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Nucleic Acids Research Dec 2023Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for...
Anti-CRISPR (Acr) proteins are encoded by mobile genetic elements to overcome the CRISPR immunity of prokaryotes, displaying promises as controllable tools for modulating CRISPR-based applications. However, characterizing novel anti-CRISPR proteins and exploiting Acr-related technologies is a rather long and tedious process. Here, we established a versatile plasmid interference with CRISPR interference (PICI) system in Escherichia coli for rapidly characterizing Acrs and developing Acr-based technologies. Utilizing the PICI system, we discovered two novel type II-A Acrs (AcrIIA33 and AcrIIA34), which can inhibit the activity of SpyCas9 by affecting DNA recognition of Cas9. We further constructed a circularly permuted AcrIIA4 (cpA4) protein and developed optogenetically engineered, robust AcrIIA4 (OPERA4) variants by combining cpA4 with the light-oxygen-voltage 2 (LOV2) blue light sensory domain. OPERA4 variants are robust light-dependent tools for controlling the activity of SpyCas9 by approximately 1000-fold change under switching dark-light conditions in prokaryotes. OPERA4 variants can achieve potent light-controllable genome editing in human cells as well. Together, our work provides a versatile screening system for characterizing Acrs and developing the Acr-based controllable tools.
Topics: Humans; CRISPR-Associated Protein 9; CRISPR-Cas Systems; Gene Editing; Plasmids; Bacterial Proteins; Escherichia coli
PubMed: 37930830
DOI: 10.1093/nar/gkad995 -
Nature Communications Jun 2024Phage predation is generally assumed to reduce microbial proliferation while not contributing to the spread of antibiotic resistance. However, this assumption does not...
Phage predation is generally assumed to reduce microbial proliferation while not contributing to the spread of antibiotic resistance. However, this assumption does not consider the effect of phage predation on the spatial organization of different microbial populations. Here, we show that phage predation can increase the spread of plasmid-encoded antibiotic resistance during surface-associated microbial growth by reshaping spatial organization. Using two strains of the bacterium Escherichia coli, we demonstrate that phage predation slows the spatial segregation of the strains during growth. This increases the number of cell-cell contacts and the extent of conjugation-mediated plasmid transfer between them. The underlying mechanism is that phage predation shifts the location of fastest growth from the biomass periphery to the interior where cells are densely packed and aligned closer to parallel with each other. This creates straighter interfaces between the strains that are less likely to merge together during growth, consequently slowing the spatial segregation of the strains and enhancing plasmid transfer between them. Our results have implications for the design and application of phage therapy and reveal a mechanism for how microbial functions that are deleterious to human and environmental health can proliferate in the absence of positive selection.
Topics: Plasmids; Escherichia coli; Bacteriophages; Drug Resistance, Bacterial; Anti-Bacterial Agents; Conjugation, Genetic
PubMed: 38926498
DOI: 10.1038/s41467-024-49840-7 -
Microbiology Spectrum Aug 2023Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments....
Plasmids contribute to microbial diversity and adaptation, providing microorganisms with the ability to thrive in a wide range of conditions in extreme environments. However, while the number of marine microbiome studies is constantly increasing, very little is known about marine plasmids, and they are very poorly represented in public databases. To extend the repertoire of environmental marine plasmids, we established a pipeline for the assembly of plasmids in the marine environment by analyzing available microbiome metagenomic sequencing data. By applying the pipeline to data from the Red Sea, we identified 362 plasmid candidates. We showed that the distribution of plasmids corresponds to environmental conditions, particularly, depth, temperature, and physical location. At least 7 of the 362 candidates are most probably real plasmids, based on a functional analysis of their open reading frames (ORFs). Only one of the seven has been described previously. Three plasmids were identified in other public marine metagenomic data from different locations all over the world; these plasmids contained different cassettes of functional genes at each location. Analysis of antibiotic and metal resistance genes revealed that the same positions that were enriched with genes encoding resistance to antibiotics were also enriched with resistance to metals, suggesting that plasmids contribute site-dependent phenotypic modules to their ecological niches. Finally, half of the ORFs (50.8%) could not be assigned to a function, emphasizing the untapped potential of the unique marine plasmids to provide proteins with multiple novel functions. Marine plasmids are understudied and hence underrepresented in databases. Plasmid functional annotation and characterization is complicated but, if successful, may provide a pool of novel genes and unknown functions. Newly discovered plasmids and their functional repertoire are potentially valuable tools for predicting the dissemination of antimicrobial resistance, providing vectors for molecular cloning and an understanding of plasmid-bacterial interactions in various environments.
