CRISPR-Cas systems in enterococci

Abstract

Enterococci are members of the microbiota of humans and other animals. They can also be found in the environment, associated with food, healthcare infections, and hospital settings. Due to their wide distribution, they are inserted in the One Health context. The selective pressure caused by the extensive use of antimicrobial agents in humans, animals, and agriculture has increased the frequency of resistance to various drugs among enterococcal species. CRISPR-Cas system, an important prokaryotic defense mechanism against the entry of mobile genetic elements, may prevent the acquisition of genes involved in antimicrobial resistance and virulence. This system has been increasingly used as a gene editing tool, which can be used as a way to recognize and inactivate genes of interest. Here, we conduct a review on CRISPR systems found in enterococci, considering their occurrence, structure and organization, mechanisms of action and use as a genetic engineering technology. Type II-A CRISPR-Cas systems were shown to be the most frequent among enterococcal species, and the orphan CRISPR2 was the most commonly found system (54.1%) among enterococcal species, especially in Enterococcus faecalis. Distribution of CRISPR systems varied among species. CRISPR systems had 1 to 20 spacers, with size between 23 and 37 bp and direct repeat sequences from 25 to 37 bp. Several applications of the CRISPR-Cas biotechnology have been described in enterococci, mostly in vitro, using this editing tool to target resistance- and virulence-related genes.

Introduction

Enterococci are Gram-positive bacteria that are usually found as members of the microbiota of humans and other animals. They can be also found in the environment, as well as associated with food. Due to their wide distribution, they are inserted in the One Health context. In humans, enterococcal species cause opportunistic infections, which mostly include soft tissue infection, urinary tract infection, and bacteremia [1]. Despite being part of the human intestinal microbiota, enterococci have emerged as some of the main pathogens of healthcare-related infections (HAI), especially in hospital settings [2]. More than 60 enterococcal species are currently described with a validly published and correct name (https://lpsn.dsmz.de/genus/enterococcus). Enterococcus faecalis and Enterococcus faecium are the most frequent species in humans and they are frequently associated with antimicrobial resistance, especially E. faecium [2].

To treat severe enterococcal infections, such as endocarditis, a beta-lactam (e.g., ampicillin) and an aminoglycoside at high concentration (e.g., gentamicin) are used synergistically to achieve a bactericidal effect. In case of resistance, vancomycin is the main alternative [3]. However, vancomycin-resistant enterococci (VRE) have been detected since the 1980’s [4] and these strains are currently considered a major global public health concern in terms of antibiotic resistance. VRE have been classified as a serious threat in the United States and vancomycin-resistant E. faecium belongs to the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter spp.), which includes the most relevant multidrug-resistant bacterial pathogens [5, 6]. Vancomycin-resistant E. faecium is also included in the World Health Organization (WHO) list of bacterial priority pathogens for which new antibiotics are urgently needed. This antibiotic-resistant species is of particular importance due to its ability to transmit resistance elements across the One Health spectrum [7]. Therefore, finding new or alternative therapeutic options for the treatment or control of enterococcal infections is of great interest.

Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (Cas) proteins, known as CRISPR-Cas systems, act as a prokaryotic adaptive immune system against mobile genetic elements, such as bacteriophages and plasmids. Once the genetic material (i.e., DNA or RNA) of the exogenous element enter the bacterial cell, the CRISPR-Cas system can recognize, process, and incorporate a fragment of the exogenous DNA into the 5’-end of the CRISPR locus [8–10]. CRISPR systems are mostly located on chromosomes, but they can also be found in plasmids, such as for Lactococcus lactis [11, 12].

The most common methods used to detect CRISPR systems in bacteria are PCR, sequencing or in silico PCR of the genome. After sequencing, sequences are usually subjected to the CRISPRFinder or CRISPRCasFinder programs, which identify structures similar to CRISPR sequences [13, 14].

The CRISPR-Cas systems are classified into class 1 (types I, III, and IV) and class 2 (types II, V, and VI), and 33 subtypes [15]. The classification is based on the different types of effector Cas proteins, in which the action mechanism of class 1 systems is based on the participation of a complex of proteins, while class 2 systems act by the effect of only one major Cas protein (Cas9, Cas12 or Cas13) [16]. CRISPR arrays may be found with or without associated cas genes. When cas genes are absent, CRISPR is usually not able to provide genome defense without the presence of another type of CRISPR, since the core function of the system is performed by the cas genes [17]. Some Cas proteins are detected in specific types of CRISPR system, such as Cas3 for type I, Cas9 for type II, Cas10 for type III, Cas12 for type V, and Cas13 for type VI [15, 18, 19].

A CRISPR-Cas system needs to recognize the foreign DNA or RNA and assemble the machinery before it can cleave the targeted nucleic acid. Therefore, a functional CRISPR system generally acts following three phases: adaptation (or acquisition), important to create a genetic memory; expression (or biogenesis), defined by expression of CRISPR RNA (crRNA), and interference, in which the DNA or RNA cleavage occurs [16, 20–22].

