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Journal of Virology Jul 2023Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an...
Nonstructural protein 13 (nsp13), the helicase of SARS-CoV-2, has been shown to possess multiple functions that are essential for viral replication, and is considered an attractive target for the development of novel antivirals. We were initially interested in the interplay between nsp13 and interferon (IFN) signaling, and found that nsp13 inhibited reporter signal in an IFN-β promoter assay. Surprisingly, the ectopic expression of different components of the RIG-I/MDA5 pathway, which were used to stimulate IFN-β promoter, was also mitigated by nsp13. However, endogenous expression of these genes was not affected by nsp13. Interestingly, nsp13 restricted the expression of foreign genes originating from plasmid transfection, but failed to inhibit them after chromosome integration. These data, together with results from a runoff transcription assay and RNA sequencing, suggested a specific inhibition of episomal but not chromosomal gene transcription by nsp13. By using different truncated and mutant forms of nsp13, we demonstrated that its NTPase and helicase activities contributed to the inhibition of episomal DNA transcription, and that this restriction required direct interaction with episomal DNA. Based on these findings, we developed an economical and convenient high-throughput drug screening method targeting nsp13. We evaluated the inhibitory effects of various compounds on nsp13 by the expression of reporter gene plasmid after co-transfection with nsp13. In conclusion, we found that nsp13 can specifically inhibit episomal DNA transcription and developed a high-throughput drug screening method targeting nsp13 to facilitate the development of new antiviral drugs. To combat COVID-19, we need to understand SARS-CoV-2 and develop effective antiviral drugs. In our study, we serendipitously found that SARS-CoV-2 nsp13 could suppress episomal DNA transcription without affecting chromosomal DNA. Detailed characterization revealed that nsp13 suppresses episomal gene expression through its NTPase and helicase functions following DNA binding. Furthermore, we developed a high-throughput drug screening system targeting SARS-CoV-2 nsp13. Compared to traditional SARS-CoV-2 drug screening methods, our system is more economical and convenient, facilitating the development of more potent and selective nsp13 inhibitors and enabling the discovery of new antiviral therapies.
Topics: Humans; SARS-CoV-2; COVID-19; Nucleoside-Triphosphatase; RNA Helicases; Viral Nonstructural Proteins; DNA Helicases; Antiviral Agents; DNA; Plasmids
PubMed: 37347173
DOI: 10.1128/jvi.00512-23 -
Science Advances Mar 2024Suprachoroidal nonviral gene therapy with biodegradable poly(β-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented...
Suprachoroidal nonviral gene therapy with biodegradable poly(β-amino ester) nanoparticles (NPs) provides widespread expression in photoreceptors and retinal pigmented epithelial (RPE) cells and therapeutic benefits in rodents. Here, we show in a human-sized minipig eye that suprachoroidal injection of 50 μl of NPs containing 19.2 μg of GFP expression plasmid caused GFP expression in photoreceptors and RPE throughout the entire eye with no toxicity. Two weeks after injection of 50, 100, or 200 μl, there was considerable within-eye and between-eye variability in expression that was reduced 3 months after injection of 200 μl and markedly reduced after three suprachoroidal injections at different locations around the eye. Reduction of bacterial sequences in the expression plasmid resulted in a trend toward higher expression. These data indicate that nonviral suprachoroidal gene therapy with optimized polymer, expression plasmid, and injection approach has potential for treating photoreceptors throughout the entire retina of a human-sized eye.
Topics: Animals; Humans; Swine; Swine, Miniature; Retina; Plasmids; Genetic Therapy; Nanoparticles
PubMed: 38457512
DOI: 10.1126/sciadv.adl3576 -
Nature Microbiology Oct 2023Since 2016, Yemen has been experiencing the largest cholera outbreak in modern history. Multidrug resistance (MDR) emerged among Vibrio cholerae isolates from cholera...