Topics: Indian Ocean; Plasmids; Anti-Bacterial Agents; Metagenomics; Metagenome
PubMed: 37395658
DOI: 10.1128/spectrum.00400-23 -
Microbiology Spectrum Aug 2023Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (). is a...
Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (). is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, -carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of exclusively on IncX4 plasmids. was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS-like element. Based on the genetic diversity of E. coli isolates, transmission of the resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn transposon on the IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described -carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, , has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of and its transmission between different niches.
Topics: Animals; Humans; Colistin; Escherichia coli; Anti-Bacterial Agents; Poultry; Escherichia coli Proteins; Plasmids; Genomics; Drug Resistance, Bacterial; Microbial Sensitivity Tests
PubMed: 37358464
DOI: 10.1128/spectrum.01015-23 -
Nucleic Acids Research Jul 2023Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M...
Many novel traits such as antibiotic resistance are spread by plasmids between species. Yet plasmids have different host ranges. Restriction-modification systems (R-M systems) are by far the most abundant bacterial defense system and therefore represent one of the key barriers to plasmid spread. However, their effect on plasmid evolution and host range has been neglected. Here we analyse the avoidance of targets of the most abundant R-M systems (Type II) for complete genomes and plasmids across bacterial diversity. For the most common target length (6 bp) we show that target avoidance is strongly correlated with the taxonomic distribution of R-M systems and is greater in plasmid genes than core genes. We find stronger avoidance of R-M targets in plasmids which are smaller and have a broader host range. Our results suggest two different evolutionary strategies for plasmids: small plasmids primarily adapt to R-M systems by tuning their sequence composition, and large plasmids primarily adapt through the carriage of additional genes protecting from restriction. Our work provides systematic evidence that R-M systems are important barriers to plasmid transfer and have left their mark on plasmids over long evolutionary time.
Topics: DNA Restriction-Modification Enzymes; Plasmids; Bacteria; Adaptation, Physiological; Drug Resistance, Microbial
PubMed: 37254807
DOI: 10.1093/nar/gkad452 -
International Journal of Nanomedicine 2023Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors....
INTRODUCTION
Chimeric antigen receptor (CAR) cell therapy represents a hallmark in cancer immunotherapy, with significant clinical results in the treatment of hematological tumors. However, current approved methods to engineer T cells to express CAR use viral vectors, which are integrative and have been associated with severe adverse effects due to constitutive expression of CAR. In this context, non-viral vectors such as ionizable lipid nanoparticles (LNPs) arise as an alternative to engineer CAR T cells with transient expression of CAR.
METHODS
Here, we formulated a mini-library of LNPs to deliver pDNA to T cells by varying the molar ratios of excipient lipids in each formulation. LNPs were characterized and screened in vitro using a T cell line (Jurkat). The optimized formulation was used ex vivo to engineer T cells derived from human peripheral blood mononuclear cells (PBMCs) for the expression of an anti-CD19 CAR (CAR-CD19BBz). The effectiveness of these CAR T cells was assessed in vitro against Raji (CD19) cells.
