The Science behind Biofilm: Unraveling Enterococcus Genus’ Remarkable Ability to Produce Microbial Communities

Abstract

The remarkable ability of Enterococcus to produce microbial communities, specifically biofilms, is a topic of interest in scientific research. Biofilms formed by Enterococcus species, are known to contribute to their survival in extreme environments and their involvement in persistent bacterial infections. The aim of this chapter is to provide a comprehensive understanding of the mechanisms underlying biofilm formation in clinically important species such as E. faecalis and the increasingly drug-resistant but less well-studied E. faecium. Enterococcus forms biofilms through a complex interaction between genes and virulence factors such as DNA release, cytolysin, pili, secreted antigen A, and microbial surface components that recognize adhesive matrix molecules (MSCRAMMs). Quorum sensing mediated by peptide pheromones targets gene expression and regulation and is essential for the coordination of biofilm formation. Furthermore, control over extracellular DNA (eDNA) release has been shown to be crucial for biofilm formation. In E. faecalis, autolysin N-acetylglucosaminidase and proteases such as serine protease and gelatinase are important players in this process, influencing biofilm development and virulence. The study of biofilm formation in Enterococcus can provide insights into the pathogenesis of opportunistic infections and their prevention and provide directions for future anti-biofilm therapeutic research.

1. Introduction

The genus Enterococcus comprises a ubiquitous group of Gram-positive and facultative anaerobic bacteria. Enterococcus spp., once considered as a harmless commensal of the intestine, have now emerged as medically important pathogens and are associated with both community-acquired and nosocomial infections [1].

These bacteria are commonly found in the human gastrointestinal tract and are responsible for a number of infections such as urinary tract infections, bacteremia, endocarditis, wound infections (from burns or surgical incisions), abdominal and biliary tract infections, and infections caused by catheters and medical implants [2, 3].

Enterococci are resilient and versatile species that can survive under harsh conditions, making them well-adapted to the healthcare environment [4]. Most enterococcal infections are caused by Enterococcus faecalis and Enterococcus faecium, both of which exhibit intrinsic resistance to common antibiotics such as trimethoprim-sulfamethoxazole, aminoglycosides, cephalosporins, and clindamycin [1].

Furthermore, due to an exceptionally flexible genome, these two species are able to rapidly develop resistance to other antibiotics, including high-level ampicillin, vancomycin, and aminoglycoside resistance [5], either by horizontal gene transfer or by mutation [6]. According to the World Health Organization (WHO), vancomycin-resistant E. faecium is a high-priority pathogen that necessitates the development of novel antimicrobial therapies [7, 8].

The fact that Enterococcus is overly resistant to antibiotics is not the only characteristic that puts human and animal health at risk. Additionally, they have a high capacity for environmental persistence [9]. They are also well-known for their genome plasticity, which enables them to effortlessly acquire, maintain, and spread genetic traits in other Gram-positive bacteria in addition to their remarkable ability to form biofilms [10].

2. Virulence factors and biofilm

Biofilms are complex and dynamic forms of highly organized microbial communities that have sparked significant interest in recent years due to bacteria’s remarkable ability to survive in biofilms, development of infections, and virulence [11]. Among the bacteria capable of forming these microbial communities, the Enterococcus genus stands out for its widespread distribution in various environments and its increasing prevalence in hospital-acquired infections. To effectively combat these infections, a thorough understanding of the underlying processes and mechanisms behind biofilm formation in this genus is crucial [12]. In this chapter, we discuss the steps, molecular mechanisms, and pathogenic factors involved in enterococcal biofilm formation. By elucidating the science of biofilm formation in the genus Enterococcus, we hope to pave the way for the fight and future advances in the field of dealing with this bacterium.

The virulence factors in Enterococcus species, particularly Enterococcus faecalis and Enterococcus faecium, include gelatinase (encoded by the gelE gene), aggregation substance (asa1 gene), and enterococcal surface protein (esp gene) [13, 14, 15]. These factors are associated with biofilm formation and other virulence properties. The gelE, asa1, and esp genes have been identified in clinical and commensal Enterococcus isolates, with varying prevalence rates [16, 17]. Gelatinase, in particular, plays a significant role in biofilm formation and is associated with the initial attachment, microcolony formation, and biofilm maturation stages, contributing to the survival of Enterococcus in various environments and its involvement in persistent bacterial infections [11, 12, 18]. The presence of the gelE gene and gelatinase activity has been observed in a high percentage of Enterococcus strains, indicating its relevance in biofilm formation among these isolates [13, 19]. Biofilm formation by Enterococcus species is a significant concern as it contributes to antimicrobial resistance, virulence, evasion of the host immune system, and the presence of resistant bacteria in healthcare facilities [20]. The presence of virulence factors, including gelatinase, and their role in biofilm formation are important considerations in understanding the pathogenicity and epidemiology of Enterococcus species.

3. Biofilm formation

Biofilm formation is a complex process that involves several stages, including initial attachment, extracellular polymeric substance (EPS) production, maturation, and dispersion of the biofilm. The biofilm structure provides bacteria with the ability to tolerate harsh environmental conditions and resist antimicrobial agents, making biofilm-related diseases difficult to prevent and cure. Many mechanisms, including limited diffusion of antimicrobial agents through the biofilm, communication between the antimicrobial agents and the biofilm matrix, enzyme-mediated resistance, levels of metabolic activity within the biofilm, genetic adaptation, efflux pumps, and outer membrane structure, are thought to contribute to biofilms’ resistance to antimicrobial agents [21, 22, 23]. Additionally, quorum sensing influences the development of biofilms and regulates the production of virulence factors, contributing to biofilm resistance [24].

Therefore, Enterococcus forms biofilms through a complex interaction between genes and virulence factors, such as DNA release, cytolysin, pili, secreted antigen A, and microbial surface components that recognize adhesive matrix molecules (MSCRAMMs) [11]. Peptide pheromones such as cOB1, Ccf, and Cpd play a vital role in coordinating biofilm development through quorum sensing, targeting gene expression and regulation. One essential element in the formation of biofilms has been identified as the control of extracellular DNA (eDNA) release. In E. faecalis proteases like serin protease and gelatinase, as well as the autolysin N-acetylglucosaminidase, are important participants in this process that affect the development of biofilms and virulence (Figures 1 and 2) [25].

Figure 1.

Virulence factor and biofilm-associated proteins. (a) E. faecalis and (b) E. faecium.

Figure 2.

The protein of virulence factors and biofilm formation in enterococcus species.

3.1 Biofilm-related secreted virulence factors

3.1.1 Gelatinase

Gelatinase is an important virulence factor in E. faecalis that contributes to biofilm formation and interactions with other microbes and the host immune system [11, 13]. The long-standing model for gelatinase production is that it is positively regulated by the Fsr quorum sensing system [26]. However, two more factors that control the expression and activity of gelatinase in E. faecalis have been found in recent studies. Gelatinase expression requires Bph phosphatase in an Fsr-dependent mechanism, whereas gelatinase activity requires the membrane-anchored protein foldase PrsA in an Fsr-independent mechanism [26].

Gelatinase is encoded by the gelE gene and is an extracellular zinc metalloprotease [26]. Gelatinase is one of the key virulence factors involved in the complex interplay of genes and factors that contribute to biofilm development autolysis, interactions with other microbes, the host immune system, antibiotic resistance, and virulence [11, 27, 28]. Gelatinase is associated with the initial attachment, microcolony formation, and biofilm maturation stages, contributing to the survival of Enterococcus in various environments and its involvement in persistent bacterial infections [29]. This protein’s primary roles include the maturation of enterocin O16 and the hydrolase activity against various small peptides, collagen, hemoglobin, gelatin, complement components C3, C3a, and C5a, endothelin-1, and casein that imply a potential part in the pathophysiology of E. faecalis [30]. The presence of the gelE gene and gelatinase activity has been observed in a high percentage of Enterococcus strains, indicating its relevance in biofilm formation among these isolates [31, 32]. Gelatinase promotes the aggregation of the cells in microcolonies, which constitutes the initial step of biofilm formation [33]. Gelatinase production in E. faecalis is influenced by various environmental factors such as culture medium, temperature, pH, divalent cations, and carbon sources [34, 35].