Since 2016, Yemen has been experiencing the largest cholera outbreak in modern history. Multidrug resistance (MDR) emerged among Vibrio cholerae isolates from cholera patients in 2018. Here, to characterize circulating genotypes, we analysed 260 isolates sampled in Yemen between 2018 and 2019. Eighty-four percent of V. cholerae isolates were serogroup O1 belonging to the seventh pandemic El Tor (7PET) lineage, sub-lineage T13, whereas 16% were non-toxigenic, from divergent non-7PET lineages. Treatment of severe cholera with macrolides between 2016 and 2019 coincided with the emergence and dominance of T13 subclones carrying an incompatibility type C (IncC) plasmid harbouring an MDR pseudo-compound transposon. MDR plasmid detection also in endemic non-7PET V. cholerae lineages suggested genetic exchange with 7PET epidemic strains. Stable co-occurrence of the IncC plasmid with the SXT family of integrative and conjugative element in the 7PET background has major implications for cholera control, highlighting the importance of genomic epidemiological surveillance to limit MDR spread.
Topics: Humans; Cholera; Vibrio cholerae O1; Yemen; Plasmids; Genomics
PubMed: 37770747
DOI: 10.1038/s41564-023-01472-1 -
The Journal of Antimicrobial... Sep 2023Resistance genes can be genetically transmitted and exchanged between commensal and pathogenic bacterial species, and in different compartments including the...
OBJECTIVES
Resistance genes can be genetically transmitted and exchanged between commensal and pathogenic bacterial species, and in different compartments including the environment, or human and animal guts (One Health concept). The aim of our study was to evaluate whether subdosages of antibiotics administered in veterinary medicine could enhance plasmid transfer and, consequently, resistance gene exchange in gut microbiota.
METHODS
Conjugation frequencies were determined with Escherichia coli strains carrying IncL- (blaOXA-48) or IncI1-type (blaCTX-M-1) plasmids subjected to a series of subinhibitory concentrations of antibiotics used in veterinary medicine, namely amoxicillin, ceftiofur, apramycin, neomycin, enrofloxacin, colistin, erythromycin, florfenicol, lincomycin, oxytetracycline, sulfamethazine, tiamulin and the ionophore narasin. Treatments with subinhibitory dosages were performed with and without supplementation with the antioxidant edaravone, known as a mitigator of the inducibility effect of several antibiotics on plasmid conjugation frequency (PCF). Expression of SOS-response associated genes and fluorescence-based reactive oxygen species (ROS) detection assays were performed to evaluate the stress oxidative response.
RESULTS
Increased PCFs were observed for both strains when treating with florfenicol and oxytetracycline. Increased expression of the SOS-associated recA gene also occurred concomitantly, as well as increased ROS production. Addition of edaravone to the treatments reduced their PCF and also showed a decreasing effect on SOS and ROS responses for both plasmid scaffolds.
CONCLUSIONS
We showed here that some antibiotics used in veterinary medicine may induce transfer of plasmid-encoded resistance and therefore may contribute to the worldwide spread of antibiotic resistance genes.
Topics: Animals; Humans; Anti-Bacterial Agents; Oxytetracycline; Edaravone; Reactive Oxygen Species; Escherichia coli; Plasmids; Drug Resistance, Microbial; Gene Transfer, Horizontal
PubMed: 37486104
DOI: 10.1093/jac/dkad226 -
BMC Microbiology Dec 2023The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.
OBJECTIVE
The purpose of this study is to reduce the spread of the vanA gene by curing the vanA-harboring plasmid of vancomycin-resistant using the CRISPR-Cas9 system.
METHODS
Two specific spacer sequence (sgRNAs) specific was designed to target the vanA gene and cloned into plasmid CRISPR-Cas9. The role of the CRISPR-Cas system in the plasmid elimination of drug-resistance genes was verified by chemically transformation and conjugation delivery methods. Moreover, the elimination efficiency in strains was evaluated by plate counting, PCR, and quantitative real-time PCR (qPCR). Susceptibility testing was performed by broth microdilution assay and by Etest strips (bioMérieux, France) to detect changes in bacterial drug resistance phenotype after drug resistance plasmid clearance.
RESULTS
In the study, we constructed a specific prokaryotic CRISPR-Cas9 system plasmid targeting cleavage of the vanA gene. PCR and qPCR results indicated that recombinant pCas9-sgRNA plasmid can efficiently clear vanA-harboring plasmids. There was no significant correlation between sgRNA lengths and curing efficiency. In addition, the drug susceptibility test results showed that the bacterial resistance to vancomycin was significantly reduced after the vanA-containing drug-resistant plasmid was specifically cleaved by the CRISPR-Cas system. The CRISPR-Cas9 system can block the horizontal transfer of the conjugated plasmid pUC19-vanA.