CRISPR-Cas systems have been widely researched for biotechnological applications, particularly since a genome-editing tool based on the CRISPR-Cas system from Streptococcus pyogenes was developed [23–25]. Potential applications include diagnostic and control of infectious diseases, genetic improvement of plants and animals in agriculture and livestock, and treatment of genetic disorders and/or genetic-related diseases (e.g., cancer, thalassemia, sickle cell disease, Leber’s congenital amaurosis etc.) [26]. In genome editing, the CRISPR-Cas technology can replace other methods, such as zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN), due to lower complexity, higher efficiency and the possibility of easily programming several targets concomitantly [27].

Considering the relevance of enterococci under the One Health perspective and the potential applications of the CRISPR-Cas technology, we aimed to conduct a review to provide a comprehensive overview of the current literature on CRISPR-Cas systems in enterococci, detailing the frequency, structure and organization of CRISPR systems, mechanisms of action and gene editing in enterococci.

Mechanisms of action of CRISPR-Cas system in Enterococcus spp.

The first phase of CRISPR-Cas mechanism of action is the adaptation stage. In type II-A CRISPR-Cas systems, the most common CRISPR type found among enterococcal species, Cas1 and Cas2 are major proteins that work at the spacer acquisition phase, together with Csn2. These proteins recognize a specific sequence of the invader genetic material, called protospacer adjacent motif (PAM), and selects the protospacer. Enterococcus spp. usually encodes a type II CRISPR-Cas9 system with a canonical PAM sequence of NGG (where N is any nucleotide) located downstream to the target sequence in mobile genetic elements. The selected protospacer is incorporated as a spacer sequence into the CRISPR locus by the Cas proteins and another repeat sequence is synthesized [28, 29]. Of note, Cas1 and Cas2 can be found in almost all CRISPR types and PAM sequences can vary or are even not required depending on the type of CRISPR-Cas system. In the genus Enterococcus, the cas1 gene is usually about 865-bp long [30] and is considered the universal CRISPR marker and the most used during the investigation for the presence of the CRISPR-Cas systems [31]. The csn2 gene was shown to be regulated by the presence of Ca⁺² to perform its oligomerization and double-stranded DNA binding. The presence of the ion permits more rigidity in Csn2 structure [31].

The second phase is the expression stage where the CRISPR array is transcribed into an initial RNA form (pre-crRNA) that includes all the repeat and spacer sequences. This pre-crRNA is then processed to produce mature crRNA, which contains single spacer sequences. Following, the trans-encoded small RNA (tracrRNA) and the mature crRNA form a duplex [20, 32, 33].

Finally, in the interference stage, the tracrRNA:crRNA duplex and the Cas endonuclease, such as Cas9 in type II-A, form a ribonucleoprotein (RNP) complex which recognizes the PAM sequence and cleaves the exogenous DNA [21, 31]. In this phase, the csn1 gene is also required for the silencing mediated by crRNA in type II-A CRISPR-Cas [31].

As previously mentioned, the most common type of CRISPR system in enterococci is the type II-A, and it is mostly composed of CRISPR1-Cas, CRISPR2 and CRISPR3-Cas. CRISPR1-Cas and CRISPR3-Cas rely on the action of their own Cas proteins, but CRISPR2 is an orphan locus, which means it does not have cas genes. Still, CRISPR2 is a conserved locus and it can provide genome defense in the presence of Cas proteins encoded by CRISPR1-Cas. Unlike CRISPR2, CRISPR1-Cas and CRISPR3-Cas systems are less frequently observed in multidrug-resistant bacteria, since they can actively function as a protection against mobile genetic elements, consequently working against the acquisition of resistance genes [34].

To assess if loss-of-function mutations in the CRISPR array under antibiotic selection may cause a disabled CRISPR protection, serial passages in the presence and absence of antibiotics followed by deep sequencing were used to analyze a population of E. faecalis possessing a functional CRISPR3-Cas. Frequency of erythromycin-resistant cells decreased over the course of serial passage in BHI in most wild-type (WT) strains. When exposed to BHI with erythromycin, the WT strains had the CRISPR3 spacer 6 (identical to the repB gene of the model pheromone-responsive plasmid pAD1) deleted and variation in cas9 sequence. Erythromycin selection caused CRISPR3 array mutations in WT strains, compromising CRISPR-Cas defense. To check if this selection occurs in nature, genome data found in literature of an E. faecalis strain with a frameshift mutation in CRISPR3 cas9 gene were used, demonstrating that loss-of-function mutation in CRISPR-Cas permits plasmid acquisition or plasmid maintenance, since it had both CRISPR3 system and a plasmid encoding linezolid resistance [35].

Regarding the targets of the CRISPR-Cas systems of enterococci, phage and plasmid sequences have been identified [32, 36]. A study found identity between the spacers encountered at the CRISPR locus and known pheromone responsive plasmids and phage sequences, but not for transposons [32]. In addition, spacers with homology to usual enterococcal phages, such as phiEf11, phiFL3A and SAP6, were identified in an E. faecalis isolate [36].