The gelatinase gene in E. faecalis encodes a prepropolypeptide or prezymogen. The mature gelatinase is a polypeptide with 318 amino acid residues and a molecular mass of approximately 34.05 kDa. It was synthesized as a 509-amino-acid prepropolypeptide, which is subject to cleavage of the 192 amino acids at the amino-terminal end comprising the presequence or signal sequence and the prosequence [11, 36]. In order to fully activate the protease activity of gelatinase, processing involves the removal of 14 C-terminal amino acid residues [37].

Gelatinase and serine protease, encoded by gelE and sprE, respectively, are regulated positively by the Fsr quorum-sensing system, which are part of an Agr-like system in E. faecalis [38]. Both gelE and sprE are located adjacent to the Fsr genes and are regulated by a common promoter [26]. The Fsr quorum-sensing system controls biofilm development through regulating the production of gelatinase [39].

Gelatinase requires the removal of its 14 C-terminal amino acid in order to demonstrate its protease activity. The Fsr quorum-sensing system controls the gelatinase gelE gene, which is a component of the gelE-sprE operon, along with two other loci (ef1097 and FsrABDC). The two-component system that drives the transcription of gelE consists of a membrane-bound histidine kinase (HK) that recognizes extracellular cues, potentially harmful substances, or other factors that alter the extracellular environment’s pH, osmolarity, or redox status. In response, HK autophosphorylates itself. The second component is a response regulator (RR), which controls DNA transcriptions and thereby triggers cellular responses [27, 40]. These proteases work together with the Fsr quorum-sensing system to promote virulence, tissue deterioration, and biofilm formation [28, 37, 38]. In addition to the Fsr system, two other factors have been identified to regulate gelatinase expression and activity in E. faecalis OG1RF [41].

Gelatinase is regulated by a quorum-sensing system mediated by an autoinducing peptide called gelatinase biosynthesis-activating pheromone (GBAP) [42]. After GBAP’s structure-activity relationship was investigated, it was discovered that agonist activity involves the entire ring region of GBAP, while there is no strict recognition of the tail region’s side chains [43]. The alanine substitution of Phe or Trp in GBAP abolishes their receptor-binding abilities and GBAP agonist activities, suggesting the importance of these two aromatic side chains in receptor interaction and activation [44].

In general, regulation of Fsr is mediated by the QS system in Enterococcus spp. The small lactone gelatinase biosynthesis-activating pheromone (GBAP) is produced by FsrB processing and exporting the FsrD propeptide, which is encoded by FsrD [44, 45, 46]. One of two components of a regulatory system, FsrC, phosphorylates the intracellular response regulator, FsrA, in response to extracellular GBAP [45]. The production of biofilm is subsequently induced by FsrA through the expression of the genes ef1097, ef1097b, the Fsr locus, gelE (which codes for a gelatinase), and sprE (which codes for a serine protease) [39, 47].

These findings expand the current model for gelatinase production in E. faecalis and provide important insights into the regulatory networks involved in gelatinase expression [48]. Gelatinase-mediated disruption of the intestinal epithelium is also implicated in disease pathogenesis, highlighting the importance of understanding gelatinase function in Enterococcus (Figure 3) [33].

Figure 3.

Gelatinase production in E. faecalis. The Fsr quorum-sensing system and its regulation in E. faecalis are subjects of interest. Upon export and processing of the FsrD propeptide (which is encoded by FsrD), a small lactone known as gelatinase biosynthesis-activating pheromone (GBAP) is produced through the involvement of FsrB. FsrC, on the other hand, is a component of a two-component regulatory system that responds to extracellular GBAP, leading to the phosphorylation of FsrA, the intracellular response regulator. Thus, the expression of the genes Fsr locus, gelE (which encodes a gelatinase), ef1097, ef1097b, and sprE (which encodes a serine protease) is induced. The pre-proprotein, consisting of 170 amino acids, and encoded by ef1097, undergoes cleavage (with the removal of the N-terminal 34 amino acids) and is then transported through the sec-dependent pathway. It is during this process that the precursor is further modified by gelatinase, resulting in the formation of enterocin O16.

3.1.2 Cytolysin

Cytolysin is a virulence factor produced by E. faecalis that is associated with biofilm formation [49]. This protein participates in quorum sensing, a peptide pheromone-mediated process of intercellular communication that includes Cb, Ccf, and Cpd. Therefore, through regulating and focusing on gene expression, quorum sensing plays a critical role in biofilm formation [50]. This process is essential for the development of multidrug-resistant properties in pathogenic bacteria like E. faecalis [51].

The release of cytokines triggers the dlt gene of LTA, which improves autolytic activity and bacterial envelope properties, contributing to biofilm formation [11, 49]. Cytolysin, activated by the cylLL and cylLS genes, enhances the survival ability of E. faecalis and may play a role in biofilm formation [52]. Additionally, gelatinase and serine protease are key players in biofilm development and virulence in E. faecalis [53]. The regulation of extracellular DNA (eDNA) release, mediated by autolysin and proteases, also influences biofilm development in E. faecalis [54]. Overall, the presence of cytolysin and its interaction with other virulence factors contribute to the biofilm-forming ability of Enterococcus species, including E. faecalis.

Cytolysin is a protein closely related to lantibiotics, a class of bacteriocins that have lanthionine (Certain strains of E. faecalis produce a structurally unique bacterial toxin called cytolysin and has a distant relationship to the lantibiotics, a class of bacteriocins.) [52], and methyllanthionine, dehydroalanine, and dehydrobutyrine structures with different stereochemistries in the same peptide [55]. Cytolysin and aggregation substance (AS) are both encoded on the pAD1 sex pheromone plasmid [56]. The pAD1 plasmid is a 60-kb conjugative plasmid found in E. faecalis that encodes a mating response to the peptide sex pheromone cAD1 and a cytolytic exotoxin [57, 58]. The cytolytic exotoxin contributes to the virulence of E. faecalis. The AS, also known as aggregation substance, is an adhesin that mediates cell-to-cell contact between mating partners, leading to efficient plasmid transfer. The AS is responsible for both adhesions to host tissues and aggregation of E. faecalis cells [59].

Cytolysin’s function is to kill other bacteria, with a particular emphasis on Gram-negative bacteria and eukaryotic cells like red blood cells. In solid media, hemolysis is observed, but not in liquid cultures [11]. Cell death is required for the formation of biofilms. Extracellular DNA is created, which is necessary for the synthesis of biofilms [60].

The genes responsible for cytolysin synthesis in Enterococcus are organized into an operon, which includes eight genes: cylR1, cylR2, cylLL, cylLS, cylM, cylB, cylA, and cylI [28, 61]. The cytolysin operon in E. faecalis transcribed polycistronically and contains two promoters, PL and PR, which regulate the transcription of genes related to toxin production and regulation, respectively [62]. The PL promoter regulates the transcription of the structural genes, while the PR promoter regulates the transcription of the regulatory genes, cylR1 and cylR2, which are transcribed divergently [37]. The remaining six genes, cylLL, cylLS, cylM, cylB, cylA, and cylI, encode structural and functional proteins [63].

Two proteins, CylR1 and CylR2, are responsible for the repression of the cytolysin operon, which is then derepressed through a quorum-sensing mechanism that involves the secreted autoinducer CylLS″ [64]. The active cytolysin subunits, CylLL″ and CylLS″, are synthesized ribosomally as nonidentical peptides, posttranslationally modified, then secreted and activated [65].

After the transcription and translation of the cylLL and cylLS genes in E. faecalis, the resulting peptides undergo a series of changes both intracellularly and extracellularly [11]. Intracellularly, the peptides undergo several modifications, including dehydrogenation of serine and threonine, cyclization reactions, and proteolytic cleavage [66]. The CylM enzyme (that is synthesized by the 5th gene in this operon) catalyzes the dehydrogenation of serine and threonine, generating CylLL* and CylLS*, which are further modified by CylB (encoded by the 6th gene in the operon). CylB has two domains [11]. The C-terminal domain of CylB acts as an ATP-binding transporter, exporting the subunits out of the cell, while the N-terminal domain acts as a proteolytic site, cleaving the peptides outside the cell to form CylLL′ and CylLS′ [67]. ATP is needed to transform CylLS*, but not needed to transform CylLL*. The extracellular protease CylA (encoded by 7th gene in the operon) removes six amino acid residues from both CylLL and CylLS, making them active toxin subunits. These extracellular changes play a role in the virulence and biofilm-forming capabilities of E. faecalis. Protein CylI (encoded by 8th gene in the operon), is the immunity protein that to act as a defense mechanism against autolysis by interacting with other subunits [28].

