Investigating the Genomic Background of CRISPR-Cas Genomes for CRISPR-Based Antimicrobials. - Related Documents




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923501.0000Investigating the Genomic Background of CRISPR-Cas Genomes for CRISPR-Based Antimicrobials. CRISPR-Cas systems are an adaptive immunity that protects prokaryotes against foreign genetic elements. Genetic templates acquired during past infection events enable DNA-interacting enzymes to recognize foreign DNA for destruction. Due to the programmability and specificity of these genetic templates, CRISPR-Cas systems are potential alternative antibiotics that can be engineered to self-target antimicrobial resistance genes on the chromosome or plasmid. However, several fundamental questions remain to repurpose these tools against drug-resistant bacteria. For endogenous CRISPR-Cas self-targeting, antimicrobial resistance genes and functional CRISPR-Cas systems have to co-occur in the target cell. Furthermore, these tools have to outplay DNA repair pathways that respond to the nuclease activities of Cas proteins, even for exogenous CRISPR-Cas delivery. Here, we conduct a comprehensive survey of CRISPR-Cas genomes. First, we address the co-occurrence of CRISPR-Cas systems and antimicrobial resistance genes in the CRISPR-Cas genomes. We show that the average number of these genes varies greatly by the CRISPR-Cas type, and some CRISPR-Cas types (IE and IIIA) have over 20 genes per genome. Next, we investigate the DNA repair pathways of these CRISPR-Cas genomes, revealing that the diversity and frequency of these pathways differ by the CRISPR-Cas type. The interplay between CRISPR-Cas systems and DNA repair pathways is essential for the acquisition of new spacers in CRISPR arrays. We conduct simulation studies to demonstrate that the efficiency of these DNA repair pathways may be inferred from the time-series patterns in the RNA structure of CRISPR repeats. This bioinformatic survey of CRISPR-Cas genomes elucidates the necessity to consider multifaceted interactions between different genes and systems, to design effective CRISPR-based antimicrobials that can specifically target drug-resistant bacteria in natural microbial communities.202235692726
921410.9998Enabling genetic analysis of diverse bacteria with Mobile-CRISPRi. The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans(1-6). However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.201930617347
923420.9998CRISPR provides acquired resistance against viruses in prokaryotes. Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.200717379808
920030.9998Application of the CRISPR/Cas System for Generation of Pathogen-Resistant Plants. The use of the CRISPR/Cas9 prokaryotic adaptive immune system has led to a breakthrough in targeted genome editing in eukaryotes. The CRISPR/Cas technology allows to generate organisms with desirable characteristics by introducing deletions/insertions into selected genome loci resulting in the knockout or modification of target genes. This review focuses on the current state of the CRISPR/Cas use for the generation of plants resistant to viruses, bacteria, and parasitic fungi. Resistance to DNA- and RNA-containing viruses is usually provided by expression in transgenic plants of the Cas endonuclease gene and short guide RNAs (sgRNAs) targeting certain sites in the viral or the host plant genomes to ensure either direct cleavage of the viral genome or modification of the plant host genome in order to decrease the efficiency of virus replication. Editing of plant genes involved in the defense response to pathogens increases plants resistance to bacteria and pathogenic fungi. The review explores strategies and prospects of the development of pathogen-resistant plants with a focus on the generation of non-transgenic (non-genetically modified) organisms, in particular, by using plasmid (DNA)-free systems for delivery of the Cas/sgRNA editing complex into plant cells.201830878030
921540.9998Bacterial type IV secretion systems and spread of antimicrobial resistance: a study of potential inhibitors to T4SS-based resistance spread. Antimicrobial resistance (AMR) is a major global health threat, mainly driven by the rapid spread of resistance genes through horizontal gene transfer (HGT). The Type IV Secretion System (T4SS) acts as a crucial molecular machinery that facilitates this process, allowing bacteria to transfer DNA, effector proteins, and virulence factors. This review systematically explores the structural and functional diversity of T4SS, its role in spreading AMR, and current methods for its inhibition. T4SS consists of a multi-protein complex that spans bacterial membranes, mediating conjugative plasmid transfer, host-pathogen interactions, and bacterial competition. Key components include ATPases, pilus structures, and membrane-associated proteins that show both conserved features and species-specific adaptations. These traits enable functional specialization across Gram-positive and Gram-negative bacteria, significantly