CONCLUSION
In conclusion, our study demonstrated that CRISPR-Cas9 achieved plasmid clearance and reduced antimicrobial resistance. The CRISPR-Cas9 system could block the horizontal transfer of plasmid carrying vanA. This strategy provided a great potential to counteract the ever-worsening spread of the vanA gene among bacterial pathogens and laid the foundation for subsequent research using the CRISPR-Cas9 system as adjuvant antibiotic therapy.
Topics: Vancomycin; RNA, Guide, CRISPR-Cas Systems; Vancomycin Resistance; CRISPR-Cas Systems; Anti-Bacterial Agents; Plasmids; Bacterial Proteins
PubMed: 38049763
DOI: 10.1186/s12866-023-03136-w -
Current Genetics Dec 2023pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible...
pET expression plasmids are widely used for producing recombinant proteins in Escherichia coli. Selection and maintenance of cells harboring a pET plasmid are possible using either a Tn3.1-type genetic fragment (which encodes a ß-lactamase and confers resistance to ß-lactam antibiotics) or a Tn903.1-type genetic fragment (which encodes an aminoglycoside-3'-phosphotransferase and confers resistance aminoglycoside antibiotics). Herein we have investigated how efficiently pET plasmids are maintained using these two fragments. The study reveals that pET plasmids are efficiently maintained with both Tn3.1 and Tn903.1 genetic fragments prior to the induction of recombinant protein production, and over short induction times (i.e., 2 h). However, over longer induction times (i.e., 20 h), the efficiency of plasmid maintenance depends on the host strain used, and the type of antibiotic selection cassette used. Based on our collective observations, we have 2 general tips for efficiently maintaining pET plasmids during recombinant production experiments. Tip #1: Use a strain with lowered levels of the T7 RNA polymerase, such as C41(DE3). pET plasmids will be efficiently maintained over long induction times with both the Tn3.1 and Tn903.1 genetic fragments, regardless of whether antibiotics are present during cultivation. Tip #2: If a strain with higher levels of T7 RNA polymerase strain is necessary, such as BL21(DE3)), keep induction times short or use a plasmid containing a Tn903.1-type fragment and select with kanamycin.
Topics: Plasmids; Escherichia coli; Recombinant Proteins; Anti-Bacterial Agents; Aminoglycosides
PubMed: 37938343
DOI: 10.1007/s00294-023-01276-0 -
The ISME Journal Oct 2023Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within...
Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids' ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a model system consisting of a synthetic and stable five-species community and a broad host-range plasmid, engineered to carry different payloads. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species specific. Addition of growth partners increased the cost of a high-payload plasmid to a focal strain, and accordingly, plasmid loss from the focal species occurred over a shorter time frame. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.
Topics: Microbiota; Plasmids; Ecology; Anti-Bacterial Agents
PubMed: 37558861
DOI: 10.1038/s41396-023-01487-w -
Zoological Research Sep 2023Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic...
Conjugative transfer of antibiotic resistance genes (ARGs) by plasmids is an important route for ARG dissemination. An increasing number of antibiotic and nonantibiotic compounds have been reported to aid the spread of ARGs, highlighting potential challenges for controlling this type of horizontal transfer. Development of conjugation inhibitors that block or delay the transfer of ARG-bearing plasmids is a promising strategy to control the propagation of antibiotic resistance. Although such inhibitors are rare, they typically exhibit relatively high toxicity and low efficacy and their mechanisms of action are inadequately understood. Here, we studied the effects of dihydroartemisinin (DHA), an artemisinin derivative used to treat malaria, on conjugation. DHA inhibited the conjugation of the IncI2 and IncX4 plasmids carrying the mobile colistin resistance gene ( ) by more than 160-fold in , and more than two-fold (IncI2 plasmid) in a mouse model. It also suppressed the transfer of the IncX3 plasmid carrying the carbapenem resistance gene by more than two-fold . Detection of intracellular adenosine triphosphate (ATP) and proton motive force (PMF), in combination with transcriptomic and metabolomic analyses, revealed that DHA impaired the function of the electron transport chain (ETC) by inhibiting the tricarboxylic acid (TCA) cycle pathway, thereby disrupting PMF and limiting the availability of intracellular ATP for plasmid conjugative transfer. Furthermore, expression levels of genes related to conjugation and pilus generation were significantly down-regulated during DHA exposure, indicating that the transfer apparatus for conjugation may be inhibited. Our findings provide new insights into the control of antibiotic resistance and the potential use of DHA.