RESULTS
LNPs formulated with different molar ratios of excipient lipids efficiently delivered pDNA to Jurkat cells with low cytotoxicity compared to conventional transfection methods, such as electroporation and lipofectamine. We show that CAR-CD19BBz expression in T cells was transient after transfection with LNPs. Jurkat cells transfected with our top-performing LNPs underwent activation when exposed to CD19 target cells. Using our top-performing LNP-9-CAR, we were able to engineer human primary T cells to express CAR-CD19BBz, which elicited significant specific killing of CD19 target cells in vitro.
CONCLUSION
Collectively, our results show that LNP-mediated delivery of pDNA is a suitable method to engineer human T cells to express CAR, which holds promise for improving the production methods and broader application of this therapy in the future.
Topics: Humans; Excipients; Leukocytes, Mononuclear; Plasmids; Nanoparticles; DNA; Lipids
PubMed: 37873551
DOI: 10.2147/IJN.S424723 -
Microbiology Spectrum Aug 2023In this study, we screened the gene cluster among 1,541 samples obtained from patients, healthy individuals, companion animals, pigs, chicken, and pork and chicken meat...
In this study, we screened the gene cluster among 1,541 samples obtained from patients, healthy individuals, companion animals, pigs, chicken, and pork and chicken meat in Yangzhou, China. As a result, nine strains from humans, animals, and foods were positive for , which was located on plasmids or the chromosome. Seven different sequence types (STs) were identified, including ST15 ( = 2), ST580, ST1944, ST2294, ST5982, ST6262 ( = 2), and ST6265. All the positive strains were clustered into two distinct clades, and they shared a 24,087-bp core structure of , bounded by IS in the same orientation. IS could facilitate rapid and wide dissemination of in from various sources. Tigecycline has been regarded as one of the last-resort antibiotics available for the treatment of infections caused by carbapenem-resistant . The plasmid-mediated resistance-nodulation-division-type efflux pump gene cluster - is a newly identified tigecycline resistance determinant. In this study, we revealed that has disseminated among Klebsiella pneumoniae strains from poultry, food markets, and patients. It is critical to strengthen continuous monitoring, and control measures should be implemented to prevent the further dissemination of .
Topics: Humans; Animals; Swine; Tigecycline; Klebsiella pneumoniae; Klebsiella Infections; Drug Resistance, Bacterial; Anti-Bacterial Agents; Plasmids; Multigene Family; Microbial Sensitivity Tests
PubMed: 37378518
DOI: 10.1128/spectrum.05364-22 -
The Journal of Investigative Dermatology Jan 2024Cutibacterium acnes is a commensal bacterium on the skin that is generally well-tolerated, but different strain types have been hypothesized to contribute to the disease...
Cutibacterium acnes is a commensal bacterium on the skin that is generally well-tolerated, but different strain types have been hypothesized to contribute to the disease acne vulgaris. To understand how some strain types might contribute to skin inflammation, we generated a repository of C. acnes isolates from skin swabs of healthy subjects and subjects with acne and assessed their strain-level identity and capacity to stimulate cytokine release. Phylotype II K-type strains were more frequent on healthy and nonlesional skin of subjects with acne than those isolated from lesions. Phylotype IA-1 C-type strains were increased on lesional skin compared with those on healthy skin. The capacity to induce cytokines from cultured monocyte-derived dendritic cells was opposite to this action on sebocytes and keratinocytes and did not correlate with the strain types associated with the disease. Whole-genome sequencing revealed a linear plasmid in high-inflammatory isolates within similar strain types that had different proinflammatory responses. Single-cell RNA sequencing of mouse skin after intradermal injection showed that strains containing this plasmid induced a higher inflammatory response in dermal fibroblasts. These findings revealed that C. acnes strain type is insufficient to predict inflammation and that carriage of a plasmid could contribute to disease.
Topics: Animals; Mice; Humans; Skin; Acne Vulgaris; Dermatitis; Propionibacterium acnes; Plasmids; Inflammation; Cytokines
PubMed: 37478901
DOI: 10.1016/j.jid.2023.05.029 -
BMC Microbiology Nov 2023Carbapenemase-producing Klebsiella pneumoniae (CPKP) is one of the most dangerous multidrug-resistant (MDR) pathogens in human health due to its widespread circulation...