contributing to the spread of vital resistance genes like extended-spectrum β-lactamases and carbapenemases via mobile genetic elements. Several approaches have been developed to inhibit T4SS and combat AMR. Small molecules targeting ATPase activity or protein interactions are promising, as are natural phytochemicals that interfere with conjugation. Bacteriophage therapy provides another strategy by specifically targeting plasmid-carrying bacteria. Host immune responses, such as innate immune recognition and secretory immunoglobulins, also show potential to influence T4SS activity. Although progress has been made, challenges remain, especially in developing selective inhibition methods that do not harm beneficial microbiota or host cells. Future research should focus on high-resolution structural studies to support rational drug design and preclinical testing of combination therapies that include T4SS inhibitors with existing antibiotics. Gaining a deeper understanding of T4SS regulation and host-pathogen interactions will be vital for creating targeted AMR strategies that also maintain ecological balance.202540956426
921850.9998CRISPR-Cas System: A New Dawn to Combat Antibiotic Resistance. Antimicrobial resistance (AMR) can potentially harm global public health. Horizontal gene transfer (HGT), which speeds up the emergence of AMR and increases the burden of drug resistance in mobile genetic elements (MGEs), is the primary method by which AMR genes are transferred across bacterial pathogens. New approaches are urgently needed to halt the spread of bacterial diseases and antibiotic resistance. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), an RNA-guided adaptive immune system, protects prokaryotes from foreign DNA like plasmids and phages. This approach may be essential in limiting horizontal gene transfer and halting the spread of antibiotic resistance. The CRISPR-Cas system has been crucial in identifying and understanding resistance mechanisms and developing novel therapeutic approaches. This review article investigates the CRISPR-Cas system's potential as a tool to combat bacterial AMR. Antibiotic-resistant bacteria can be targeted and eliminated by the CRISPR-Cas system. It has been proven to be an efficient method for removing carbapenem-resistant plasmids and regaining antibiotic susceptibility. The CRISPR-Cas system has enormous potential as a weapon against bacterial AMR. It precisely targets and eliminates antibiotic-resistant bacteria, facilitates resistance mechanism identification, and offers new possibilities in diagnostics and therapeutics.202438605260
826260.9998Advances in CRISPR-Cas systems for human bacterial disease. Prokaryotic adaptive immune systems called CRISPR-Cas systems have transformed genome editing by allowing for precise genetic alterations through targeted DNA cleavage. This system comprises CRISPR-associated genes and repeat-spacer arrays, which generate RNA molecules that guide the cleavage of invading genetic material. CRISPR-Cas is classified into Class 1 (multi-subunit effectors) and Class 2 (single multi-domain effectors). Its applications span combating antimicrobial resistance (AMR), targeting antibiotic resistance genes (ARGs), resensitizing bacteria to antibiotics, and preventing horizontal gene transfer (HGT). CRISPR-Cas3, for example, effectively degrades plasmids carrying resistance genes, providing a precise method to disarm bacteria. In the context of ESKAPE pathogens, CRISPR technology can resensitize bacteria to antibiotics by targeting specific resistance genes. Furthermore, in tuberculosis (TB) research, CRISPR-based tools enhance diagnostic accuracy and facilitate precise genetic modifications for studying Mycobacterium tuberculosis. CRISPR-based diagnostics, leveraging Cas endonucleases' collateral cleavage activity, offer highly sensitive pathogen detection. These advancements underscore CRISPR's transformative potential in addressing AMR and enhancing infectious disease management.202439266183
947570.9998Rapidly evolving genes in pathogens: methods for detecting positive selection and examples among fungi, bacteria, viruses and protists. The ongoing coevolutionary struggle between hosts and pathogens, with hosts evolving to escape pathogen infection and pathogens evolving to escape host defences, can generate an 'arms race', i.e., the occurrence of recurrent selective sweeps that each favours a novel resistance or virulence allele that goes to fixation. Host-pathogen coevolution can alternatively lead to a 'trench warfare', i.e., balancing selection, maintaining certain alleles at loci involved in host-pathogen recognition over long time scales. Recently, technological and methodological progress has enabled detection of footprints of selection directly on genes, which can provide useful insights into the processes of coevolution. This knowledge can also have practical applications, for instance development of vaccines or drugs. Here we review the methods for detecting genes under positive selection using divergence data (i.e., the ratio of nonsynonymous to synonymous substitution rates, d(N)/d(S)). We also review methods for detecting selection using polymorphisms, such as methods based on F(ST) measures, frequency spectrum, linkage disequilibrium and haplotype structure. In the second part, we review examples where targets of selection have been identified in pathogens using these tests. Genes under positive selection in pathogens have mostly been sought among viruses, bacteria and protists, because of their paramount importance for human health. Another focus is on fungal pathogens owing to their agronomic importance. We finally discuss promising directions in pathogen studies, such as detecting selection in non-coding regions.200919442589
