Targeting Peptide-Based Quorum Sensing Systems for the Treatment of Gram-Positive Bacterial Infections

Abstract

Bacteria utilize a cell density-dependent communication system called quorum sensing (QS) to coordinate group behaviors. In Gram-positive bacteria, QS involves the production of and response to auto-inducing peptide (AIP) signaling molecules to modulate group phenotypes, including pathogenicity. As such, this bacterial communication system has been identified as a potential therapeutic target against bacterial infections. More specifically, developing synthetic modulators derived from the native peptide signal paves a new way to selectively block the pathogenic behaviors associated with this signaling system. Moreover, rational design and development of potent synthetic peptide modulators allows in depth understanding of the molecular mechanisms that drive QS circuits in diverse bacterial species. Overall, studies aimed at understanding the role of QS in microbial social behavior could result in the accumulation of significant knowledge of microbial interactions, and consequently lead to the development of alternative therapeutic agents to treat bacterial infectivity. In this review, we discuss recent advances in the development of peptide-based modulators to target QS systems in Gram-positive pathogens, with a focus on evaluating the therapeutic potential associated with these bacterial signaling pathways.

Graphical Abstract

Introduction

In nature bacteria regulate a vast range of social behaviors in a synchronized manner with members of their community using a universal cell-cell signaling mechanism commonly referred to as quorum sensing (QS).^1,2 Bacteria utilize this communication system to monitor their population density in each environment and to trigger population-wide changes in gene expression when the population reaches a critical cell density.³ This population size-dependent pathway has appeared as a popular model for understanding bacterial sociality as this signaling system allows the microbial community to behave in a coordinated manner to direct processes contributing to virulence factor production, biofilm formation, competence development, and other pathogenic or symbiotic interactions.^1–5

Initiation of bacterial communication depends on the production, secretion and detection of small chemical signals called autoinducers. There are several types of autoinducers: Gram-negative bacteria generally utilize small molecules known as N-acyl-homoserine lactones (AHLs), whereas Gram-positive bacteria generally utilize peptide-based molecules named auto-inducing peptides (AIPs). These two classes of autoinducers are thought to be highly specific and are generally used for intraspecies cell-cell communication. However, it is becoming increasingly evident that these two signal classes are not only limited to intraspecies chemical language, rather, bacteria also use these signaling molecules as interspecies communication devices.^3,4,6 Moreover, auto-inducer 2 (AI-2) has been found in over 70 species of Gram-negative and Gram-positive bacteria, and has been recognized as a universal interspecies signaling molecule. QS circuits rely on the concentration of the secreted signal in the extracellular environment, and once it reaches a threshold level, the signal binds and activates a receptor protein, initiating a coordinated change in gene expression in the population.^4,5,7

There is a great deal of research showing the link between QS and bacterial pathogenicity.^5,7,8 Increased rate of antibiotic resistance development among bacteria due to the overuse and misuse of antibiotics has also been well-documented. These two factors: involvement of QS circuits in bacterial infectivity and increased rate of resistance development, offer a novel approach to traditional antimicrobials that would allow the treatment of bacterial infections through targeting of QS systems while avoiding selective pressure for resistance development. Both traditional methods and bioinformatics techniques have revealed the presence of homologues, analogues or similar QS systems that use small peptide signals across Gram-positive genera.⁹ The potential for commonality in aspects of these communication systems paves the way to identifying therapeutics that could target multiple pathogens. In addition, understanding how bacteria interact with one another using QS within polymicrobial communities can improve our current understanding of microbial community interactions and guide the design of new antibacterial therapeutics to treat bacterial co-infections. In this review, we will focus on the advances that have been made in understanding the structure and function of several peptide-based Gram-positive bacterial QS systems with the intent to highlight the therapeutic potential of these QS systems to treat microbial infections.