When target cells are not present, the subunit CylLL′ combines with CylLS′ to prevent the operon from being autoinducible. However, CylLL″ binds to the target preferentially when target cells are present (cause their dissolution through lysis, which may lead to cell death and the release of cellular contents, including DNA), allowing free CylLS″ to accumulate above the induction threshold to express high levels of cytolysin [11].

Additionally, the CylR1-CylR2 complex is a DNA-binding transmembrane complex that induces the synthesis of the cyl operon [68]. CylR1 is a transmembrane subunit and the mature CylLS′ is capable of interacting with this, leading to the expression of the operon, and acts as an autoinducer [69]. Therefore, CylR1 is necessary for the cytolysin operon to be induced; however, the exact mechanism by which the accumulation of extracellular CylLS″ is communicated, as well as CylR1’s function in transmitting this signal, remains unclear [11].

CylR2 is a DNA-binding protein that binds specifically to a 22 bp fragment of the cytolysin promoter region [70]. The CylR2 protein is activated when the quorum-sensing system, involving the accumulation of CylLS″, reaches a threshold level (activated PL), and allowing for the transcription of the cytolysin operon [65].

CylR2 works together with CylR1 in order to repress transcription of cytolysin genes [56]. Similarly, it can be hypothesized that CylR1 causes CylR2’s DNA binding specificity to be shifted to sequences next to the PR repeat when the autoinducer CylLS″ is present (Figure 4).

Figure 4.

Cytolysin in E. faecalis. Cytolysin is composed of two structural components known as CylLL (larger subunit) and CylLS (smaller subunit), which are encoded by the genes cylLL and cylLS. These core peptides, CylLL and CylLS, undergo post-translational modifications facilitated by CylM. Subsequently, these peptides undergo further processing and transportation mediated by CylB. The removal of six amino acid residues, also known as the leader peptide, from both CylLL and CylLS by the extracellular protease CylA results in the activation of these subunits as toxins. The signaling mechanism responsible for the dissociation of CylR2 from the PL promoter, triggered by the binding of extracellular CylLS to CylR1 at the membrane, plays a crucial role in transducing the signal. Additionally, CylI contributes to self-immunity against cytolysin.

3.1.3 Secreted antigen A (SagA)

Secreted antigen A (SagA) is a major secreted antigen found in E. faecium, and it appears to be essential for the growth, biofilm formation, resistance to stresses, and virulence of this bacterium [71]. In E. faecium, SagA was identified as the most abundant protein in biofilms, and its localization differed between different clades of E. faecium [72].

This stress-related protein (SagA) is a NlpC/p60-endopeptidase and peptidoglycan hydrolase, and plays a significant role in adhesion through its broad-spectrum binding to extracellular matrix (ECM) proteins, such as fibrinogen, fibronectin, laminin, type-I and type-II and type IV collagen, that generates small muropeptides [73].

The peptidoglycan composition and hydrolase activity of E. faecium can activate nucleotide-binding oligomerization domain-containing protein 2 (NOD2) in mammalian cells [74]. E. faecium, a commensal bacterium, and its secreted peptidoglycan hydrolase (SagA) have been found to enhance intestinal barrier function and limit bacterial pathogenesis by generating smaller muropeptides that effectively activate NOD2 in mammalian cells [75].

Additionally, the binding of SagA to the extracellular matrix is related to its role in adhesion and biofilm formation. E. faecium strains from clade A1 and B showed that SagA was the most abundant protein in biofilms, and its presence was essential for biofilm formation in most strains [72]. These findings suggest that the binding of SagA to the extracellular matrix plays a crucial role in adhesion and biofilm formation in different pathogens [76].

3.2 Biofilm-related surface proteins

3.2.1 Pili

Pili are large biopolymers comprised of subunits assembled into helix-like structures anchored in the bacterial outer membrane [77]. The pili of E. faecalis and E. faecium play a crucial role in the motility, attachment of the bacteria to host cells, conjugation, host cell invasion, DNA and protein secretion, biofilms formation, the development of pathogenicity, and antibiotic resistance [78].

The endocarditis- and biofilm-associated pili (Ebp) pili are important for the initial attachment of E. faecalis to surfaces, as well as for the formation of biofilms and the dissemination of genetic material within bacterial populations [79]. The Ebp are an important factor for biofilm formation in vitro and in vivo. The Ebp of E. faecalis are composed of several subunits, including EbpA, EbpB, EbpC, and EbpD [80]. EbpA is the major pilin subunit, while EbpB and EbpC are minor pilin subunits that contribute to the stability and assembly of the pilus structure [81]. EbpD is a sortase enzyme that catalyzes the covalent linkage of the pilin subunits during pilus assembly [82]. The Ebp pili are composed of repeating units of the EbpA subunit, which form a helical structure that extends from the bacterial surface [83].

Additionally, Ebp pili are important for the mobilization of plasmids carrying antibiotic resistance or virulence factors, providing E. faecalis with fitness and survival advantages [84]. Ebp proteins have been shown to be essential for biofilm formation in vitro, as well as in experimental animal models of endocarditis and urinary tract infections [85].

The expression of pili genes in E. faecalis, particularly the Ebp genes, involves a complex regulatory network. The Ebp operon, which encodes the Ebp pili, is regulated by various factors, including the upstream EbpR gene, the rnjB gene, and the Fsr quorum-sensing system [86]. The rnjB gene has been identified as an activator of pilin gene expression at the mRNA level. Additionally, the Ebp locus is unique to enterococci, and its expression is regulated by a combination of genetic elements and regulatory genes, which are involved in the assembly of the structural subunits into pili [87].

Protruding from the bacterial cell wall, pili are proteinaceous, non-flagellar, covalently linked multimers with matrix protein-binding and adhesive properties [88]. Pili play a role in the colonization of host tissue, bacterial cell aggregation, and the development of microcolonies and biofilms in Gram-positive bacteria [89]. They are short, hair-like structures that can have a role in movement but are more often involved in adhesion to other bacteria, host cells, or environmental surfaces. PilB pili are often involved in conjugation, and their expression is different in each stage of bacterial growth, with a significant presence during the exponential phase [11].

In E. faecium, a single bacterium’s surface contains two distinct types of pili, called PilA and PilB, both of which aid in the creation of biofilms. PilB pili are thicker than PilA, indicating the incorporation of more pilin subunits [11].

New murein sacculi form as the cell grows, pushing the older ones with PilB proteins inside to the poles. The murein sacculi are the peptidoglycan layers that form the cell wall of bacteria, and they play a crucial role in maintaining the structural integrity of the cell. During cell division, the cell grows, and new murein sacculi are synthesized. As the cell grows, the old murein sacculi are pushed to the poles, and the newly synthesized peptidoglycan layers are formed between the two new cells. The PilB proteins are translocated around the cross wall and deposited in the murein sacculi during their separation [90]. The expression of PilB pili is regulated by a complex network, and their distinct characteristics and roles make them important factors in biofilm formation and bacterial behavior [91]. PilB pili are thicker than PilA, indicating the incorporation of more pilin subunits. Additionally, PilB pili are often involved in conjugation, and their expression is different in each stage of bacterial growth, with a significant presence during the exponential phase [11]. On the other hand, PilA pili are absent during cell division and are involved in adhering the bacteria to a substrate or to another cell. These pili are involved in the pathogenesis of E. faecium, as they are expressed in specific conditions and contribute to the bacterium’s ability to adhere to surfaces and form biofilms [92].

PilA-type pili are only expressed when cells are grown on solid media; they are not expressed in broth culture. Temperature influences the regulation of PilA-type pili’s surface expression [93]. Conditional expression of pili may play a role in the pathogenesis of E. faecium, as evidenced by the specific enrichment of pilin gene clusters in hospital-acquired E. faecium isolates [94]. PilA-type pili play a role in the formation of biofilms by E. faecium, which is a crucial aspect of enterococcal pathogenesis [95].

