Engineering a CRISPR Interference System To Repress a Class One Integron in Escherichia coli

Engineering a CRISPR Interference System To Repress a Class One Integron in Escherichia coli

ABSTRACT Microbial multidrug resistance poses a huge threat to human health. Bacterial acquisition of multidrug resistance relies primarily on class one integron-involved horizontal gene transfer of antibiotic resistance genes. To date, no strategies other than the use of antibiotics can efficiently cope with multidrug resistance. Here, we report that an engineered CRISPR interference system can markedly reduce multidrug resistance by blocking a class one integron in Escherichia coli. Using CRISPR interference to block plasmid R three eight eight class one integron, E. coli recombinants showed halted growth upon exposure to relevant antibiotics. A microplate alamarBlue assay showed that both subgenomic RNAs R three and R six led to eight- and thirty-two-fold decreases in half-maximal inhibitory concentrations for trimethoprim and sulfamethoxazole, respectively. Reverse transcription and quantitative PCR revealed that the strain employing subgenomic RNA R six exhibited ninety-seven percent and eighty-four percent decreases in the transcriptional levels of the dfrB two cassette and sul one, two typical antibiotic resistance genes, respectively. Quantitative PCR analysis also demonstrated that the strain recruiting subgenomic RNA R three showed a ninety-six percent decrease in the transcriptional level of int one, and a conjugation assay revealed a one thousand-fold decrease in horizontal gene transfer rates of antibiotic resistance genes. Overall, the subgenomic RNA R three targeting the thirty-one base pairs downstream of the Pc promoter on the int one nontemplate strand outperformed other subgenomic RNAs in reducing integron activity. Furthermore, this CRISPR interference system is reversible, genetically stable, and titratable by varying the concentration of the inducer. To our knowledge, this is the first report on exploiting a CRISPR interference system to reduce the class one integron in E. coli. This study provides valuable insights for future development of CRISPR interference-based antimicrobial agents and cellular therapy to suppress multidrug resistance.

Recent years have witnessed the severe threat of antibiotic-resistant pathogens to human health. In the United States, approximately two million patients each year are infected with antibiotic-resistant pathogens, resulting in at least twenty-three thousand fatalities, and this situation is getting worse. Antibiotics have been extensively harnessed to combat pathogen infections. However, overuse of them accelerates the evolution of microbial multidrug resistance, and this situation necessitates combinatorial use of antibiotics. Multidrug resistance is largely attributed to horizontal gene transfer of antibiotic resistance genes, and horizontal gene transfer is typically accomplished by mobile genetic elements through transformation, conjugation, and transduction. Among all types of mobile genetic elements, mobile integrons are commonly found in clinical settings and other circumstances and played a crucial role in the early rise of multidrug resistance among clinically relevant bacteria in the nineteen sixties. Indeed, increasing evidence has shown that the transmission of antibiotic resistance genes among Gram-negative pathogens is frequently brought by the mobile integron-involved horizontal gene transfer of antibiotic resistance genes.

MECHANISMS OF RESISTANCE

Mobile integrons usually work with transposons, insertion sequences, and conjugative plasmids and participate in the acquisition, expression, and dissemination of antibiotic resistance genes embedded in gene cassettes. Thus, integrons contribute to the transmission of bacterial antibiotic resistance. Structurally, almost all integrons are composed of three parts: one, an int one gene (driven by a native promoter Pint), which encodes an Int one integrase belonging to tyrosine recombinase family; two, a primary recombination site, att one, which serves as both the recognition site of Int one integrase and the receptor site for gene cassettes; and three, a Pc promoter within the int one coding sequence, which drives the transcription of gene cassettes inserted at the att one site. The gene cassette usually harbors an open reading frame surrounded by an integrase-specific recombination site, attC. Typically, the integration of the gene cassette into integron is fulfilled by Int one integrase through site-specific recombination between att one and attC or between two attC sites. Once integrated, gene cassettes are expressed under the control of the Pc promoter and transcribed only in a direction opposite to that of int one. In addition, when subjected to stimuli (e.g., antibiotic selective pressure), the gene cassette located between two adjacent attC sites might be excised by Int one integrase, resulting in the rearrangement of internal gene cassettes or gene capture by other integrons. The entire process involves integration, expression, and excision of gene cassettes, leading to physiological alternations or even novel genetic traits adapting to environments.