To understand the Cas1-Cas2 spacer integration mechanism, four structural snapshots were captured from type II-A Cas1 and Cas2 of E. faecalis during the integration process. The complex binds to a 30-bp protospacer sequence, searches for half-sites, relates to the leader side of CRISPR direct repeat sequence and promotes a nucleophilic attack to connect one end of the leader sequence to the 3’ end of the protospacer [37]. Another study conducted experiments to define the kinetic framework of this mechanism, showing that the system cannot find the CRISPR repeat by itself, needing help from the leader sequence adjacent to the repeat, which acts by stabilizing the integration by the leader-side and the spacer-side. The RNA transcription settles the postsynaptic complex (PSC) helping direct the integration, since PSC could cease the production of RNA, affecting CRISPR activity. The resolution of PSC is essential to the efficient action of CRISPR-Cas system [38].

Using the anti-CRISPR acr genes found in enterococci, a demonstration of the inhibition action of this mechanism against CRISPR-Cas systems in E. faecalis was investigated by using CRISPR-Cas to target a plasmid expressing acrIIA16, acrIIA17, and acrIIA19 genes [39]. It was shown that the presence of the genes blocks the action of CRISPR1-Cas and confers a modest protection against the action of CRISPR3-Cas in the strain during plasmid conjugation, increasing the horizontal gene transfer frequency. It was suggested that it acts on sgRNA, ApoCas9 (apoenzyme Cas9, the inactive part of the enzyme that requires cofactors) or both. The genes encoding for anti-CRISPR proteins can be found in some mobile genetic elements, being able to inhibit CRISPR action in several cells [39].

Organization and structure of CRISPR-Cas systems in Enterococcus spp.

Some variations in the number and size of spacers were observed in enterococci. CRISPR system is usually composed of 4 to 10 palindromic direct repeat sequences with size of 25 to 45 bp separated by 1 to 42 spacers, of 23 to 37 bp [17, 30, 36, 40–46].

There are some differences in the structure of CRISPR among the various type II-A subtypes. Although the palindromic repeats of CRISPR1-Cas and CRISPR2 are identical, which may suggest that their function is connected, there is a distinction in the CRISPR3-Cas sequence when compared to CRISPR2 and CRISPR1-Cas in E. faecalis [32]. The orphan CRISPR2 structure consists only of spacers and repeats, without any accessory genes, and so it cannot perform any function by itself, while CRISPR1-Cas and CRISPR3-Cas are complete [47]. CRISPR4, a more recently described CRISPR system, was found to have similar repeat sequences as CRISPR1-Cas and CRISPR2, being called CRISPR4.1 and CRISPR4.2, respectively, depending on the type of CRISPR it relates. However, the end of the repeat sequence and the spacers of CRISPR4 differ from the others. Usually associated cas genes were not detected in CRISPR4 [17]. Figure 1 illustrates the structure of different types of CRISPR systems found in enterococcal species.

Fig. 1.

Organization of CRISPR loci from the different CRISPR subtypes found in enterococcal species [Adapted from Palmer and Gilmore [32]; Hullahalli et al. [17]; Jiang et al. [90]; Zheng et al. [91]; Tao et al. [30])

CRISPR loci constantly appear in a conserved position on the chromosome. In E. faecalis, CRISPR1-Cas was found between open reading frames (ORFs) homologues of the reference strains V583 EF0672 and EF0673, CRISPR2 between EF2063 and EF2061, and CRISPR3-Cas between EF1760 and EF1759 [32, 41, 48, 49]. An unusual sequence of 184 bp, similar to CRISPR1-Cas, was found in one strain between EF0672 and EF0673 genes, identified as a type II-C locus (CRISPR-Cas7) [49].

Regarding the presence of cas genes, some differences were found between the types of CRISPR encountered on different enterococcal species. A type I-C CRISPR-Cas arrangement was found in two pathogenic Enterococcus cecorum strains. This type is not common in enterococci, but it has already been described in streptococci [50, 51]. Another strain had a similar type I-C CRISPR-Cas, but it lacked a cas2 gene, suggesting it is not active [50]. In E. lacertideformus genome, three CRISPR systems and two cas clusters were identified. One cluster, characterized as type II-A, had cas1, cas2 and csn2 genes flanking its sequence. The second cluster was located upstream to the first one and had cas1 and cas2 genes [52]. In a comparative study of E. faecalis and E. faecium genomes from the NCBI database, cas1, cas2, and cas9 genes were detected in both species. One E. faecalis isolate harbored two cas9 gene variants – cas9 and cas9.1 [53]. In another analysis of 110 enterococcal genomes extracted from NCBI, 46 genomes had a cas gene cluster and the CRISPR-Cas systems found were divided into types II-A (80.4%), II-C (10.9%), I-C (4.4%), and I-B (2.2%). One E. faecalis strain displayed both types, II-A and II-C, simultaneously [30].

In a metagenomic analysis of jalebi batter, a naturally fermented cereal-based food from India, E. hirae was found to harbour nine CRISPR-Cas gene clusters. The clusters included genes cas1_TypeII, cas2, cas2_TypeI-II-III, cas3_TypeI, cas3_TypeI, cas4_TypeI-II, cas9_TypeII, CAS-TypeIIA, and csn2_TypeIIA [54]. In another study, two cas3 genes in E. faecalis belonging to type I CRISPR system were detected in isolates recovered from home-made fermented soybean food [55].