3.2.2 Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs)

Microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) play a significant role in the biofilm formation of Enterococcus species. MSCRAMMs are increasingly associated with multi-drug-resistant strains.

MSCRAMMs are a family of surface-anchored bacterial adhesins that bind to host molecules, particularly collagen, and mediate the initial attachment of bacteria to host tissue, playing a critical role in the establishment of infection [11]. Examples of MSCRAMMs include Ace, Acm, and Scm [96]. Ace in E. faecalis, and Acm and Scm in E. faecium are collagen-binding MSCRAMM [97]. The amino acid sequence of these surface-anchored proteins typically consists of leucine, proline, glycine, and any other amino acid. Ace is a collagen-binding MSCRAMM protein that inhibits the adhesion of enterococcal bacteria to extracellular matrix proteins [98]. Acm is a cell-wall-anchored collagen adhesin produced by some isolates of E. faecium [94]. Scm is a second collagen adhesin of E. faecium that binds to collagen type V efficiently and differs from Acm in its binding specificities [10]. These proteins, along with other virulence factors and antibiotic resistance, are important during enterococcal infection. The presence of these surface proteins in the majority of E. faecalis isolates suggests their requirement during infection and their potential as targets for therapy or vaccine development.

3.2.3 Ace

The Ace protein is involved in biofilm formation in E. faecalis. The Ace gene in E. faecalis encodes an adhesin that binds to collagen (types I and IV) and laminin [99]. Ace is a collagen-binding MSCRAMM from E. faecalis that is thought to mediate the pathogen’s attachment to host tissues through its interactions with collagen and laminin. It is a major player in simulated faecalis infections [100]. Ace is expressed conditionally after growth in serum or in the presence of collagen [101]. The expression of Ace is significantly induced by high temperature (culture at 46°C) and in vivo through a variety of pathways, depending on the surroundings that E. faecalis encountered when infecting [102]. Patients’ serum has been shown to contain antibodies against Ace in cases of E. faecalis infection [103].

Ace protein is positively regulated by the ArgR family transcription factors AhrC and ArgR2 [104, 105]. These transcription factors activate the expression of EbpR, which in turn activates the transcription of the pilus structural genes, including the Ebp locus encoding adhesive pili [104]. The expression of Ace is also enhanced by AhrC and ArgR2, but it is not dependent on EbpR [106]. Additionally, the GrvRS two-component regulatory system plays a major role in controlling Ace expression and E. faecalis virulence [107].

Ace expression in E. faecalis is dependent on both external and internal factors. External factors include the presence of bile salts and serum, while internal factors include the putative Ers box [11]. Ace is expressed conditionally after growth in serum or in the presence of collagen [101, 108]. The expression of Ace is significantly induced by high temperature (culture at 46°C) and in vivo by various mechanisms, according to the environmental conditions encountered by E. faecalis during infection. Patients’ serum has been shown to contain antibodies against Ace in cases of faecalis infection [109].

Research has demonstrated that in rat models, the administration of monoclonal anti-Ace antibodies (mAb), more especially mAb70, decreases E. faecalis infections. In a rat infective endocarditis model, pretreatment with mAb70 significantly reduced E. faecalis aortic valve infection [110].

The N1N2 subdomains of collagen-binding adhesins in E. faecalis bind to collagen through a mechanism called the “Collagen Hug” mechanism [111]. This mechanism involves the N1N2 subdomains adopting a two-domain structure, with N2 being the binding subdomain, and using the “Collagen Hug” mechanism for binding to collagen. The N1N2 subdomains of Ace in E. faecalis have been shown to bind collagen with high affinity [112, 113]. The binding mechanism is initiated when the collagen triple helix interacts with the N1N2 subdomains, leading to structural rearrangements within the N1 domain and the insertion of the C-terminal extension of the N2 domain into the N1 domain by β-strand complementation, forming a “latch” [113, 114, 115].

3.2.4 Acm

The Acm protein in E. faecium is encoded by the Acm gene, which stands for adhesion to collagen of E. faecium. The Acm protein is a cell wall-anchored collagen adhesin that mediates adherence to collagen. It is composed of an A domain, various B sequences, and a C-terminal sequence that enables anchoring to the peptidoglycan layer [71, 116].

The N-terminal part of the Acm protein in E. faecium interacts with both integrins and enterococcal surface proteins, while the C-terminal region is bound to the cell wall. The N-terminal domain of the thermo-regulated surface protein PrpA of E. faecium has also been found to interact with fibrinogen, fibronectin, and platelets [117]. The N-terminal part of the Acm protein interacts with integrins and enterococcal surface proteins, while the C-terminal region is responsible for binding to the cell wall.

Three subdomains make up the Acm A domain: N1, which corresponds to aa 29–150; N2, which corresponds to aa 151–346; and N3, which corresponds to aa 347–529 [118]. The specific subdomains of the A domain are also identified, further confirming the interaction of the N-terminal part with collagen and other proteins [119].

The N1N2 subdomains bind with affinity to collagen, through a mechanism called the collagen hug mechanism [113]. The binding mechanism is initiated when the collagen triple helix interacts with the N1N2 subdomains, leading to structural rearrangements within the N1 domain and the insertion of the C-terminal extension of the N2 domain into the N1 domain by β-strand complementation, forming a “latch” [120]. The amount of Acm protein expressed on the cell surface is correlated with the pathogen’s adherence levels [121].

The amount of surface Acm correlates with collagen adherence in E. faecium isolates derived from clinical samples [122]. The Acm protein was detected predominantly in clinically derived isolates, and levels of collagen adherence were highly correlated with the amount of cell surface Acm [123]. This correlation is significant as Acm is a collagen adhesin that enhances the initial adherence of E. faecium to damaged heart valves, and human antibodies against Acm have been shown to inhibit collagen adherence of E. faecium [124, 125].

Collagen type I adherence was found to be highly statistically significantly correlated with a clinical origin serving as the isolate’s source [126, 127]. The presence of an uninterrupted Acm gene and surface expression of Acm were confirmed in the diverse collection of isolates, indicating the involvement of Acm in collagen adherence [11, 128].

In patients at risk of E. faecium infections, the use of particular antibodies against Acm would facilitate the inhibition of collagen adherence and is therefore a promising therapeutic and preventive strategy [129]. Antibodies against the high-affinity binding subdomains of Acm inhibited collagen adherence of E. faecium cells [129].

3.2.5 Scm

Scm (second collagen adhesin of E. faecium), previously known as Fms10, is a collagen adhesin produced by E. faecium [98]. Scm binds to collagen type V and fibrinogen, and it is one of the 15 predicted microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) in E. faecium [10]. Scm has a much greater affinity for collagen type V than Acm, another collagen adhesin in E. faecium [119].

E. faecium can tailor its adherence phenotype to the specific tissue it is growing in by using its two types of collagen adhesins [98]. Type V collagen is prevalent in the intestinal submucosa [130], which is abundant in the intestinal submucosa, and fibrinogen, allowing the bacterium to fine-tune its adherence phenotype to better suit the given tissue [131]. This adhesion capability is important for the colonization and persistence of E. faecium in different tissues, making Scm a potential target in the treatment of E. faecium-producing biofilm infections. Therefore, Scm might be a key factor that causes E. faecium’s resistance and persistence in the gastrointestinal (GI) tract [132] and Scm is a potential target in the treatment of E. faecium-producing biofilm infections [11].

3.2.6 Aggregation substance (AS)

A glycoprotein on the bacterial surface that resembles hair is called the aggregation substance (AS) [133]. When exposed to a sex pheromone, cAD1, it is synthesized on “older” structures of the Enterococcus cell wall from the asa1 gene, which is found on a sex pheromone plasmid, pAD1 [134]. The two cells can now exchange the pAD1 plasmid, which contains the asa1 gene that codes for AS, once the AS is expressed on the cell surface and interacts with the EBS (enterococcal-binding substance) [79].

The aggregation substance (AS) contains two Arg-Gly-Asp (RGD) integrin-binding motifs, which are known to ligate integrins and play a role in AS-mediated binding to eukaryotic cells [135].