921780.9998Role of CRISPR-Cas systems and anti-CRISPR proteins in bacterial antibiotic resistance. The emergence and development of antibiotic resistance in bacteria is a serious threat to global public health. Antibiotic resistance genes (ARGs) are often located on mobile genetic elements (MGEs). They can be transferred among bacteria by horizontal gene transfer (HGT), leading to the spread of drug-resistant strains and antibiotic treatment failure. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) is one of the many strategies bacteria have developed under long-term selection pressure to restrict the HGT. CRISPR-Cas systems exist in about half of bacterial genomes and play a significant role in limiting the spread of antibiotic resistance. On the other hand, bacteriophages and other MGEs encode a wide range of anti-CRISPR proteins (Acrs) to counteract the immunity of the CRISPR-Cas system. The Acrs could decrease the CRISPR-Cas system's activity against phages and facilitate the acquisition of ARGs and virulence traits for bacteria. This review aimed to assess the relationship between the CRISPR-Cas systems and Acrs with bacterial antibiotic resistance. We also highlighted the CRISPR technology and Acrs to control and prevent antibacterial resistance. The CRISPR-Cas system can target nucleic acid sequences with high accuracy and reliability; therefore, it has become a novel gene editing and gene therapy tool to prevent the spread of antibiotic resistance. CRISPR-based approaches may pave the way for developing smart antibiotics, which could eliminate multidrug-resistant (MDR) bacteria and distinguish between pathogenic and beneficial microorganisms. Additionally, the engineered anti-CRISPR gene-containing phages in combination with antibiotics could be used as a cutting-edge treatment approach to reduce antibiotic resistance.202439149034
939890.9998Effectiveness of CRISPR-Cas in Sensitizing Bacterial Populations with Plasmid-Encoded Antimicrobial Resistance. The spread of bacteria resistant to antibiotics poses a serious threat to human health. Genes that encode antibiotic resistance are often harbored on plasmids, extra-chromosomal DNA molecules found in bacteria. The emergence of multiresistance plasmids is particularly problematic and demands the development of new antibiotics and alternative strategies. CRISPR-Cas derived tools with their sequence specificity offer a promising new approach to combating antibiotic resistance. By introducing CRISPR-Cas encoding plasmids that %specifically target antibiotic resistance genes on plasmids, the susceptibility of bacteria to conventional antibiotics can be restored. However, genetic variation within bacterial populations can hinder the effectiveness of such CRISPR-Cas tools by allowing some mutant plasmids to evade CRISPR-mediated cleaving or gene silencing. In this study, we develop a model to test the effectiveness of CRISPR-Cas in sensitizing bacterial populations carrying resistance on non-transmissible plasmids and assess the success probability of a subsequent treatment with conventional antibiotics. We evaluate this probability according to the target interference mechanism, the copy number of the resistance-encoding plasmid, and its compatibility with the CRISPR-Cas encoding plasmid. Our results identify promising approaches to revert antibiotic resistance with CRISPR-Cas encoding plasmids: A DNA-cleaving CRISPR-Cas system on a plasmid incompatible with the targeted plasmid is most effective for low copy numbers, while for resistance plasmids with higher copy numbers gene silencing by CRISPR-Cas systems encoded on compatible plasmids is the superior solution.202540985758
9671100.9998Genome-scale genetic manipulation methods for exploring bacterial molecular biology. Bacteria are diverse and abundant, playing key roles in human health and disease, the environment, and biotechnology. Despite progress in genome sequencing and bioengineering, much remains unknown about the functional organization of prokaryotes. For instance, roughly a third of the protein-coding genes of the best-studied model bacterium, Escherichia coli, currently lack experimental annotations. Systems-level experimental approaches for investigating the functional associations of bacterial genes and genetic structures are essential for defining the fundamental molecular biology of microbes, preventing the spread of antibacterial resistance in the clinic, and driving the development of future biotechnological applications. This review highlights recently introduced large-scale genetic manipulation and screening procedures for the systematic exploration of bacterial gene functions, molecular relationships, and the global organization of bacteria at the gene, pathway, and genome levels.201222517266