Some specific E. faecalis strains have been studied in detail. Strain OG1RF was shown to have two CRISPR systems, CRISPR1-Cas and CRISPR2. Both of them had seven repeat sequences of 37 bp with a 29 bp spacer (total of 14). OG1RF has the presence of four cas genes (cas1, cas2, csn1, and csn2) and a single copy of the repeat sequence in the same CRISPR array. An uncommon attribute found in OG1RF is the presence of a gene downstream to the system, which encodes a transmembrane protein [47]. Three identical spacers were found in 42 strains of the sequence type ST40; two of these spacers were also found in the strain OG1RF [36].

In a comparative study of various enterococcal species, different CRISPR locus structures were identified. Enterococcus thailandicus had the largest CRISPR array [45]. During a genomic investigation with food-related and probiotic Enterococcus strains, one E. raffinosus isolate was found to have more CRISPR arrays, including four gene cassettes, one of which was the largest found in the study, with a sequence of 1,215 bp [44].

An evaluation of Cas1 (CRISPR1) found similarity in the phylogeny of this protein among different species, suggesting a horizontal gene transfer of CRISPR arrays or conservation of CRISPR-Cas systems with high similarity between the species. The cas1 genes identified in E. faecalis and E. hirae differ from the others, showing species-level evolution [56].

Regarding the distribution of CRISPR system in isolates from different origins, a study analyzed E. faecalis genomes from NCBI Refseq (as of September 1, 2021) and from a recent large-scale One Health investigation of enterococci [57]. A consistent prevalence of CRISPR1-Cas and CRISPR3-Cas was observed in strains from human and animal sources. Of the 401 animal-derived strains, 110 (27.6%) strains had CRISPR1-Cas, and 38 (9.5%) strains had CRISPR3-Cas. Human strains presented similar frequencies, with 485 (29.4%) and 140 (8.5%) of the 1,653 strains with CRISPR1-Cas and CRISPR3-Cas, respectively [57].

Presence and absence of CRISPR-Cas systems in Enterococcus spp.

Class 2 type II-A is the prevalent CRISPR-Cas system within the genus Enterococcus, and it uses the Cas9 protein as an effector. Cas9 is a multidomain protein with different functions, such as DNA cleavage and crRNA maturation [24]. The most common type II-A CRISPR in enterococci is CRISPR2 detected in 1,262 (56.4%) of 2,236 isolates of Enterococcus spp., followed by CRISPR1-Cas and CRISPR3-Cas in 757 (33.9%) and 226 (10.1%) isolates, respectively. Hullahalli and colleagues [17] described a different CRISPR locus named CRISPR4, with a direct repeat sequence similar to CRISPR1-Cas and CRISPR2, in three (0.1%) isolates of E. faecalis.

By examining the diversity of the most studied Enterococcus species, we observed that CRISPR systems tend to be more frequent in E. faecalis [30, 32, 40, 46, 48, 53, 58–61]. Across all articles, the number of CRISPR systems detected in 3,283 E. faecalis isolates was 3,129 (95.3%). This number refers to all four type II CRISPR systems (CRISPR1-Cas, CRISPR2, CRISPR3-Cas, and CRISPR4) and also the times they appeared in concomitance in a single isolate.

Some species, such as E. malodoratus and E. sanguinicola, or certain strains within a given species do not have a CRISPR-Cas system, showing ability to easily modify their genome, a feature known as genomic plasticity. With this ability, these bacteria are able to develop new characteristics which may help their survival and adaptation to the environment, including the capacity to develop antimicrobial resistance [1].

Table 1 shows the frequencies of the different types of CRISPR found in several enterococcal species, while Table 2 presents the occurrence of CRISPR systems in articles that either did not specify the CRISPR type or did not belong to the category of type II-A.

Table 1.

Distribution of CRISPR1-Cas, CRISPR2, CRISPR3-Cas and CRISPR4 in enterococcal species in the articles included in this review