Aspartic acid, glycine, and arginine make up the three amino acid sequences known as RGD, which aids in adhesion. This motif interacts with the lipoteichoic acid-like substance Enterococcus-binding substance (EBS) and the β subunit of integrins of eukaryotic cells, primarily macrophages and epithelial cells [116].

The aggregation substance (AS) has a signal domain with 43 amino acids and a proline-rich sequence at the C-terminal, which is crucial for integration into the bacterial cell wall and membrane [11].

The AS is involved in adhesion, aggregation, and antibiotic resistance, and it is a key factor in biofilm production by Enterococcus species. The AS is a significant component of the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs) family, which are bacterial adhesins capable of binding predominantly to collagen and other extracellular matrix elements [136].

The Enterococcus-binding substance (EBS), which is similar in structure to lipoteichoic acid, also interacts with the β subunit of the integrins of eukaryotic cells, mainly macrophages and epithelial cells [137]. The accurate statement is that the RGD sequence comprises three amino acids that mediate the binding of numerous ligands to eukaryotic integrin receptors. The protein interacts with the β subunit of integrins in eukaryotic cells, specifically in macrophages and epithelial cells, as well as the Enterococcus-binding substance (EBS), which shares structural similarities with lipoteichoic acid.

The N-terminal domain of the AS protein (amino acids 44–331) is surface-exposed and has been shown to interact with integrins and enterococcal surface proteins [138]. The C-terminal region of the AS protein contains an LPXTG motif, which is responsible for anchoring the protein to the cell wall [139].

An essential virulence factor for the development of infective endocarditis has been identified as the N-terminal portion of the aggregation substance (AS) protein from E. faecalis [140, 141].

It has been demonstrated that the AS protein increases adherence and internalization into several cell types, most likely by means of the N-terminal domain-mediated integrin binding. The N-terminal signal peptide directs secretory proteins into the endoplasmic reticulum of eukaryotes or the periplasmic space of prokaryotes [141].

The N-terminal part of the aggregation substance (AS) protein from E. faecalis interacts with both integrins and enterococcal surface proteins, while the C-terminal region is bound to the cell wall. When donor and recipient cells transfer genes, the AS protein, a large (137 kDa) surface-expressed protein that is encoded by pheromone-responsive conjugative plasmids, is required for the formation of large-cell aggregates [142]. Since AS encourages adherence to cultured pig kidney tubular cells and internalization into cultured intestinal epithelial cells, there are several lines of evidence that support its role as a virulence factor [138]. The N-terminal domain of the AS protein (amino acids 44–331) is surface-exposed and has been shown to interact with integrins and enterococcal surface proteins [138]. The C-terminal region of the AS protein contains an LPXTG motif, which is responsible for anchoring the protein to the cell wall [143].

According to experimental research, biofilm formation is substantially reduced when N-terminal amino acids are removed in comparison to their C-terminal mutant counterparts [11]. This suggests that the N-terminal part of the protein plays a crucial role in biofilm formation.

The AS protein from E. faecalis helps with bacterial aggregation and is involved in the formation of large-cell aggregates during gene transfer between donor and recipient cells [68]. The F− bacterium secretes small peptides called sex pheromones, which are important for intercellular communication among Gram-positive bacteria, including enterococcal sex pheromone systems [144, 145]. E. faecalis strains lacking plasmids release a group of heat-stable, protease-sensitive pheromones that are specific to donors with different conjugative plasmids. In response, bacteria harboring a specific plasmid produce a proteinaceous adhesin that promotes the formation of mating aggregates with adjacent recipients [146]. The N-terminal signal peptides direct secretory proteins into the endoplasmic reticulum of eukaryotes or the periplasmic space of prokaryotes.

The AS protein from E. faecalis helps with bacterial aggregation and is involved in the formation of large-cell aggregates during gene transfer between donor and recipient cells. The F− bacterium secretes small peptides called sex pheromones, which are important for intercellular communication among Gram-positive bacteria, including enterococcal sex pheromone systems. When the F+ bacterium detects these pheromones, it initiates transcription of the asa1 gene and displays AS on its cell surface. Once the AS is expressed on the cell surface, it interacts with EBS, and conjugation occurs, after which both bacteria possess the pAD1 plasmid [11].

The F+ bacterium responds to the sex pheromones released by the F− bacterium by activating the transcription of the asa1 gene, leading to the production of the aggregation substance (AS) protein on the cell surface. Subsequently, when AS interacts with EBS, conjugation occurs, leading to the transfer of the pAD1 plasmid, after which both bacteria possess the pAD1 plasmid. This process is part of the intercellular communication and gene transfer mechanisms observed in E. faecalis, involving the synthesis and expression of AS in response to sex pheromones, ultimately leading to conjugation and plasmid transfer [11].

The expression of the aggregation substance (AS) protein in E. faecalis leads to bacterial clumping and higher antibiotic resistance, which helps in biofilm formation. According to experimental research, biofilm formation is substantially reduced when N-terminal amino acids are removed in comparison to their C-terminal mutant counterparts. The AS protein is a glycoprotein that resembles hair and is found on the surface of bacteria. It is produced by the asa1 gene located on the sex pheromone plasmid pAD1, on the “older” structures of the Enterococcus cell wall when exposed to the sex pheromone cAD1. It has been demonstrated that the AS protein increases adherence and internalization into several cell types, most likely by means of the N-terminal domain-mediated integrin binding [11]. The AS protein plays a crucial role in biofilm formation and antibiotic resistance in E. faecalis.

The aggregation substance (AS) of E. faecalis has been shown to promote adherence, phagocytosis, and intracellular survival of the bacterium within human macrophages. It is encoded on sex pheromone plasmids and is a surface-bound glycoprotein that mediates aggregation between bacteria, thereby facilitating the formation of large-cell aggregates during gene transfer between donor and recipient cells. The AS protein has been found to significantly augment adherence and internalization by macrophages via an interaction with the integrin macrophage-1 antigen (Mac-1). This interaction allows AS-positive enterococci to outlive phagocytosis significantly better than AS-negative strains [147]. Additionally, the AS protein has been implicated as an important virulence factor for the development of infective endocarditis and has been shown to promote adherence to cultured pig kidney tubular cells and internalization into cultured intestinal epithelial cells. The N-terminal domain of the AS protein has been identified as a surface-exposed region that is involved in these interactions. The AS protein not only facilitates bacterial aggregation but also plays a significant role in promoting adherence, phagocytosis, and intracellular survival within macrophages, contributing to the pathogenesis of E. faecalis infections.

The RGD motifs of the aggregation substance (AS) are recognized by the beta subunits of integrins, particularly αvβ3, αvβ5, αvβ6, αvβ8, α5β1, and αIIbβ3 [148]. These integrins are known to bind the RGD-recognition sequence and are involved in mediating cell adhesion, migration, and signaling processes by recognizing specific binding sites, typically small peptide sequences such as RGD motifs [148]. The RGD motifs within the AS protein are important for integrin binding and have been shown to play a role in promoting adherence, phagocytosis, and intracellular survival of E. faecalis within human macrophages [11].

The RGD motifs of the aggregation substance (AS) are known to ligate integrins, particularly CD18 and CD11b on macrophages, which helps with bacterial integration [149]. The RGD motifs are important for integrin binding and have been shown to play a role in promoting adherence, phagocytosis, and intracellular survival of E. faecalis within human macrophages [150, 151]. The same RGD region also helps in extracellular matrix binding [152]. The AS protein contains an N-terminal domain, a variable region, a central domain, and a C-terminal domain that is responsible for anchoring the protein to the cell wall [153]. The RGD motifs within the AS protein play a crucial role in mediating interactions with host cells and promoting bacterial survival, contributing to the pathogenesis of E. faecalis infections.

The presence of the aggregation substance (AS) on the surface of E. faecalis increases the adherence to both fibronectin (8-fold) and thrombospondin (4-fold) [11]. The RGD motifs within the AS protein are important for integrin binding and have been shown to play a role in promoting adherence, phagocytosis, and intracellular survival of E. faecalis within human macrophages [154]. The main integrins recognized by the RGD motifs are CD18 and CD11b on macrophages, which help with bacterial integration [155]. The same RGD region also helps in extracellular matrix binding [156]. The RGD motifs within the AS protein play a crucial role in mediating interactions with host cells and promoting bacterial survival, contributing to the pathogenesis of E. faecalis infections.