BACKGROUND
Carbapenemase-producing Klebsiella pneumoniae (CPKP) is one of the most dangerous multidrug-resistant (MDR) pathogens in human health due to its widespread circulation in the nosocomial environment. CPKP carried by companion dogs, which are close to human beings, should be considered a common threat to public health. However, CPKP dissemination through companion animals is still under consideration of major diagnosis and surveillance systems.
METHODS
Two CPKP isolates which were genotyped to harbor bla -encoding IncX3 plasmids, were subjected to the whole-genome study. Whole bacterial DNA was isolated, sequenced, and assembled with Oxford Nanopore long reads and corrected with short reads from the Illumina NovaSeq 6000 platform. The whole-genome structure and positions of antimicrobial resistance (AMR) genes were identified and visualized using CGView. Worldwide datasets were downloaded from the NCBI GenBank database for whole-genome comparative analysis. The whole-genome phylogenetic analysis was constructed using the identified whole-chromosome SNP sites from K. pneumoniae HS11286.
RESULTS
As a result of the whole-genome identification, 4 heterogenous plasmids and a single chromosome were identified, each carrying various AMR genes. Multiple novel structures were identified from the AMR genes, coupled with mobile gene elements (MGE). The comparative whole-genome epidemiology revealed that ST378 K. pneumoniae is a novel type of CPKP, carrying a higher prevalence of AMR genes.
CONCLUSIONS
The characterized whole-genome analysis of this study shows the emergence of a novel type of CPKP strain carrying various AMR genes with variated genomic structures. The presented data in this study show the necessity to develop additional surveillance programs and control measures for a novel type of CPKP strain.
Topics: Animals; Dogs; Humans; Klebsiella pneumoniae; Anti-Bacterial Agents; Phylogeny; Klebsiella Infections; Drug Resistance, Multiple, Bacterial; beta-Lactamases; Plasmids; Carbapenem-Resistant Enterobacteriaceae; Microbial Sensitivity Tests
PubMed: 37924028
DOI: 10.1186/s12866-023-03074-7 -
PLoS Pathogens Apr 2024Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear...
Lyme disease is a tick-borne infection caused by the spirochete Borrelia (Borreliella) burgdorferi. Borrelia species have highly fragmented genomes composed of a linear chromosome and a constellation of linear and circular plasmids some of which are required throughout the enzootic cycle. Included in this plasmid repertoire by almost all Lyme disease spirochetes are the 32-kb circular plasmid cp32 prophages that are capable of lytic replication to produce infectious virions called ϕBB-1. While the B. burgdorferi genome contains evidence of horizontal transfer, the mechanisms of gene transfer between strains remain unclear. While we know that ϕBB-1 transduces cp32 and shuttle vector DNA during in vitro cultivation, the extent of ϕBB-1 DNA transfer is not clear. Herein, we use proteomics and long-read sequencing to further characterize ϕBB-1 virions. Our studies identified the cp32 pac region and revealed that ϕBB-1 packages linear cp32s via a headful mechanism with preferential packaging of plasmids containing the cp32 pac region. Additionally, we find ϕBB-1 packages fragments of the linear chromosome and full-length plasmids including lp54, cp26, and others. Furthermore, sequencing of ϕBB-1 packaged DNA allowed us to resolve the covalently closed hairpin telomeres for the linear B. burgdorferi chromosome and most linear plasmids in strain CA-11.2A. Collectively, our results shed light on the biology of the ubiquitous ϕBB-1 phage and further implicates ϕBB-1 in the generalized transduction of diverse genes and the maintenance of genetic diversity in Lyme disease spirochetes.
Topics: Humans; Borrelia burgdorferi; Bacteriophages; Plasmids; Lyme Disease; Genomics; DNA
PubMed: 38558079
DOI: 10.1371/journal.ppat.1012122