9205110.9997Resistance induction based on the understanding of molecular interactions between plant viruses and host plants. BACKGROUND: Viral diseases cause significant damage to crop yield and quality. While fungi- and bacteria-induced diseases can be controlled by pesticides, no effective approaches are available to control viruses with chemicals as they use the cellular functions of their host for their infection cycle. The conventional method of viral disease control is to use the inherent resistance of plants through breeding. However, the genetic sources of viral resistance are often limited. Recently, genome editing technology enabled the publication of multiple attempts to artificially induce new resistance types by manipulating host factors necessary for viral infection. MAIN BODY: In this review, we first outline the two major (R gene-mediated and RNA silencing) viral resistance mechanisms in plants. We also explain the phenomenon of mutations of host factors to function as recessive resistance genes, taking the eIF4E genes as examples. We then focus on a new type of virus resistance that has been repeatedly reported recently due to the widespread use of genome editing technology in plants, facilitating the specific knockdown of host factors. Here, we show that (1) an in-frame mutation of host factors necessary to confer viral resistance, sometimes resulting in resistance to different viruses and that (2) certain host factors exhibit antiviral resistance and viral-supporting (proviral) properties. CONCLUSION: A detailed understanding of the host factor functions would enable the development of strategies for the induction of a new type of viral resistance, taking into account the provision of a broad resistance spectrum and the suppression of the appearance of resistance-breaking strains.202134454519
9233120.9997The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Bacteria and Archaea have developed several defence strategies against foreign nucleic acids such as viral genomes and plasmids. Among them, clustered regularly interspaced short palindromic repeats (CRISPR) loci together with cas (CRISPR-associated) genes form the CRISPR/Cas immune system, which involves partially palindromic repeats separated by short stretches of DNA called spacers, acquired from extrachromosomal elements. It was recently demonstrated that these variable loci can incorporate spacers from infecting bacteriophages and then provide immunity against subsequent bacteriophage infections in a sequence-specific manner. Here we show that the Streptococcus thermophilus CRISPR1/Cas system can also naturally acquire spacers from a self-replicating plasmid containing an antibiotic-resistance gene, leading to plasmid loss. Acquired spacers that match antibiotic-resistance genes provide a novel means to naturally select bacteria that cannot uptake and disseminate such genes. We also provide in vivo evidence that the CRISPR1/Cas system specifically cleaves plasmid and bacteriophage double-stranded DNA within the proto-spacer, at specific sites. Our data show that the CRISPR/Cas immune system is remarkably adapted to cleave invading DNA rapidly and has the potential for exploitation to generate safer microbial strains.201021048762
9240130.9997CRISPR-Cas-Mediated Phage Resistance Enhances Horizontal Gene Transfer by Transduction. A powerful contributor to prokaryotic evolution is horizontal gene transfer (HGT) through transformation, conjugation, and transduction, which can be advantageous, neutral, or detrimental to fitness. Bacteria and archaea control HGT and phage infection through CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immunity. Although the benefits of resisting phage infection are evident, this can come at a cost of inhibiting the acquisition of other beneficial genes through HGT. Despite the ability of CRISPR-Cas to limit HGT through conjugation and transformation, its role in transduction is largely overlooked. Transduction is the phage-mediated transfer of bacterial DNA between cells and arguably has the greatest impact on HGT. We demonstrate that in Pectobacterium atrosepticum, CRISPR-Cas can inhibit the transduction of plasmids and chromosomal loci. In addition, we detected phage-mediated transfer of a large plant pathogenicity genomic island and show that CRISPR-Cas can inhibit its transduction. Despite these inhibitory effects of CRISPR-Cas on transduction, its more common role in phage resistance promotes rather than diminishes HGT via transduction by protecting bacteria from phage infection. This protective