Peptide-Based QS mechanisms in Gram-positive bacteria

Based on features of the peptide signaling molecules and their receptors, Gram-positive bacterial QS signaling pathways have been classified into two groups. The peptide signaling molecules, which are central to QS activation, are ribosomally synthesized as precursor peptides and oftentimes undergo post-translational modifications. Following transcription and translation, the precursor peptide is generally processed to the mature peptide signal and secreted into the extracellular environment via a dedicated ATP-binding cassette (ABC) transporter. These mature peptide signals can be linear or macrocyclic.¹⁰ As the cell density increases, the concentration of processed peptide signal increases, and once it reaches a certain threshold concentration, the peptide binds to its cognate receptor, either in a self-signaling pathway or a two-component pathway. The self-signaling pathway is known as the RRNPP protein family, which stands for Rap (Bacillus subtilis), Rgg (Streptococcus), NprR (Bacillus cereus), PlcR (B. cereus), PrgX (Enterococcus faecalis),^11,12 as these are the response regulators that were identified from various founding species of this circuit class. In this system class, either during or following export, the peptide signal is processed to its mature form. Then, the mature peptide signal is being imported back into the cell by a transmembrane oligopeptide permease (Opp) transporter where it directly binds to its cytoplasmic response regulator, resulting in upregulation of the corresponding QS genes (Figure 1b).

Figure 1: Generalized Common QS circuits in Gram-positive bacteria.

a) TCSTS-like QS circuits involve the transduction of mature peptide signal across the membrane without physical transport of the signal; b) RRNPP-like QS circuits involve the physical import of the mature peptide signal before it binds to and activates the cytosolic transcription factor/receptor.

The other most widely studied pathway, which was first described in Streptococcus pneumoniae (Com system) and later in many other Gram-positive bacteria, including Staphylococcus aureus (Agr system), and E. faecalis (Fsr system), is commonly referred to as two-component signal transduction system (TCSTS).⁵ This pathway is comprised of a membrane-bound histidine kinase receptor and a separate response regulator that is associated with the induction of QS-dependent phenotypes. In this pathway, the peptide signaling molecule is primarily encoded as precursor peptide and then modified post-translationally. The mature peptide is secreted into the outside of the cell and upon reaching a threshold concentration, it is recognized by the specific histidine kinase receptor. The activated receptor then phosphorylates the downstream response regulator. The activated regulator upregulates the transcription of specific bacterial genes associated with group behaviors and phenotypes as well as transcription of the respective genes related to the AIP secretion pathway itself (Figure 1a). TCSTS-based QS systems have been found to regulate a myriad of group behaviors in many Gram-positive species.

PlcR-PapR QS system in B. cereus

B. cereus is a food-poisoning pathogen and is genetically close to two other human pathogens: B. anthracis and B. thuringiensis.¹³ Some strains of B. cereus can colonize the intestines and cause more severe diseases, such as endophthalmitis or meningitis, through the production and secretion of several virulence factors, including hemolysins, phospholipases, proteases, and enterotoxins.^13,14 In B. cereus, the expression of virulence factors is controlled by a QS system that consists of an intracellular PapR-derived AIP (Table 1) and the transcription factor PlcR (Phospholipase C Regulator).¹⁵ The initiation of the QS signaling pathway begins with the production of the PapR precursor peptide, which needs to be transported outside the cell by a membrane carrier protein and processed by the secreted neutral protease B (NprB) into the mature AIP signal. The processed AIP is reimported back into the cell through the Opp, where it binds to PlcR, stimulating the expression of several genes involved in the secretion of virulence factors (Figure 2).^13–15 Previous studies showed that inactivation of the plcR gene reduces the secretion of virulence factors but cannot fully eliminate virulence factor secretion. This is due to the involvement of several additional QS systems in the regulation of virulence factors.^8,14

Table 1. Signaling peptides of Gram-positive bacteria.

In the peptide sequences, cysteine (red) is marked to highlight the residue required for thiolactone ring formation. Macrocyclic part marked in parenthesis.

