Abstract
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.IMPORTANCE Enterococcus 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.
Highlights
Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections
The well-studied type II CRISPR-Cas system consists of a DNA endonuclease (Cas9) that is guided to the bacterial chromosome by a short 20-nucleotide single guide RNA, where they generate a double-stranded DNA break by recognizing a 2- to 6-bp DNA sequence called a protospacer-adjacent motif (PAM) that immediately follows the targeted gene sequence [14]
Barcoded guide RNA sequences with a dead” Cas9 (dCas9) handle under the control of the same nisA promoter were synthesized as gBlocks (IDT, USA) and cloned into the pGCP123 expression vector by an In-Fusion reaction to generate pGCP123-single guide RNA (sgRNA) [8, 23, 29]
Summary
Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. 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. Built upon streptococcal dCas, we developed an 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. This tool is optimized to study genes important for biofilm initiation, maturation, and maintenance and can be used to perturb preformed biofilms. Because S. pyogenes dCas performance varies in different species, with low knockdown efficiency and proteotoxicity in Mycobacterium tuberculosis, for example, dCas from other species such as Streptococcus thermophilus have been effectively used for CRISPRi [24, 25]
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