Species	Number of isolates	CRISPR system (%)				Reference
		CRISPR1-Cas	CRISPR2	CRISPR3-Cas	CRISPR4
E. faecalis	16	43.8%	0	0	-	[47]
E. faecium	7	0	42.9%	0	-	[62]
E. faecalis	48	25%	100%	8.3%	-	[32]
E. faecium	8	37.5%	0	0	-
E. faecalis	52	32.7%	90.4%	1.9%	-	[48]
E. faecium	10	0	0	0	-
E. faecalis	78	33.3%	0	33.3%	-	[77]
E. faecalis	88	52.3%	95.5%	0	-	[40]
E. hirae	107	42.1%	49.5%	0	-
E. faecium	10	0	0	0	-	[78]
E. faecalis	42	97.6%	100%	0	-	[36]
E. faecalis	228	11.8%	100%	6.6%	1.3%	[17]
E. faecalis	110	23.6%	0	0	-	[56]
E. hirae	78	73.1%	0	0	-
E. durans	56	7.1%	0	0	-
E. faecium	14	7.1%	0	0	-
E. casseliflavus	7	14.3%	0	0	-
E. sulfureus	2	50%	0	0	-
E. mundtii	2	0	0	0	-
E. sanguinicola	1	0	0	0	-
E. malodoratus	4	0	0	0	-
E. termitis	1	0	0	0	-
E. faecalis	325	60.6%	99.1%	2.5%	-	[43]
E. faecalis	67	44.8%	88.1%	8.9%	-	[58]
E. faecium	53	5.7%	32.7%	1.9%	-
E. faecium	2	0	0	0	-	[79]
E. faecalis, E. faecium, E. gallinarum, E. casseliflavus, and E. durans	180	23.9%	24.4%	34.4%	-	[80]
E. faecalis	74	97.3%	100%	0	-	[49]
E. faecalis	1	0	100%	0	-	[41]
E. faecalis	161	13%	55.3%	17.4%	-	[81]
E. faecalis	1	0	100%	0	-	[42]
E. lacertideformus	1	100%	100%	100%	-	[52]
E. faecalis	1	100%	100%	100%	-	[82]
E. faecalis	144	10.4%	53.5%	18.8%	-	[63]
E. faecalis	50	50%	78%	36%	-	[59]
E. faecium	35	28.6%	22.9%	34.3%	-
E. faecalis	22	72.7%	9.1%	40.9%	-	[60]
E. faecium	22	22.7%	13.6%	36.4%	-
E. faecalis	62	1.6%	16.1%	0	-	[66]
E. faecium	38	0	26.3%	2.6%	-
E. faecium	8	37.5%	0	62.5%	-	[83]
E. faecalis	7	0	0	0	-	[84]
E. faecium	13	0	0	0	-

-, not investigated

Table 2.

Frequency of CRISPR-Cas systems by species in the articles included in this review

Species	Number of isolates	Presence of CRISPR system (%)	Reference
E. cecorum	3	100%	[50]
E. faecalis	10	90%	[61]
E. faecium	20	0
E. faecalis	15	6.7%	[85]
E. faecalis	5	60%	[44]
E. faecium	13	61.5%
E. hirae	1	100%
E. mundtii	3	33.3%
E. durans	1	0
E. malodoratus	1	0
E. raffinosus	1	100%
E. faecalis	1	100%	[45]
E. faecium	3	0
E. hirae	10	90%
E. durans	1	100%
E. villorum	2	100%
E. gallinarum	1	0
E. casseliflavus	2	0
E. thailandicus	1	100%
E. faecalis	20	75%	[65]
E. faecalis	24	87.5%	[46]
E. faecium	11	72.7%
E. gallinarum	2	0
E. casseliflavus	2	100%
E. faecalis	1,591	75.4%	[53]
E. faecium	1,981	4.9%
E. faecium	2,223	91.6%	[64]
E. durans	1	100%	[86]
E. faecalis	40	92.5%	[30]
E. faecium	36	11.1%
E. avium	2	50%
E. cecorum	3	66.7%
E. durans	6	33.3%
E. hirae	10	70%
E. mundtii	6	50%
E. silesiacus	1	100%
E. thailandicus	1	100%
Enterococcus sp.	5	20%
E. lactis	2	100%	[87]
E. faecium	12	100%	[88]
E. mundtii	1	0	[89]

CRISPR-Cas system and the acquisition of virulence and antimicrobial resistance genes in Enterococcus spp.

The CRISPR-Cas system is an important mechanism that enables the study of important processes that occur in bacteria and plays a significant role in preventing the acquisition of resistance and virulence genes. A functional CRISPR-Cas system can be an active barrier against the acquisition of several genes, and are often absent in multidrug-resistant microorganisms [34]. This was demonstrated, for example, by Van Schaik and colleagues [62], who found several resistance genes in E. faecium isolates that contained only the CRISPR2 orphan locus, as well as by Palmer and Gilmore [32], who detected CRISPR2 in all E. faecalis isolates in concomitance with the presence of resistance genes, showing that this locus alone does not confer protection.

Several studies reported a correlation between the presence of antimicrobial resistance [e.g., aac(6’)-Ie –aph(2″)-Ia aminoglycoside resistance gene, erm(B) macrolide resistance gene, tet(M) tetracycline resistance gene, vanA vancomycin resistance gene etc.] and/or virulence [e.g., aggregation substance (asa1), cytolysin (cyl), enterococcal surface protein (esp), glycosyl hydrolase (hyl) etc.] genes and the absence of CRISPR-Cas systems. However, sometimes the opposite has also been found, with a positive correlation between the presence of CRISPR systems and resistance and virulence genes [48, 59, 63].

In addition, it seems to have a lack of species-specific mechanisms, since 17 (40%) spacers were shared by the genomes of E. hirae and E. faecalis. This finding supports the occurrence of gene exchange between bacterial species in the intestinal microbiota [40].

The absence of a functional CRISPR-Cas system has been associated with plasmid acquisition, presence of prophage regions, antimicrobial resistance and virulence genes [42, 52, 53, 63]. This association can be seen, for example, in the most frequently studied E. faecalis reference strains, OG1RF and V583. While the strain OG1RF has a functional CRISPR-Cas system without the presence of any prophage regions or plasmids, V583 lacks a functional locus and has three plasmids and seven prophage regions, besides being multidrug-resistant [32, 47]. One strain of E. lacertideformus had only one incomplete prophage region, in agreement with the presence of a functional CRISPR [52].

