequent demise of Tn6330 warrant further studies.
Abstract: Background The mcr-1 gene has been widely reported in both bacterial chromosomes and plasmids, while its stability in these genetic materials is not well understood. Objectives Our aim was to characterize the stability and dynamics of Tn6330 elements in both a plasmid and the chromosome in a single bacterial population. Methods Plasmid-borne and chromosomal Tn6330 were characterized by PCR, conjugation, S1-PFGE, stability assay, single-molecule long-read sequencing and bioinformatics analysis. Results Tn6330 was simultaneously detected in both a plasmid and the chromosome of a clinical Escherichia coli strain. The plasmid was found to comprise the IncFIB replicon and a phage-like replicon, as well as two integrons that harboured various mobile elements and resistance genes including mcr-1, floR, blaTEM-1b and strAB. Both plasmid-borne and chromosomal Tn6330 transposons could be re-organized into a circular intermediate that played a role in transmission of the mcr-1 gene. Tn6330 was found to be very stable in both the plasmid and chromosome after 30 passages of 12 h with or without colistin selective pressure. The decayed structure of Tn6330 in the genuine single DNA molecules of bacterial populations, although occurring at a very low frequency, could be detected for the first time, in which Tn6330 was degraded into a single ISApl1 element. Conclusions Long-read sequencing technology is a good tool to study the evolution and stability of genetic elements in bacteria. The ultrastability of an mcr-1-encoding element in a bacterial plasmid and chromosome renders it unlikely to be eradicated quickly by the reduced use of colistin, and factors leading to the frequent demise of Tn6330 warrant further studies.
TL;DR: It is demonstrated that the E2 protein targets Topo1 to the viral origin, and this depends on acetylation of K111, which is highly conserved among almost all papillomavirus E2 proteins and is critical for viral DNA replication.
Abstract: Human papillomavirus (HPV) E2 proteins are integral for the transcription of viral genes and the replication and maintenance of viral genomes in host cells. E2 recruits the viral DNA helicase E1 to the origin. A lysine (K111), highly conserved among almost all papillomavirus (PV) E2 proteins, is a target for P300 (EP300) acetylation and is critical for viral DNA replication (E. J. Quinlan, S. P. Culleton, S. Y. Wu, C. M. Chiang, et al., J Virol 87:1497-1507, 2013, https://doi.org/10.1128/JVI.02771-12; Y. Thomas and E. J. Androphy, J Virol 92:e01912-17, 2018, https://doi.org/10.1128/JVI.01912-17). Since the viral genome exists as a covalently closed circle of double-stranded DNA, topoisomerase 1 (Topo1) is thought to be required for progression of the replication forks. Due to the specific effect of K111 mutations on DNA unwinding (Y. Thomas and E. J. Androphy, J Virol 92:e01912-17, 2018, https://doi.org/10.1128/JVI.01912-17), we demonstrate that the E2 protein targets Topo1 to the viral origin, and this depends on acetylation of K111. The effect was corroborated by functional replication assays, in which higher levels of P300, but not its homolog CBP, caused enhanced replication with wild-type E2 but not the acetylation-defective K111 arginine mutant. These data reveal a novel role for lysine acetylation during viral DNA replication by regulating topoisomerase recruitment to the replication origin.IMPORTANCE Human papillomaviruses affect an estimated 75% of the sexually active adult population in the United States, with 5.5 million new cases emerging every year. More than 200 HPV genotypes have been identified; a subset of them are linked to the development of cancers from these epithelial infections. Specific antiviral medical treatments for infected individuals are not available. This project examines the mechanisms that control viral genome replication and may allow the development of novel therapeutics.
TL;DR: The study characterized IncI 1 positive Salmonella isolates and provided evidence about the potential contributions of IncI1 plasmids virulence phenotypes and areas where they do not, and should allow for more focused efforts to assess the impact of plasmIDS on bacterial pathophysiology and human health.