Among five classes of mobile integrons, class one integrons are the most disseminated type in commensals and pathogens of humans and animals and have been found in other ecosystems. Structurally, the three prime conserved segment in the class one integron comprises three elements: qacE forty-one (cationic compound disinfectant resistance gene; GenBank accession number NG zero four eight zero four two), sul one (sulfamethoxazole resistance gene; GenBank accession number WP zero zero zero two five nine zero three one), and orf five (open reading frame with unknown function). It is extremely challenging to eliminate class one integrons as their mobility allows them to move onto other recipients to ensure their persistence. To date, no efficient approach has been developed to block class one integrons. One exception is struvite in combination with a biochar amendment, which has been shown to suppress class one integrons in phyllosphere and rhizosphere soils. Other exceptions are treatments of residual wastewater solids, such as thermophilic anaerobic digestion, alkaline stabilization, and pasteurization. These approaches can thwart class one integrons in wastewater solids-amended soil. So far, at least one hundred thirty-two antibiotic resistance gene cassettes have been identified which confer resistances to almost all types of antibiotics. Therefore, it is highly desirable to come up with an approach suppressing class one integrons to counteract the devastating effects of multidrug resistances.

CRISPR technology opens avenues for genome editing and gene regulation. In sharp contrast to successful application in fungi, especially Saccharomyces cerevisiae, CRISPR editing in bacteria is problematic due mainly to the lack of corresponding DNA repair mechanisms. Derived from CRISPR-Cas nine, CRISPR interference consists mainly of one or several subgenomic RNAs and a catalytically dead Cas nine protein lacking endonuclease activity. With the goal of knockdown rather than knockout of a gene, CRISPRi technology does not rely on the cellular innate DNA repair machinery and thus is functional in microbes lacking a DNA repair mechanism. The CRISPRi system has been widely harnessed to retard the initiation or elongation of gene transcription. Notably, the CRISPRi system can simultaneously upregulate or downregulate multiple genes due to the RNAs which lead dCas nine to desired genomic loci. Considering that integron-assisted MDR involves a series of genes, CRISPRi may simultaneously interfere with multiple ARGs and thus hold great potential for prevention of bacterial infection and MDR treatment. For instance, simply by encapsulating the dCas nine and a panel of RNAs into nanoparticles and incubating these with bacteria for transformation, a wide range of ARGs could be inhibited, achieving desired therapeutic effects.

Given the above information, we conjectured that the CRISPRi system may efficiently reduce MDR by blocking class one integrons. Following this assumption, we developed a CRISPRi system in Escherichia coli C six hundred to curb the class one integron on the conjugative plasmid R three hundred eighty-eight. Reverse transcription and quantitative PCR analysis were performed to assess the ability of a CRISPRi system to repress the IntI one integrase gene and ARGs. A microplate alamarBlue assay and growth measurement were performed to dissect the performance of the CRISPRi system in mitigating antibiotic resistance arising from the class one integron. Conjugation assays were carried out to disentangle the impacts of the CRISPRi system on HGT of ARGs associated with the IntI one integrase-mediated integration of ARG cassettes. A titration experiment was performed to determine whether the activity of the CRISPRi system can be controlled by varying the concentration of the inducer. Finally, serial subculture experiments were conducted to investigate the reversibility and hereditary stability of a CRISPRi system.

Overall, this study aims to develop a CRISPRi system capable of repressing class one integron-aided MDR of E. coli and other bacteria.