Among the 622 spacers detected in 110 enterococcal strains (E. faecalis, n = 40; E. faecium, n = 36; E. avium, n = 2; E. cecorum, n = 3; E. durans, n = 6; E. hirae, n = 10; E. mundtii, n = 6; E. silesiacus, n = 1; E. thailandicus, n = 1; and Enterococcus sp., n = 5), 517 spacers were different from each other and had homology to plasmids (9; 1.7%), to phages (37; 7.2%), to target their own bacterial genomes (126; 24.4%), and to target non-self bacterial genomes (345; 66.7%). Most (75%) plasmids had homology with enterococcal plasmids, and the remaining (25%), with Clostridium perfringens plasmids [30].

A genome analysis of E. faecium strains found several antimicrobial resistance genes and prophage regions, which is in agreement with the absence of cas genes in the isolates [62]. Additionally, a median number of 13 antimicrobial resistance genes was found in E. faecium strains lacking CRISPR-Cas, whereas strains with the system had a median number of only three resistance genes [64]. Studies on E. faecalis isolates from root canals of patients showed that strains without CRISPR formed stronger biofilms and caused more significant lesions [65].

No correlation was found between the presence of CRISPR1-Cas and antimicrobial resistance, but isolates without CRISPR3-Cas had higher minimum inhibitory concentration (MIC) values to vancomycin. The same was observed for gentamicin MIC values when in lack of CRISPR2 and CRISPR3-Cas [63].

The aph(3’)-IIIa aminoglycoside resistance gene was detected in 10 of 15 CRISPR-negative isolates, without statistical correlation, but a statistical correlation was found between the aac(6’)-Ie–aph(2″)-Ia aminoglycoside resistance gene and CRISPR loci. Among vancomycin-resistant enterococci strains, no correlation between CRISPR loci and vanA gene was found. The presence of CRISPR was related to efaA and gelE virulence genes, but the hyl gene was related to the absence of CRISPR systems [60].

A high frequency (74.2%) of asa1 virulence gene in E. faecalis was found among CRISPR-positive isolates, followed by the esp gene (62.9%). In E. faecium, the presence of the esp gene was more common (92.1%) among CRISPR-positive isolates [66].

In the absence of CRISPR systems, the presence of the aac(6’)-Ii aminoglycoside resistance gene (62.8%), the efmA efflux pump gene (41.2,%), the eatAv multidrug resistance gene (29.4%), and the vanC vancomycin resistance gene (7.9%) in 110 isolates was significantly higher than those containing confirmed CRISPR arrays (10.2%, 1.7%, 8.5%, and 0, respectively). The frequency of the aac(6’)-Ii gene (50%) and the efmA gene (32.8%) when in presence of orphan CRISPR systems was significantly higher than in those containing CRISPR systems with cas genes (13% and 2.9%, respectively). In addition, the presence of the aac(6’)-Ii (45.1%) and efmA (29.6%) genes in the group without complete CRISPR-Cas system was more common than in the group with the complete CRISPR-Cas system (15.4% and 2.6%, respectively) [30]. In contrast, nine out of 10 E. faecalis belonging to the lineage ST40 had functional CRISPR-Cas and multiple acquired resistance genes [61].

Regarding the function of CRISPR system against pheromone-responsive plasmids (PRP), experiments using the E. faecalis strain T11 that has a CRISPR3-Cas and an orphan CRISPR2 were performed [34]. The process was done by deleting the cas9 gene in the CRISPR3-Cas system, which increased conjugation and, consequently, plasmid acquisition. These experiments showed that CRISPR2 is not functional against PRP acquisition and only works when associated with a cas9 gene derived from a different CRISPR-Cas [34].

In another study, the same strain and also PRP were used to evaluate the interactions of conjugative plasmids with CRISPR-Cas elements [67]. Although the dissemination of the plasmids has been blocked by CRISPR-Cas in vivo using a mouse model of intestinal dysbiosis, the in-vitro results showed that PRP can escape the action of the system even in different conditions (different biofilm settings, donor-to-recipient ratios, presence of bacteriocin). These studies show that CRISPR-Cas defense function can be more effective in vivo, producing defective transconjugants or not producing them at all, combined with a rapid elimination by the intestine [67].

To test the efficacy of CRISPR-Cas in sterilized manure, conjugation assays were performed using two different E. faecalis recipients, T11RF (rifampin-fusidic acid-resistant derivative of strain T11) with a functional CRISPR-Cas system and T11RF Δcas9 with a deletion of cas9 gene, which promotes a non-functional defense against plasmid pAM714. The experiment was done in three different conditions, using BHI agar, BHI broth, and liquefied autoclaved dairy manure. In the absence of functional CRISPR-Cas system, a higher frequency of plasmids was observed in all three conditions, showing the role of the system in the acquisition of plasmids, especially in manure [57].

CRISPR-Cas use for gene editing in Enterococcus spp.