Abstract: Mobile genetic elements, such as plasmids, can potentially increase the ability of bacteria to infect and persist in vertebrate host cells. IncI1 plasmids are widely distributed in Salmonella from food animal sources and associated with clinically important strains. These plasmids often encode antimicrobial resistance; however, little is known about their impact on the virulence of Salmonella strains. To assess the potential impact of the plasmids on virulence, 43 IncI1-positive Salmonella isolates from human and animal sources were subjected to whole genome sequence (WGS) analyses and evaluated for their abilities to invade and persist for 48 h in Caco-2 human intestinal epithelial cells, form biofilms and encode bacteriocins. Draft WGS data were submitted to predict the presence of virulence and antimicrobial resistance genes, plasmid replicon types present, conduct plasmid multilocus sequence typing (pMLST), and core genome MLST (cgMLST) in the isolates. Caco-2 cells were infected with Salmonella strains and incubated for both one and 48 h for the invasion and persistence assays, respectively. Additionally, Salmonella isolates and IncI1 plasmid carrying transconjugants (n = 12) generated in Escherichia coli were assessed for their ability to produce biofilms and bacteriocin inhibition of growth of other bacteria. All Salmonella isolates infected Caco-2 cells and persisted in the cells at 48 hrs. Persistent cell counts were observed to be significantly higher than invasion assay cell counts in 26% of the isolates. Among the IncI1 plasmids, there were 18 pMLST types. Nearly 35% (n = 15) of Salmonella isolates produced biofilms; however, none of the IncI1-positive transconjugants produced increased biofilms compared to the recipient. Approximately 65% (n = 28) of isolates and 67% (n = 8) of IncI1-positive transconjugants were able to inhibit growth of at least one E. coli strain; however, none inhibited the growth of strains from species other than E. coli. The study characterized IncI1 positive Salmonella isolates and provided evidence about the potential contributions of IncI1 plasmids virulence phenotypes and areas where they do not. These findings should allow for more focused efforts to assess the impact of plasmids on bacterial pathophysiology and human health.
TL;DR: Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence, and it is shown that multipleplasmid-borne TA systems have a prominent role in preserving plasmID integrity and ensuring the maintenance of virulence genes in free-living conditions.
Abstract: Pseudomonas syringae is a γ-proteobacterium causing economically relevant diseases in practically all cultivated plants. Most isolates of this pathogen contain native plasmids collectively carrying many pathogenicity and virulence genes. However, P. syringae is generally an opportunistic pathogen primarily inhabiting environmental reservoirs, which could exert a low selective pressure for virulence plasmids. Additionally, these plasmids usually contain a large proportion of repeated sequences, which could compromise plasmid integrity. Therefore, the identification of plasmid stability determinants and mechanisms to preserve virulence genes is essential to understand the evolution of this pathogen and its adaptability to agroecosystems. The three virulence plasmids of P. syringae pv. savastanoi NCPPB 3335 contain from one to seven functional stability determinants, including three highly active toxin-antitoxin systems (TA) in both pPsv48A and pPsv48C. The TA systems reduced loss frequency of pPsv48A by two orders of magnitude, whereas one of the two replicons of pPsv48C likely confers stable inheritance by itself. Notably, inactivation of the TA systems from pPsv48C exposed the plasmid to high-frequency deletions promoted by mobile genetic elements. Thus, recombination between two copies of MITEPsy2 caused the deletion of an 8.3 kb fragment, with a frequency of 3.8 ± 0.3 × 10− 3. Likewise, one-ended transposition of IS801 generated plasmids containing deletions of variable size, with a frequency of 5.5 ± 2.1 × 10− 4, of which 80% had lost virulence gene idi. These deletion derivatives were stably maintained in the population by replication mediated by repJ, which is adjacent to IS801. IS801 also promoted deletions in plasmid pPsv48A, either by recombination or one-ended transposition. In all cases, functional TA systems contributed significantly to reduce the occurrence of plasmid deletions in vivo. Virulence plasmids from P. syringae harbour a diverse array of stability determinants with a variable contribution to plasmid persistence. Importantly, we showed that multiple plasmid-borne TA systems have a prominent role in preserving plasmid integrity and ensuring the maintenance of virulence genes in free-living conditions. This strategy is likely widespread amongst native plasmids of P. syringae and other bacteria.