Species	Peptide regulator family	QS System	Peptide Name	Peptide Sequence
Bacillus cereus	RRNPP family	papR-plcR	PapR I(PapR7)	ADLPFEF
			PapR II	SDMPFEF
			PapR III	NEVPFEF
			PapR IV	SDLPFEH
			PapRa	CSIPYEY
		nprR-nprX	NprRB	SKPDIVG
Bacillus anthracis Bacillus thuringiensis	RRNPP family	nprR-nprX	NprRB	SKPDI SDIYG
Staphylococcus aureus	TCSTS (Agr)	agrBDCA	Sa AIP 1	YST(CDFIM)
			Sa AIP 2	GVNA(CSSLF)
			Sa AIP 3	IN(CDFLL)
			Sa AIP 4	YST(CYFIM)
Streptococcus pneumoniae	TCSTS(Com)	comCDE	CSP1	EMRLSKFFRDFILQRKK
			CSP2	EMRISRIILDFLFLRKK
	Rgg family	rgg-shp	SHP	DIIIIVGG
Streptococcus mitis	TCSTS(Com)	comCDE	S. mitis-CSP-2	EIRQTHNIFFNFFKRR
	Rgg family	rgg-shp	SHP	DIIIVGG
Streptococcus pyogenes	Rgg family	rgg2-shp2	SHP2	DILIIVGG
		rgg3-shp3	SHP3	DIIIIVGG
Streptococcus agalactiae	Rgg family	rovS- shp2	SHP1520	DILIIVGG
Streptococcus dysgalactiae	Rgg family	rgg-shp	SHP	DILIIVGG
subsp. equisimilis			SHP	DILIIVGG
Streptococcus porcinus	Rgg family	rgg-shp	SHP	DIIIIAGG
Streptococcus thermophilus	Rgg family	rgg-shp	SHP	DIIIIVGG

Figure 2: B. cereus PlcR-PapR QS system.

The precursor peptide PapR is secreted and is then processed to the mature AIP by the extracellular protease, NprB. Opp transports the mature AIP back into the cell and then the intracellular AIP binds to the transcription factor, PlcR. The resultant PlcR-AIP complex upregulates virulence factor production and activates the expression of papR.

In sporulating Bacillus, a similar signaling pathway, the NprR-NprX cell–cell communication system, has been shown to regulate sporulation and the expression of virulence genes, including enterotoxins, phospholipases, proteases and chitinases.¹¹ Like the PlcR-PapR QS system, this system is also comprised of the NprR transcriptional regulator, whose activity depends on the signaling peptide, NprX. The imported mature signal activates the NprR regulator, resulting in the expression of an extracellular protease gene, nprA, during the sporulation process.^11,16,17 This QS system was found to be strain specific, with possible cross talk between some phylogenetic groups.¹⁷

Rgg/SHP QS systems in Streptococci

The Rgg (regulator gene of glucosyltransferase) and SHP (short hydrophobic peptide) cell-to-cell communication system is widespread in the streptococcus genus and has been gaining increased attention due to its involvement in controlling a variety of functions in several species of streptococci.^5,18 The Rgg transcriptional regulator was first described in S. gordonii and later found in nearly all streptococci species.^19–21 Rgg regulators are regulated by re-internalized SHPs acting as pheromones. This QS system regulates different streptococcal phenotypes, such as bacteriocin production in S. mutans,²¹ as well as the production of the exotoxin SpeB, virulence regulation and biofilm development in S. pyogenes.^22–24 Moreover, this pheromone system is widely utilized by members of the mitis group of streptococci, and an Rgg/SHP system (Figure 3 ) regulating surface polysaccharide expression in S. pneumoniae has been reported.²⁵ In this QS system, first the SHP precursor peptide is produced, processed and exported with the help of the PptAB transporter system and the membrane protease Eep. The mature peptide, SHP, is then transported from the extracellular environment to the cytosol through an Opp. Once the mature peptide is bound by the Rgg regulator, the Rgg:SHP complex activates the pheromone-feedback loop and an operon consisting of 12 genes. Activation of this regulon upregulates polysaccharide synthesis and downregulates biofilm formation (Figure 3).

Figure 3: S. pneumoniae Rgg/SHP QS system.

The SHP precursor peptide is processed and exported by PptAB and Eep. When the SHP concentration reaches a certain threshold, the mature peptide is imported by an Opp and binds to the Rgg regulator. The resultant Rgg:SHP complex upregulates polysaccharide synthesis and downregulates biofilm formation.

Interspecies cross-communication has been found in some of these systems, making such systems an important tool to study bacterial communities, which, in turn assists in the development of new anti-virulence strategies to treat bacterial infections.^26–28

QS in S. aureus (Agr system)

S. aureus is a human pathogen responsible for bacteremia, sepsis, endocarditis, toxic shock syndrome and a wide spectrum of infections of skin and soft tissues. The outbreaks of multidrug-resistant S. aureus strains, known as methicillin-resistant S. aureus (MRSA) makes treatment of S. aureus extremely difficult.^9,29,30 This pathogen secretes a wide range of virulence determinants to attack the host, including various tissue degrading enzymes, pore-forming toxins, and immune evasion factors. The accessory gene regulator (agr) operon-encoded QS system regulates the expression of different virulence factors and is directly related to the pathogenicity of this Gram-positive bacterium.^9,30,31