CRISPR-Cas has been extensively used in several experiments to perform gene editing in enterococcal species. Although different nucleases can be applied to gene editing, Cas9 is the most frequently used enzyme. When used for gene editing, Cas9 is guided by a single-guide RNA (sgRNA) that works as a fusion of crRNA and tracrRNA to cleave the target sequence [24]. The cleavage caused by the system in the targeted sequence can be repaired by two methods, depending on the type of cell: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). NHEJ reconnects the two dissociated ends by inserting or deleting a few base pairs (InDels), potentially causing inactivation; and in HDR, a donor DNA that is homologous to the target is incorporated through recombination [25]. In theory, enterococci lack a NHEJ repair mechanism, and they can only escape CRISPR-Cas lethal effect by using an HDR template model. Thus, it is possible to modify any gene of interest in enterococci [68, 69].

The functionality of CRISPR-Cas was tested in a vast range of isolates, by creating a cloning vector containing PRP to introduce different protospacers with the CRISPR1/CRISPR2 NGG PAM sequence in genetically diverse E. faecalis to determine conjugation frequencies [70]. The experiment resulted in a reduction in conjugation frequency caused by CRISPR interference (CRISPRi). Other experiments were done to confirm the function connection between CRISPR1-Cas and CRISPR2 and to examine this interaction in multidrug-resistant strains. A cas9 gene, its promoter and a tracrRNA were introduced in multidrug-resistant strains that lacked CRISPR1-Cas. In these strains, a decrease in conjugation frequencies was also observed. The researchers tested whether the degeneracy of the terminal repeat of CRISPR1 and CRISPR2 could cause a loss of function of the terminal spacer caused by the disruption of the crRNA and tracrRNA association. By replacing the terminal repeat of CRISPR1 and CRISPR2 with a direct repeat, and the loss of function was confirmed. Furthermore, the CRISPR2 locus was activated in multidrug-resistant E. faecalis strains to prevent plasmid acquisition. In addition, it was proved that CRISPR-Cas system can be used in vitro to inactivate antimicrobial resistance genes in specific strains. A self-targeting construct was created by inserting a CRISPR2 locus targeting erm(B) gene located in a PRP in the strain used, providing an assay that can be used as an in vitro strategy to alter E. faecalis populations [70].

CRISPR tolerance is a phenomenon defined as the ability of mobile genetic elements to be maintained transiently in the bacteria before being targeted by the CRISPR system. This is demonstrated by lower acquisition of targeted or self-targeted constructs [71, 72]. An advantage of this process relies on the fact that, if a mobile genetic element is favorable to bacterial cell survival, it is able to proliferate and persist longer in the bacterial cell. It was first proved that CRISPR self-targeting (which leads to chromosomal DNA breaks) can be deleterious, but not lethal in E. faecalis, since the isolates presented a growth defect. To prove that the increase in CRISPR components can increase the self-targeting lethality, cas9 expression was enhanced and a decrease in conjugation frequency of transconjugants that accept CRISPR targeting was noticed, which were shown to be mutants with inactivated CRISPR-Cas. With these results, a CRISPR-mediated genome editing tool was elaborated based on a plasmid that encodes a homologous recombination template that would result in a deletion on the vanB gene [70, 71].

To achieve depletion of antimicrobial resistance, CRISPR-Cas was used to target erm(B) and tet(M) genes in an E. faecalis strain [73]. For the delivery of the CRISPR machinery, a PRP was used. PRPs are usually chosen as vectors in gene editing using CRISPR experiments because they easily reach high frequencies of conjugation, have a limited host range, and spread easily in the species population. After conjugation of the plasmid containing CRISPR to the strain, a reduction in the generation of erythromycin- and tetracycline-resistant transconjugants was observed in vitro. A multidrug-resistant E. faecalis strain was used to test the effectiveness of the tool and the delivery of CRISPR targeting erm(B) resulted in a decrease in overall erythromycin resistance in the population. In-vivo experiments were also performed, and compared to the in-vitro experiments, less conjugation occurred; however, there was a trend for decrease in erythromycin resistance in the recipient population. Furthermore, donor cells carrying antimicrobial CRISPR-Cas became immune against the acquisition of resistance genes, suggesting a probiotic usage of these strains [73].

A Lactobacillus reuteri gene editing strategy using CRISPR-Cas9 was adapted in a vancomycin-resistant enterococci strain of E. faecium, based on the high recombination frequency characteristic of the species [68]. A plasmid vector was used to deliver CRISPR within the cell. The aim was to promote a deletion on the lacL gene, which encodes a beta-galactosidase subunit, and to insert the green fluorescent protein (gfp) gene to the mrsC macrolide resistance gene via HDR template model. The approach showed to be efficient, the processing time was reduced, and the experiments can be expanded to other species [68].

