OPEN A CRISPR-Cas nine system protecting E. coli against acquisition of antibiotic resistance genes

OPEN A CRISPR-Cas nine system protecting E. coli against acquisition of antibiotic resistance genes

Antimicrobial resistance is an increasing problem worldwide, and new treatment options for bacterial infections are direly needed. Engineered probiotics show strong potential in treating or preventing bacterial infections. However, one concern with the use of live bacteria is the risk of the bacteria acquiring genes encoding for antimicrobial resistance or virulence factors through horizontal gene transfer, and the transformation of the probiotic into a superbug. Therefore, we developed an engineered CRISPR-Cas nine system that protects bacteria from horizontal gene transfer. We synthesized a CRISPR locus targeting eight antimicrobial resistance genes and cloned this with the Cas nine and transacting tracrRNA on a medium copy plasmid. We next evaluated the efficiency of the system to block horizontal gene transfer through transformation, transduction, and conjugation. Our results show that expression of the CRISPR-Cas nine system successfully protects E. coli MG one six five five from acquiring the targeted resistance genes by transformation or transduction with two to three logs of protection depending on the system for transfer and the target gene. Furthermore, we show that the system blocks conjugation of a set of clinical plasmids, and that the system is also able to protect the probiotic bacterium E. coli Nissle one nine one seven from acquiring antimicrobial resistance genes.

Antimicrobial resistant bacterial infections pose a significant and growing global threat, with an estimated burden of over one point two million deaths worldwide in twenty nineteen directly caused by resistant bacteria, and millions more indirectly. Antimicrobial resistance arising de novo from spontaneous mutations typically provides resistance by reduced antibiotic uptake, increased antibiotic efflux through upregulation of endogenous pump systems, or structural alteration of the target molecule. Additionally, common resistance mechanisms acquired through horizontal gene transfer include antibiotic-specific efflux pumps, alternate metabolic pathways, enzymatic modifications of antibiotic targets, or direct enzymatic inactivation of antibiotics. The intense use of antibiotics worldwide has promoted the mobilization and horizontal transfer of such resistance genes to many pathogenic bacterial species, making horizontal gene transfer the most important factor driving antimicrobial resistance.

Horizontally transferred genes constitute a large part of most bacterial genomes, and the transfer of genes encoding for antimicrobial resistance or virulence factors is important for the emergence of new pathogenic strains. Genes spread amongst microbial communities via different modes of horizontal gene transfer, namely: transformation (direct uptake of DNA from the extracellular environment into bacterial cells), transduction (mediated by phage packaging of host genetic material), and conjugation (direct transfer of plasmids between bacterial cells via conjugative machinery).

With the emergence of multidrug-resistant pathogenic strains, new approaches are needed to combat this rising global health crisis. A promising alternative to traditional antibiotics is the use of probiotic bacteria. Probiotics, defined as live microorganisms that confer health benefits upon ingestion, possess the potential to combat pathogenic bacteria. Extensive research has explored the use of probiotics in treating various gastrointestinal diseases. Among others, irritable bowel syndrome, antibiotic-associated diarrhea, and antibiotics-induced Clostridioides difficile disease have shown positive effects when treated with probiotics. However, the precise mechanisms of how different probiotics protect the host from pathogenic bacteria are not entirely understood. Filling this knowledge void will be important for increasing the efficacy of probiotics, but requires significant efforts from the research community. In the meantime, genetically engineered probiotics allow a more defined way to treat specific diseases, with reported applications ranging from cancer and wound healing to conditions like phenylketonuria, inflammatory bowel disease, and infection. One of the most frequent starting strains for such approaches is the commercially available probiotic strain E. coli Nissle one nine one seven, which is excellent at colonizing the human gut and has been associated with many beneficial traits even without genetic modification. Numerous attempts to genetically engineer E. coli Nissle to prevent diseases ranging from hangover to cancer exist. But a critical concern in probiotic use, including the use of engineered E. coli Nissle, is the potential acquisition of virulence factors and antimicrobial resistance genes from pathogenic strains through horizontal gene transfer within the human gut. Likewise, the ability of probiotic strains to transfer their genetic traits to other gut bacteria warrants careful consideration. Consequently, it is important to develop means to safeguard probiotic strains against horizontal gene transfer while also implementing measures to biocontain potential probiotic strains.