The monoclonal antibodies against CD18 and CD11b reduce bacterial integration, prohibiting bacterial dissemination throughout the organism [157]. CD18 and CD11b are beta subunits of integrins that are recognized by the RGD motifs within the aggregation substance (AS) protein of E. faecalis [11]. The AS protein has been shown to increase adherence to both fibronectin and thrombospondin, and the RGD motifs within the AS protein are important for integrin binding [158]. Therefore, monoclonal antibodies against CD18 and CD11b can reduce bacterial integration by inhibiting the interaction between integrins and the RGD motifs within the AS protein, which is crucial for bacterial adherence and survival.

RGDS (RGD serine)-containing peptides have been shown to latch onto the integrins and enterococcal-binding substance (EBS) better than RGD, thus entering into competition with the aggregation substance (AS) [159]. The RGD motifs within the AS protein are important for integrin binding and have been shown to play a role in promoting adherence, phagocytosis, and intracellular survival of E. faecalis within human macrophages. The main integrins recognized by the RGD motifs are CD18 and CD11b on macrophages, which help with bacterial integration. Therefore, RGDS-containing peptides can compete with the RGD motifs within the AS protein for integrin binding, potentially reducing bacterial adherence and survival [11].

3.3 Quorum-sensing molecules

In Enterococcus, the quorum-sensing process involves the production and release of pheromones, which are tiny signal molecules that trigger a coordinated response among bacteria within the community. They are secreted or excreted chemical factors that trigger a social response in members of the same species.

Both Gram-negative and Gram-positive bacteria use cell-to-cell communication, including the use of pheromones for intercellular signaling. In Gram-negative bacteria, quorum sensing is mediated by small diffusible molecules called autoinducers, such as N-acyl homoserine lactones, which regulate various processes such as bioluminescence, virulence factor secretion, and biofilm formation [160]. On the other hand, in Gram-positive bacteria, cell-to-cell communication is often mediated by peptide-based pheromones, which control a wide range of functions, including competence, virulence, and other regulatory processes.

Bacteria produce autoinducers through this process, which is a population-density-dependent regulatory mechanism. As the cell density increases, these molecules accumulate in the environment. The position of these autoinducers’ receptors, which are found in the cytoplasm or on the cell surface, determines how they function [161].

In the case of E. faecalis, the quorum-sensing process is done through the pheromones. These pheromones play a key role in the population density-dependent regulatory mechanism by which bacteria produce autoinducers, and they control major virulence determinants including several virulence factors that are regulated by the Fsr quorum-sensing system, which encodes the cytolysin toxin and the conjugal transfer system of particular plasmids in E. faecalis (each pheromone triggers the conjugal transfer system of a particular plasmid) [162].

Some examples of these pheromones include Cpd, cOB1, and Ccf. These pheromones play a crucial role in the regulation of various biological processes, such as the transfer of conjugative plasmids and the control of major virulence determinants [50]. The enterococcal sex pheromone systems function through the import of signaling pheromone peptides, which are encoded by specific genes and are involved in the control of plasmid transfer and other regulatory functions [163].

Thus, this process makes it easier for genes that encourage the formation of biofilms to spread, such as adhesins that are involved in the infection’s pathogenesis.

Pheromones from bacteria, such as E. faecalis, have been shown to be chemotactic for human leukocytes, inducing the production of superoxides and the secretion of lysosomal enzymes. This chemotactic response demonstrates the ability of bacterial pheromones to influence the behavior of human leukocytes, highlighting their potential role as virulence factors and their impact on the host immune response.

The peptide pheromones in both Gram-negative and Gram-positive bacteria consist of sequences with seven to nine amino acids and are synthesized by ribosomes and secreted or excreted to elicit a particular reaction from another organism of the same species. Pheromones undergo posttranslation modifications and become activated during excretion. For instance, peptide pheromones in E. faecalis undergo posttranslational modifications, including proteolytic cleavage and lipidation, before being secreted and activating the quorum-sensing system. These modifications are essential for the proper function of the pheromones and their ability to regulate various biological processes.

3.3.1 cOB1

A sex pheromone called cOB1 was identified in E. faecalis bacteria and is composed of eight hydrophobic amino acids, which are H-Val-Ala-Val-Leu-Gly-Ala-OH [164]. It causes the E. faecalis hemolysin-bacteriocin (Hly/Bac) plasmids, pOB1 and pYI1, to conjugally transfer [11, 165]. This pheromone plays a significant role in the regulation of bacterial conjugation and the transfer of genetic material. Additionally, a study has shown that the pheromone peptide cOB1 forms amyloid-like structures, a characteristic not previously reported for such molecules [166]. cOB1 is an important signaling molecule involved in the communication and genetic exchange within E. faecalis bacterial populations and has been referred to as a “clumping-inducing agent” due to its role in mediating clumping between bacterial donors, also known as “self-clumping” [167]. This characteristic is in line with its function as a signaling molecule involved in the regulation of bacterial conjugation and genetic exchange. The term “clumping” refers to the aggregation of bacterial cells, and the pheromone’s ability to induce this phenomenon is an important aspect of its role in intercellular communication and the coordination of genetic transfer processes.

The cCF10 pheromone secreted by the recipient cells facilitates the transfer of the pCF10 plasmid from donor cells in E. faecalis [168, 169]. The cCF10 pheromone is a peptide that induces high-frequency plasmid transfer in vitro and in vivo, and it plays a crucial role in the regulation of bacterial conjugation and genetic exchange and genetic transfer processes within E. faecalis bacterial populations [170]. The cCF10 pheromone is internalized by donor cells that carry pCF10, causing the expression of aggregation substance (Asc10) that facilitates plasmid transfer [171].

Bacteria, including E. faecalis, secrete sex pheromones, which are linked to the formation of biofilms through the paracrine pheromone signaling pathway [90]. This pathway involves the secretion of pheromones that act as signaling molecules to regulate various processes, such as genetic transfer, conjugation, and biofilm formation. The pheromones are detected by specific receptors on neighboring cells, leading to the activation of downstream pathways that influence biofilm development and other collective behaviors [172]. The paracrine pheromone signaling pathway plays a crucial role in coordinating bacterial community behaviors, including biofilm formation, and represents an important mechanism for intercellular communication and regulation of group activities in bacterial populations.

There is a mechanism to prevent the autocrine activation of the paracrine pheromone signaling pathway in E. faecalis that includes iCF10 and PrgY. Plasmid pCF10 carries two genes, iCF10 and PrgY, that function to prevent self-induction by endogenous cCF10 in donor cells [163].

The CF10 pheromone is directly degraded by PrgY, whereas the short protein iCF10 functions as a competitive signaling inhibitor on the pheromone.

So, the membrane protein PrgY reduces endogenous cCF10 levels, while iCF10 acts as an inhibitor of cCF10 activity, preventing autocrine activation of the signaling pathway [11, 163]. These genes play a crucial role in regulating the pheromone signaling pathway and preventing self-induction, highlighting their significance in the coordination of bacterial conjugation and genetic exchange processes.

A blood plasma component degraded the competitive signaling inhibitor iCF10, activating the conjugation system and escalating the level of colonization in the host [173]. This degradation of iCF10 leads to the activation of the conjugation system, which is involved in the transfer of genetic material, and has been associated with an increase in the degree of colonization in vivo [11]. The degradation of iCF10 by a blood plasma component represents a significant regulatory mechanism that impacts the conjugation system and colonization in the host.

The cOB1 pheromone is a bacterial sex pheromone that induces the conjugal transfer of the E. faecalis hemolysin-bacteriocin (Hly/Bac) plasmid, pOB1 [174]. It has been shown to effectively inhibit multidrug-resistant E. faecalis V583 [175]. The secretion of this pheromone is mediated by an ABC transporter, and it is essential for the regulation of bacterial conjugation and genetic exchange processes. The species specificity of pheromonal signals and their role in mediating innate responses and individual recognition in vertebrates have also been studied.