A recombineering method using RecT protein and CRISPR-Cas gene editing technique to provide scarless bacterial mutants (i.e., without detectable modifications in the genome) was performed [74]. The researchers also used a method similar to the L. reuteri experiment and a plasmid vector encoding the S. pyogenes Cas9, which was transformed into E. faecium by electroporation, and a sgRNA targeting thymidylate synthase (thyA), an important enzyme for DNA synthesis. As a control, the sgRNA was mutated to create an off-target effect and it yielded more colonies than the experiments using thyA as a target, demonstrating that Cas9 was active in the species and killed the bacteria after transformation. The dCas9 (dead Cas9), an inactivated enzyme often used in gene editing experiments to provide functionally active domains to a specific locus in the genome, was also tested for gene knockout and it actively repressed thyA expression producing thymidine auxotrophy (strict dependence on a nutrient for growth) when cultured. RecT was utilized to introduce DNA templates with base-pair substitutions, deletions or insertions to thyA in different experiments, and the presence of the protein showed an improvement in recombineering activity since it successfully produced thymidine auxotrophs [74].

To study how E. faecalis is involved in the production of myristoleic acid, an inactivation of the E. faecalis gene encoding Acyl-CoA thioesterase (ACOT) was done by the CRISPRi mechanism, employing dCas9 and a sgRNA designed to recognize the target. The gene knockdown worked, but it did not affect bacterial growth [75].

A dual-vector CRISPRi delivery system with nisin-induced promoters was used for pathogenicity studies in E. faecalis [29]. The system included a plasmid encoding a dCas9 from S. pyogenes and a second plasmid with a sgRNA sequence targeting the gfp gene. Partial gene silencing was initially observed, but nisin pre-sensitization enhanced efficacy. Targeting the ebpA gene of the ebpABC operon, which encodes endocarditis and biofilm-associated pili, successfully silenced the entire operon and reduced biofilm formation. The two-component croR system, related to several functions, including antibiotic resistance and stress responses, was also targeted with CRISPRi, resulting in reduced bacitracin resistance in the knockout strain. Simultaneous silencing of multiple genes using the nisA promoter showed similar efficiency to single-target experiments, demonstrating the system's effectiveness for comprehensive genome editing in enterococci [29].

A study was performed with one E. faecium strain by transforming purified plasmids extracted from Escherichia coli containing the protein Cas12a and targeting genes to achieve a clean deletion of lacL, acpH, and treA [76]. ΔlacL mutant colonies showed no conversion of X-Gal due to the lack of function of the enzyme beta-galactosidase; ΔacpH mutant strains were capable of reducing Orange II, an azo dye; ΔtreA mutant strains were not able to grow in the presence of trehalose as the only carbon source. To test the ability of the system to perform gene knock-in, the fluorescent gene unaG, induced by the presence of trehalose, was inserted in a treA harboring plasmid. The generated mutant strain presented fluorescence when in the presence of trehalose; in the absence of this disaccharide, there was no growth due to the lack of unaG production. Since unaG-promoted fluorescence does not require oxygen presence, this construct can be used in anaerobic environment studies, such as in the gut [76].

Using a CRISPR antimicrobial plasmid to deplete erythromycin resistance targeting the erm(B) gene, an E. faecalis strain was cultured in manure. However, a decrease in resistance levels was not observed, possibly because of the absence of a solid surface for the bacteria to form biofilm to promote conjugation in addition to poor delivery of CRISPR [57].

As demonstrated in several studies, the CRISPR-Cas gene editing tool has been employed in enterococci to target genes associated with antimicrobial resistance, virulence, and metabolism. Despite successful results in some cases, further studies are necessary to fully realize the potential of this technology within the genus Enterococcus.

Final considerations and future perspectives

With the emergence of resistance to several antimicrobial agents among enterococcal species, new strategies to treat or control enterococcal infections, especially those caused by strains multidrug-resistant strains, are required. CRISPR-Cas systems have gained remarkable importance in the scientific community due to its application in genetic engineering. As it is a more practical, simpler and cheaper tool than other gene editing technologies, CRISPR-Cas has shown to be very promising in gene knockout and silencing [29, 57, 67, 68, 71–76]. In-vitro gene editing of enterococcal species, targeting antimicrobial resistance and virulence genes, has been successfully achieved. However, developing a strategy to be applied in vivo is more challenging, especially regarding human ethics and unknown risks.

Many studies use PCR as the method for detecting CRISPR arrays and the genetic determinants of virulence and resistance, but variations in regions where primers anneal, for example, may lead to false negative results. In addition, some studies analyzed a low number of isolates and there may be a bias related to the source of the isolates. Expanding the number of isolates investigated and using whole genome sequencing approaches would provide more robust evidences regarding the association between the presence/absence of CRISPR systems and resistance- and virulence-related genes.

In conclusion, further studies are needed to elucidate some CRISPR-Cas mechanisms and to understand their role in Enterococcus spp.. CRISPR systems might be an important tool to help combat antimicrobial resistance and decrease the virulence of pathogenic strains. Therefore, the continuous improvement of the technology and delivery systems for in-vivo purposes are of great interest.

Authors’ contributions

FPGN and LMT contributed to the overall design of the study. ASC, FFL, VLM, FMM, ABS, BAS, JLCL and FPGN performed formal analyses. ASC and FPGN drafted the manuscript. All authors read and approved the final manuscript.

All authors attest they meet the ICMJE criteria for authorship.

Declarations

Conflicts of interest

None.