effect can also increase transduction of phage-sensitive members of mixed populations. CRISPR-Cas systems themselves display evidence of HGT, but little is known about their lateral dissemination between bacteria and whether transduction can contribute. We show that, through transduction, bacteria can acquire an entire chromosomal CRISPR-Cas system, including cas genes and phage-targeting spacers. We propose that the positive effect of CRISPR-Cas phage immunity on enhancing transduction surpasses the rarer cases where gene flow by transduction is restricted.IMPORTANCE The generation of genetic diversity through acquisition of DNA is a powerful contributor to microbial evolution and occurs through transformation, conjugation, and transduction. Of these, transduction, the phage-mediated transfer of bacterial DNA, is arguably the major route for genetic exchange. CRISPR-Cas adaptive immune systems control gene transfer by conjugation and transformation, but transduction has been mostly overlooked. Our results indicate that CRISPR-Cas can impede, but typically enhances the transduction of plasmids, chromosomal genes, and pathogenicity islands. By limiting wild-type phage replication, CRISPR-Cas immunity increases transduction in both phage-resistant and -sensitive members of mixed populations. Furthermore, we demonstrate mobilization of a chromosomal CRISPR-Cas system containing phage-targeting spacers by generalized transduction, which might partly account for the uneven distribution of these systems in nature. Overall, the ability of CRISPR-Cas to promote transduction reveals an unexpected impact of adaptive immunity on horizontal gene transfer, with broader implications for microbial evolution.201829440578
9232140.9997CRISPR interference can prevent natural transformation and virulence acquisition during in vivo bacterial infection. Pathogenic bacterial strains emerge largely due to transfer of virulence and antimicrobial resistance genes between bacteria, a process known as horizontal gene transfer (HGT). Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci of bacteria and archaea encode a sequence-specific defense mechanism against bacteriophages and constitute a programmable barrier to HGT. However, the impact of CRISPRs on the emergence of virulence is unknown. We programmed the human pathogen Streptococcus pneumoniae with CRISPR sequences that target capsule genes, an essential pneumococcal virulence factor, and show that CRISPR interference can prevent transformation of nonencapsulated, avirulent pneumococci into capsulated, virulent strains during infection in mice. Further, at low frequencies bacteria can lose CRISPR function, acquire capsule genes, and mount a successful infection. These results demonstrate that CRISPR interference can prevent the emergence of virulence in vivo and that strong selective pressure for virulence or antibiotic resistance can lead to CRISPR loss in bacterial pathogens.201222901538
9620150.9997Determinants of Phage Host Range in Staphylococcus Species. Bacteria in the genus Staphylococcus are important targets for phage therapy due to their prevalence as pathogens and increasing antibiotic resistance. Here we review Staphylococcus outer surface features and specific phage resistance mechanisms that define the host range, the set of strains that an individual phage can potentially infect. Phage infection goes through five distinct phases: attachment, uptake, biosynthesis, assembly, and lysis. Adsorption inhibition, encompassing outer surface teichoic acid receptor alteration, elimination, or occlusion, limits successful phage attachment and entry. Restriction-modification systems (in particular, type I and IV systems), which target phage DNA inside the cell, serve as the major barriers to biosynthesis as well as transduction and horizontal gene transfer between clonal complexes and species. Resistance to late stages of infection occurs through mechanisms such as assembly interference, in which staphylococcal pathogenicity islands siphon away superinfecting phage proteins to package their own DNA. While genes responsible for teichoic acid biosynthesis, capsule, and restriction-modification are found in most Staphylococcus strains, a variety of other host range determinants (e.g., clustered regularly interspaced short palindromic repeats, abortive infection, and superinfection immunity) are sporadic. The fitness costs of phage resistance through teichoic acid structure alteration could make staphylococcal phage therapies promising, but host range prediction is complex because of the large number of genes involved, and the roles of many of these are unknown. In addition, little is known about the genetic determinants that contribute to host range expansion in the phages themselves. Future research must identify host range determinants, characterize resistance development during infection and treatment, and examine population-wide genetic background effects on resistance selection.201930902858