One of the many naturally occurring bacterial defence systems against horizontal gene transfer is CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins). The CRISPR array consists of short repeated sequences separated by similarly short unique sequences called spacers, often matching exogenic sequences such as phages and plasmids. The system also encodes Cas proteins to perform immunity functions. Many types of CRISPR systems (types one through six) have been identified in various bacterial and archaeal hosts. Here, we will focus on the Type two CRISPR system, which is one of the best characterized and more easily engineered than for example the type-one-E system found naturally in E. coli, where multiple proteins make up the endonuclease. The type two system consists of Cas one and two, the endonuclease Cas nine, the CRISPR array encoding guide RNAs, and the trans-activating tracrRNA. CRISPR-Cas immunity is carried out in three main stages: adaptation, expression, and interference.

During adaptation, Cas one and Cas two incorporate new spacers from incoming phage or plasmid DNA into the CRISPR array. This process can be either naïve, where spacers are acquired from a sequence not yet targeted by a spacer, requiring no interference machinery, or primed, where the interference machinery guides adaptation to a sequence that fully or partially matches a crRNA. Next, the CRISPR array and the remaining cas genes are expressed, and the pre-CRISPR RNA is processed into RNA guides by Cas nine and the tracrRNA. Each guide RNA contains a twenty-nucleotide-long guide sequence and palindromic RNA, which serves as the handle for the Cas nine nuclease to bind to. For interference, the twenty-nucleotide guide then directs the Cas nine-RNA complex to the correct DNA sequence by Watson-Crick base pairing, resulting in cleavage and elimination of the invading DNA. To protect the cell from self-destruction, the targeted sequence, or protospacer, is always associated with a protospacer adjacent motif (PAM), which for the CRISPR-Cas system derived from Streptococcus pyogenes (used here) is five prime-N-G-G. The PAM is essential for interference. Structural analyses show that interaction between Cas nine and the N-G-G motif allows melting of the double stranded DNA, providing access for the guide RNA to base pair with the DNA target. This motif is absent in the CRISPR locus, thereby protecting the DNA sequence from cleavage upon CRISPR expression. Thus, when foreign DNA enters a bacterium, through whatever mechanism, where the CRISPR RNA and Cas nine are expressed, RNA guided cleavage of the incoming DNA protects the cell against horizontal gene transfer. Investigation of CRISPR spacers found in nature, suggests that CRISPR systems provide protection against horizontal gene transfer of plasmids as well as phage. An excellent review on CRISPR systems can be found

Thirty-five. Several studies have explored the idea of using CRISPR-Cas systems to empower genetically engineered probiotics, by e.g. delivering CRISPR-Cas systems to specific pathogenic or drug-resistant bacteria to kill them or clear them of their antimicrobial resistance. In a study by Kim et al., a CRISPR-Cas nine system targeting a conserved sequence in TEM- and SHV-type extended-spectrum beta-lactamases could be delivered into extended-spectrum beta-lactamase-producing E. coli and restored antibiotic susceptibility. Similarly, Hao et al. used CRISPR-Cas nine to cure carbapenemase genes and plasmids in Enterobacteriaceae, resulting in efficient resensitizing to carbapenems. In a different approach, CRISPR interference was used to lower the expression of the efflux pump AcrAB-TolC in E. coli and thereby increased the susceptibility to rifampicin, erythromycin and tetracycline.

The successful application of CRISPR-Cas systems in elimination of antimicrobial resistance genes from bacterial populations suggest that it also can be efficient in preventing probiotic E. coli from acquiring antimicrobial resistance genes. In this study, we constructed a CRISPR-Cas nine system and evaluated its potential in protecting probiotic E. coli from acquiring antimicrobial resistance genes.

Materials and methods Strains and growth conditions

All strains used in this study are derivatives of Escherichia coli K-twelve MG one thousand six hundred fifty-five unless specified otherwise. All strains were grown in LB at thirty-seven degrees Celsius, shaking at two hundred RPM unless specified otherwise. Where appropriate, liquid cultures and one point five percent Luria agar plates were supplemented with: Kanamycin fifty milligrams per liter, Chloramphenicol twelve point five milligrams per liter, Ampicillin one hundred milligrams per liter, cefotaxime ten milligrams per liter, and diaminopimelic acid zero point three millimolar.