The signaling lipoprotein EF2496, which has the cOB1 precursor sequence, encodes the cOB1 pheromone in E. faecalis. Peptide pheromone transporter (PptAB), an ATP-binding transporter, carries cOB1 out of the cell via a zinc-dependent metalloprotease-mediated cleavage. The production and interaction of cOB1 depend on this proteolytic cleavage of the lipoprotein signal peptide. The monomeric form of cOB1 is capable of binding to the specific receptor and inducing a response, while the aggregated form does not bind to the receptor. cOB1 is a sex pheromone consisting of eight hydrophobic amino acids and influences the relationship with the host environment, as well as the conjugative transfer of plasmids. When released, cOB1 modulates the expression of genes and has been shown to induce transcriptional incompatibility between strains. The cOB1 pheromone is an important factor in the regulation of genetic transfer, host interaction, and gene expression in E. faecalis [11].

Gene expression is modulated by the cOB1 pheromone when it is released and binds to its receptor on the multidrug-resistant Enterococcus V583.

E. faecalis strain V583 is an opportunistic, nosocomial bacterial strain that possesses acquired multidrug resistance mechanisms.

The major effect of the cOB1 expressed by the commensal species on V583 is to induce transcriptional incompatibility, resulting in the killing of V583. The pTEF2 genes in multidrug-resistant E. faecalis V583 are involved in the pheromone-mediated killing of V583. The primary function of the cOB1 expressed by the commensal species on V583 is to induce transcription of the pTEF2 genes, which is the means by which the pTEF2-dependent killing is mediated by a pheromone [11]. The pheromone-mediated killing of V583 is a result of the induction of transcriptional incompatibility, leading to the inhibition of gene transfer and incompatibility between V583 and the fecal consortium, ultimately causing the killing of V583 [11].

3.3.2 Ccf

The CcfA gene is indeed responsible for the production of the cCF10 pheromone in E. faecalis [174]. The cCF10 pheromone is a paracrine peptide sex pheromone that also acts as an autocrine signal [168]. It is involved in the regulation of conjugation and biofilm formation in E. faecalis. The cCF10 pheromone is a key signaling molecule that mediates the induction of the conjugative mating response in E. faecalis and induces conjugative transfer of the E. faecalis tetracycline resistance plasmid pCF10 [172, 174].

With a molecular weight of 789, the cCF10 pheromone is composed of the amino acid sequence H-Leu-Val-Thr-Leu-Val-Phe-Val-OH. Two distinct peptides, one inhibitor (iCF10) and one pheromone (cCF10), both expressed by significant E. faecalis genes, mediate the transfer of pCF10. The iCF10 inhibitor is encoded by the iCF10 gene, which acts as an inhibitor of cCF10 activity, preventing autocrine activation of the signaling pathway. The PrgX receptor protein in E. faecalis is involved in the regulation of conjugation through its interaction with the cCF10 and iCF10 peptides. In the absence of cCF10, PrgX binds to specific target sites on the pCF10 plasmid and represses the transcription of genes involved in conjugation. However, when cCF10 is present, it alleviates this repression, allowing for the transcription of downstream genes encoding conjugation proteins. On the other hand, iCF10 acts as an inhibitor of the conjugation process by competing with cCF10 for interaction with PrgX. The balance between the cCF10 and iCF10 peptides determines the conjugative state of the cell, with cCF10 promoting conjugative transfer and iCF10 inhibiting it. The interaction of PrgX with these peptides is a key mechanism for the regulation of conjugation in E. faecalis [11].

Additionally, the Eep gene plays a role in the maturation of the iCF10 pheromone, contributing to the intricate regulatory network of peptide pheromone signaling in E. faecalis.

3.3.3 Cpd

The cPD1 pheromone, which facilitates the transfer of the pPD1 plasmid in E. faecalis, is in fact produced by the Cpd gene. The cPD1 pheromone has a molecular weight of 912 and an amino acid structure of H-Phe-Leu-Val-Met-Phe-Leu-Ser-Gly-OH [11]. This pheromone is involved in the regulation of plasmid transfer and the mating response.

The transfer of the pPD1 plasmid is initiated in response to the cPD1 pheromone, which is constitutively secreted by recipient bacteria [176]. The cPD1 pheromone binds to the TraA intercellular receptor [174]. The TraA protein encoded by the pPD1 plasmid functions as the receptor for the cPD1 pheromone, transducing its signal to initiate the mating response [11].

The recipient cells produce the iPD1 inhibitor, which can prevent the transfer of the pPD1 plasmid. The pPD1 plasmid’s conjugative transfer cannot be activated by the iPD1 inhibitor, a competitive inhibitor that binds to TraA prior to the cPD1 pheromone [70].

TraC is described as an extracellular, less-specific pheromone-binding protein, suggesting a lower affinity for cPD1 compared to TraA.

This is supported by competitive-inhibition analysis, which showed that the binding function of TraA is completely specific to the related cPD1 and iPD1, indicating that TraA is the primary receptor for these pheromones. Additionally, the production of the iPD1 inhibitor by recipient cells functions as a TraA antagonist, blocking self-induction in the recipient cells. PrgZ is a pheromone-binding protein that binds with the cCF10 pheromone, playing a role in the regulation of plasmid transfer. The similarity between PrgZ and TraC is supported by their sequence homology and their involvement in pheromone binding and transport. However, while TraC is involved in the crossing of the cell wall by the cPD1 pheromone, PrgZ is involved in the regulation of plasmid transfer by binding with the cCF10 pheromone and activating the conjugative transfer of the pCF10 plasmid. Therefore, while PrgZ and TraC share sequence homology and are involved in pheromone binding and transport, they play distinct roles in the regulation of plasmid transfer in E. faecalis. So, cPD1 and cCF10 pheromones can bind with TraC [11].

3.3.4 Eep

The Eep gene in E. faecalis plays a crucial role in various biological characteristics and virulence of these bacteria. In E. faecalis, the Eep gene is responsible for the production of sex pheromones and contributes to the survival of the bacteria in the host [177]. It also affects the production and release of diverse proteins, including those involved in cell envelope integrity and adherence to host epithelial cells. The Eep gene is associated with antibiotic resistance and the modification of cell wall polysaccharides. The presence of the Eep gene in Enterococcus isolates from urinary tract infections suggests its potential role in the pathogenicity of these infections [178].

The Eep gene in E. faecalis is involved in the maturation of the peptide pheromones cAD1 and cCF10 through a membrane protease [33]. Eep, a membrane-embedded zinc metalloprotease, further degrades the N-terminal overhang to produce pheromones [179]. The protease encoded by Eep comprises 422 amino acids and is essential for the processing of these peptide pheromones. This processing is significant for the regulation of plasmid transfer and other cell-signaling processes in E. faecalis. The Eep gene is not directly involved in quorum sensing but plays a crucial role in the production and maturation of specific peptide pheromones in E. faecalis [180, 181]. Additionally, the extracytoplasmic function (ECF) sigma factor RsiV, the anti-sigma factor for sigV, is processed more easily by the protease encoded by Eep [182], which contributes to stress resistance. The protease is also essential for the activation of the extracytoplasmic function sigma factor SigV, which contributes to lysozyme resistance in E. faecalis [183]. The role of the Eep-encoded protease in the processing of RsiV and the activation of SigV is linked to the bacterium’s ability to improve stress resistance, particularly in the presence of lysozyme, an important component of the host’s innate immune system.

E. faecalis’s intrinsic resistance to lysozyme stress is provided by more than just the full degradation of RsiV, which acts as a counterbalance to SigV activation. Rather, lysozyme causes the extracytoplasmic function (ECF) sigma factor in E. faecalis, SigV, to become activated, which in turn promotes lysozyme resistance. The protease encoded by the Eep gene, Eep, is involved in the processing of RsiV and the activation of SigV under lysozyme stress, which in turn improves the bacterium’s stress resistance, particularly in the presence of lysozyme, an important component of the host’s innate immune system [11, 165, 184]. The activation of SigV, facilitated by Eep, plays a key role in E. faecalis ‘ability to resist lysozyme stress, rather than the complete degradation of RsiV being a counterpoint to SigV activation.

Research has shown that in an Eep deletion mutant strain of E. faecalis, the anti-sigma factor RsiV is only partially degraded after lysozyme exposure, indicating that Eep is required for the complete degradation of RsiV. The complete degradation of RsiV is essential for the activation of SigV, an extracytoplasmic function (ECF) sigma factor, which contributes to lysozyme resistance in E. faecalis. The role of Eep in the processing of RsiV and the activation of SigV is linked to the bacterium’s ability to improve stress resistance, particularly in the presence of lysozyme [185].