Topics: Mice; Animals; Escherichia coli; Escherichia coli Infections; beta-Lactamases; Anti-Bacterial Agents; Plasmids
PubMed: 37551137
DOI: 10.24272/j.issn.2095-8137.2023.084 -
Virulence Dec 2024This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent...
OBJECTIVES
This study aimed at revealing the underlying mechanisms of the loss and gain of ceftazidime-avibactam susceptibility in a non-carbapenemase-producing hypervirulent (hvKp).
METHODS
Here we longitudinally recovered 3 non-carbapenemase-producing K1-ST23 hvKp strains at a one-month interval (KP29105, KP29499 and KP30086) from an elderly male. Antimicrobial susceptibility testing, whole genome sequencing, transcriptomic sequencing, gene cloning, plasmid conjugation, quantitative real-time PCR (qRT-PCR), and SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) were conducted.
RESULTS
Among the 3 hvKp strains, KP29105 was resistant to the third- and fourth-generation cephalosporins, KP29499 acquired resistance to both ceftazidime-avibactam and carbapenems, while KP30086 restored its susceptibility to ceftazidime-avibactam, imipenem and meropenem but retained low-level resistance to ertapenem. KP29105 and KP29499 carried plasmid-encoded genes and , respectively, but KP30086 lost both. Cloning of gene and conjugation experiment of -carrying plasmid showed that the transformant and transconjugant were susceptible to ceftazidime-avibactam but had a more than 8-fold increase in MICs. Supplementation with an outer membrane permeabilizer could reduce the MIC of ceftazidime-avibactam by 32 folds, indicating that porins play a key role in ceftazidime-avibactam resistance. The OmpK35 of the 3 isolates was not expressed, and the OmpK36 of KP29499 and KP30086 had a novel amino acid substitution (L359R). SDS-PAGE and qRT-PCR showed that the expression of porin OmpK36 of KP29499 and KP30086 was significantly down-regulated compared with KP29105.
CONCLUSIONS
In summary, we reported the rare ceftazidime-avibactam resistance in a non-carbapenemase-producing hvKp strain. Resistance plasmid carrying and mutated OmpK36 had a synergetic effect on the resistance.
Topics: Ceftazidime; Klebsiella pneumoniae; Azabicyclo Compounds; Drug Combinations; Anti-Bacterial Agents; Male; Microbial Sensitivity Tests; Klebsiella Infections; Humans; Bacterial Proteins; beta-Lactamases; Aged; Drug Resistance, Multiple, Bacterial; Virulence; Plasmids; Whole Genome Sequencing
PubMed: 38697754
DOI: 10.1080/21505594.2024.2348251 -
Infection, Genetics and Evolution :... Aug 2023CRISPR-Cas systems are the only RNA- guided adaptive immunity pathways that trigger the detection and destruction of invasive phages and plasmids in bacteria and... (Review)
Review
CRISPR-Cas systems are the only RNA- guided adaptive immunity pathways that trigger the detection and destruction of invasive phages and plasmids in bacteria and archaea. Due to its prevalence and mystery, the Class 1 CRISPR-Cas system has lately been the subject of several studies. This review highlights the specificity of CRISPR-Cas system III-A in Mycobacterium tuberculosis, the tuberculosis-causing pathogen, for over twenty years. We discuss the difference between the several subtypes of Type III and their defence mechanisms. The anti-CRISPRs (Acrs) recently described, the critical role of Reverse transcriptase (RT) and housekeeping nuclease for type III CRISPR-Cas systems, and the use of this cutting-edge technology, its impact on the search for novel anti-tuberculosis drugs.
Topics: CRISPR-Cas Systems; Mycobacterium tuberculosis; Bacteriophages; Plasmids; Antitubercular Agents
PubMed: 37217031
DOI: 10.1016/j.meegid.2023.105445