9617160.9997Multiplex CRISPRi System Enables the Study of Stage-Specific Biofilm Genetic Requirements in Enterococcus faecalis. Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multilevel regulation. Compared to established knockout methodologies, CRISPR interference (CRISPRi) approaches enable the rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here, we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both nontemplate and template strand targeting. Since the nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacterium interactions, and interspecies communication.IMPORTANCEEnterococcus faecalis causes multidrug-resistant life-threatening infections and is often coisolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labor-intensive allelic-exchange methods. Built upon streptococcal dCas9, we developed an easily modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation and antibiotic resistance and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.202033082254
9622170.9997Stable Neutralization of a Virulence Factor in Bacteria Using Temperate Phage in the Mammalian Gut. Elimination or alteration of select members of the gut microbiota is key to therapeutic efficacy. However, the complexity of these microbial inhabitants makes it challenging to precisely target bacteria. Here, we deliver exogenous genes to specific bacteria by genomic integration of temperate phage for long-lasting modification. As a real-world therapeutic test, we engineered λ phage to transcriptionally repress Shiga toxin by using genetic hybrids between λ and other lambdoid phages to overcome resistance encoded by the virulence-expressing prophage. We show that a single dose of engineered phage propagates throughout the bacterial community and reduces Shiga toxin production in an enteric mouse model of infection without markedly affecting bacterial concentrations. Our work reveals a new framework for transferring functions to bacteria within their native environment.IMPORTANCE With the increasing frequency of antibiotic resistance, it is critical to explore new therapeutic strategies for treating bacterial infections. Here, we use a temperate phage, i.e., one that integrates itself into the bacterial genome, to neutralize the expression of a virulence factor by modifying bacterial function at the genetic level. We show that Shiga toxin production can be significantly reduced in vitro and in the mammalian gut. Alternative to traditional applications of phage therapy that rely on killing bacteria, our genetics-based antivirulence approach introduces a new framework for treating bacterial infections.202031992629
9623180.9997Prokaryotic toxin-antitoxin systems--the role in bacterial physiology and application in molecular biology. Bacteria have developed multiple complex mechanisms ensuring an adequate response to environmental changes. In this context, bacterial cell division and growth are subject to strict control to ensure metabolic balance and cell survival. A plethora of studies cast light on toxin-antitoxin (TA) systems as metabolism regulators acting in response to environmental stress conditions. Many of those studies suggest direct relations between the TA systems and the pathogenic potential or antibiotic resistance of relevant bacteria. Other studies point out that TA systems play a significant role in ensuring stability of mobile genetic material. The evolutionary origin and relations between various TA systems are still a subject of a debate. The impact of toxin-antitoxin systems on bacteria physiology prompted their application in molecular biology as tools allowing cloning of some hard-to-maintain genes, plasmid maintenance and production of recombinant proteins.201121394325
9188190.9997CRISPR-Cas system, antibiotic resistance and virulence in bacteria: Through a common lens. CRISPR-Cas system, antibiotic resistance and virulence are different modes of survival for the bacteria. CRISPR-Cas is an adaptive immune system that can degrade foreign DNA, antibiotic resistance helps bacteria to evade drugs that can threaten their existence and virulence determinants are offensive tools that can facilitate the establishment of infection by pathogens. This chapter focuses on these three aspects, providing insights about the CRISPR system and resistance mechanisms in brief, followed by understanding the synergistic or antagonistic relationship of resistance and virulence determinants in connection to the CRISPR system. We have addressed the discussion of this evolving topic through specific examples and studies. Different approaches for successful detection of this unique defense system in bacteria and various applications of the CRISPR-Cas systems to show how it can be harnessed to tackle the increasing problem of antibiotic resistance have been put forth. World Health Organization has declared antibiotic resistance as a serious global problem of the 21st century. As antibiotic-resistant bacteria increase their footprint across the globe, newer tools such as the CRISPR-Cas system hold immense promise to tackle this problem.202133685595