Together with the arginine repressors AhrC and ArgR, Eep prevents the uptake of this amino acid or triggers its catabolism, which causes E. faecalis to form biofilms. This collaboration is essential for the regulation of biofilm-associated virulence factors and the early stages of biofilm formation. The deletion of genes encoding these proteins has been shown to impact biofilm formation and reduce the burden in urinary tract infections.

AhrC and Eep are biofilm infection-associated virulence factors in E. faecalis, and their absence has been linked to reduced virulence in experimental biofilm infection models of catheter-associated UTI and endocarditis. These proteins are essential for the regulation of biofilm-associated virulence factors and the early stages of biofilm formation, thereby impacting the severity of UTI and endocarditis [33].

3.3.5 The Fsr operon and ef1097 locus

The Fsr operon in E. faecalis is a regulatory system that controls biofilm development through the production of gelatinase and serine protease. The Fsr operon in E. faecalis is composed of four genes: FsrA (RR), FsrB, FsrD, and FsrC (HK) [11]. When the FsrC gene is transcribed in E. faecalis, it results in the production of a membrane-bound histidine kinase (HK)—FsrC [186]. This membrane-bound HK has the role of sensing an 11-amino-acid-long cyclized peptide lactone in the extracellular environment [187]. The interaction of this membrane-bound HK—FsrC—with the pheromone ligand GBAP leads to the activation (phosphorylation) of the response regulator FsrA, which in turn facilitates the transcription of various genes, including those involved in biofilm formation and virulence [28, 40]. GBAP is a peptide lactone that mediates quorum sensing in E. faecalis, and it is involved in the regulation of biofilm formation and virulence factors, including gelatinase and serine protease. When the FsrD gene is transcribed, a propeptide FsrD is formed, which undergoes further changes mediated by FsrB. A transmembrane protein that is a member of the accessory gene regulator protein B (AgrB) family is encoded by the FsrB gene in E. faecalis. FsrB processes a propeptide, FsrD, to generate the gelatinase biosynthesis-activating pheromone (GBAP), which is a cyclic peptide of 11 amino acid residues. Transmembrane FsrB is a member of the AgrB-accessory gene regulator protein class, which is comprised of accessory regulator proteins. FsrB modifies FsrD, turning it into GBAP, which is a signaling molecule that regulates the Fsr quorum-sensing system. GBAP further interacts with FsrC, which is a membrane histidine kinase that is part of the Fsr system. FsrC is involved in the activation of the response regulator and transcription factor FsrA, which controls the expression of virulence factors in E. faecalis [188].

The transmembrane protein FsrB, a member of the accessory gene regulator protein B (AgrB) family, is encoded by the FsrB gene. The response regulator FsrA is activated when FsrB phosphorylates GBAP, a lactone ring containing a short cyclic peptide of 11 amino acid residues, which is produced from a propeptide called FsrD (encoded by FsrD). As a result, the two-component regulatory system in E. faecalis, which consists of the sensor FsrC and the response regulator FsrA, reacts to the accumulated GBAP and is primarily involved in cell-cell communication. The transcription of the FsrBCD, gelE-sprE operons, and ef1097 locus—which is 800 kb upstream of the Fsr operon—is subsequently regulated by phosphorylated FsrA [38, 39]. gelE and sprE’s downstream transcriptional responses were impacted by the FsrABC mutation. The FsrB gene, through its product FsrB, plays a crucial role in the processing of FsrD to generate GBAP, which is a key component in the quorum-sensing system and the regulation of virulence in E. faecalis [42]. The interaction between the Fsr quorum-sensing system and GBAP leads to the activation (phosphorylation) of the response regulator FsrA, which belongs to the LytTR family of DNA-binding domains. FsrA then acts as a facilitator for the transcription of the ef1097 locus (which is made up of the genes ef1097 and ed1097b), FsrB, and gelE, which are involved in the regulation of biofilm formation and virulence factors in E. faecalis. The Fsr locus regulates the transcription of the gelatinase gene (gelE) and the serine protease gene (sprE), and it is an integral component in the expression of virulence-enhancing factors, including biofilm formation. These and 75 other genes, including bopD, a crucial gene for biofilm formation, are inhibited if the Fsr quorum-sensing system is compromised [180, 189].

The production of enterocin O16, an antimicrobial peptide (bacteriocin) made up of 68 C-terminal amino acids, is carried out by the E. faecalis ef1097 locus. This antimicrobial peptide is processed from the ef1097 proprotein by the gelE protease. The antimicrobial peptide enterocin O16, which is synthesized from the ef1097 locus, suppresses the growth of Lactobacillus species in E. faecalis. It has no effect on the growth of Listeria, Enterococcus, or Staphylococcus, though. Enterocin O16 has the ability to function as an antifungal peptide at elevated concentrations. This peptide mostly affects lactobacilli and has a limited inhibitory spectrum. Furthermore, since there is no immunity linked to the synthesis of enterocin O16, E. faecalis is inherently resistant to the antimicrobial peptide. The production of enterocin O16 is regulated by the Fsr quorum-sensing system, and mutations in the Fsr system affect its production. The Fsr quorum-sensing system, particularly the concerted expression of the Fsr operon and ef1097 locus, enables the production of enterocin O16, providing E. faecalis populations with antimicrobial activity in a cell density-dependent manner. The production of enterocin O16 is regulated by the Fsr quorum-sensing system, and mutations in the Fsr system affect its production. After the precursor ef1097, a 191-amino-acid peptide, is transported out of the cell via the Sec system, which cuts the peptide between Ala56 and Ser57, the antimicrobial peptide enterocin O16 is produced. After processing the precursor at amino acid 123, the gelatinase enzyme leaves behind the mature enterocin O16, a 68-amino acid peptide residue.

3.4 Regulators of extracellular DNA release (eDNA)

Extracellular DNA (eDNA) is a critical component of biofilm formation in various bacteria, including Enterococcus ssp. The release of eDNA in biofilms is primarily attributed to cell lysis, which can be triggered by different mechanisms such as phage-related genes and holin protein activity. eDNA forms a protective matrix along with polysaccharides and proteins, contributing to the structural integrity of biofilms. The presence of eDNA in biofilms provides protection against nucleases, with different enzymes showing varying degradation capabilities. Additionally, eDNA can exhibit peroxidase-like activity, contributing to the intrinsic defense mechanisms of biofilms.

eDNA is involved in the detachment and dispersion of biofilms, acts as a structural component in the extracellular matrix, and can be used as a nutrient source. Additionally, eDNA has been associated with DNA damage repair and gene transfer through transformation of competent sister bacteria. The release of eDNA is regulated by specific mechanisms such as autolysis, active secretion, and association with membrane vesicles. Additionally, the generation of eDNA is triggered in a subpopulation in response to the accumulation of quorum-sensing signals and coordinated by the population when it reaches a specific cell density. The biological function of eDNA is not limited to a single role, as it varies depending on the microorganism, but it is clearly important in biofilm formation and maintenance, as well as in other cellular processes such as gene transfer and DNA damage repair.

eDNA release in biofilm formation of Enterococcus is a complex process involving various mechanisms and regulation factors. The interaction of genes and virulence factors, such as gelatinase, cytolysin, secreted antigen A, pili, microbial surface components that recognize adhesive matrix molecules (MSCRAMMs), and DNA release, facilitates the formation of biofilms by Enterococcus species, such as E. faecalis and E. faecium [11]. Quorum sensing, mediated by peptide pheromones, plays a crucial role in coordinating biofilm development by targeting gene expression and regulation [190]. Targeting the expression and regulation of genes, quorum sensing—mediated by peptide pheromones like Cb, Ccf, and Cpd—is essential for coordinating the formation of biofilms. The regulation of eDNA release has emerged as a fundamental component in biofilm formation, with autolysin N-acetylglucosaminidase and proteases influencing biofilm development and virulence [191]. Additionally, the release of eDNA can be triggered by specific stressors, bacterial apoptosis, or fratricide-induced death, and can also be derived from host tissue cells or neutrophil extracellular traps (NETs) [12].