The Agr system is known to contain two adjacent transcripts named RNAII and RNAIII. The RNAII transcript comprises of four genes in the transcriptional order of agrBDCA. The signaling cascade begins with the production of a 46-amino acid precursor peptide of the AIP signal, AgrD. AgrB is an integral membrane protein that processes the AgrD precursor peptide into its mature cyclic form and exports the cyclic peptide as the functional signal to the extracellular environment. AgrC and AgrA form a classical bacterial TCSTS where AgrC acts as the histidine kinase sensor and AgrA is a response regulator. Upon binding of AIP to AgrC, AgrA gets activated and binds to two promoter regions (P2 for RNAII and P3 for RNAIII) to autoactivate the Agr system and upregulate RNAIII transcription (Figure 4).

Figure 4: S. aureus Agr QS system.

The precursor peptide AgrD is processed and secreted across the cell membrane as the mature AIP signal by the AgrB transporter system. Mature AIP signal then binds to the extracellular domain of the AgrC receptor. Upon binding, the histidine kinase domain of AgrC phosphorylates the response regulator, AgrA. Phosphorylated AgrA binds the P2 and P3 promoters to autoactivate the agr operon (called RNAII) and the RNAIII regulatory RNA, respectively. RNAIII promotes virulence factor production and represses the expression of rot, leading to derepression of virulence factor production.

Upregulation of toxins and virulence determinants depends on either the activation of RNAIII or AgrA.^30,32 RNAIII is the agr-induced major regulatory RNA (rRNA) molecule and is responsible for the expression of most of the virulence genes through directly affecting mRNA stability and stimulating or inhibiting mRNA translation, or indirectly through RNAIII-mediated inhibition of the Rot transcriptional regulator translation.³² The toxin repressor protein, Rot, is a member of the Sar and MarR families of transcriptional regulators that can promote or repress the expression of hundreds of toxins, proteases, adhesion factors and metabolic pathways.^29–31 This combination of direct and indirect RNAIII-mediated gene regulation transforms S. aureus cells into a hostile form capable of invasive infection.

AgrA acts as a key transcription factor and positively regulates the expression of the agr operon mainly via activating the agr P2 and P3 promoters.^9,33 AgrA can additionally activate the expression of the α- and β-phenol soluble modulins (PSMs).²⁹ PSMs are short amphipathic cytolytic peptides that have been shown to be critical for staphylococcal pathogenesis by enhancing the survival and dissemination of S. aureus in invasive infection.^9,30 PSMβs are important for staphylococcal biofilm maturation, and within the mature biofilm structures, agr-mediated PSM promoter activation can be observed.³⁴ AgrA may be the most important element in the initiation of transcription at P2 and a previous study has shown that the deletion of agrA completely abolishes both RNAIII and agr mRNA transcription.³³

The S. aureus Agr QS system has been classified into four different specificity groups. Each Agr system (referred to as Agr-I, Agr-II, Agr-III, and Agr-IV) recognizes a different AIP signal (referred to as AIP-I, AIP-II, AIP-III, and AIP-IV, Table 1). These AIP signals vary in overall peptide length (7–9 amino acids) and sequences.⁹ All the S. aureus AIP signals share several structural features: (1) The last five residues in each AIP are constrained as a thiolactone macrocycle between the side chain of a conserved cysteine residues and the C-terminal carboxylate. (2) All AIPs contain an N-terminal exocyclic “tail” that varies in length (2–4 residues). (3) All AIPs contain 2–3 hydrophobic residues at the C-terminal region (within the macrocycle) that are critical for receptor binding. Due to the differences in peptide sequences, these signaling molecules function as cross-type antagonists for AgrC activation in a process named “Agr interference”.^9,30 This phenomenon is typical in this species and leads to competition between different specificity groups of S. aureus. The involvement of Agr or analogues QS circuitries in the regulation of virulence in other Gram-positive human pathogens makes the Agr QS system an attractive anti-virulence target for the treatment of multiple pathogen infections.

QS in S. pneumoniae (Com system)

S. pneumoniae is a notorious opportunistic human pathogen that causes pneumonia, bacteremia, otitis media, and meningitis in immune compromised human hosts.^35,36 S. pneumoniae was the first species of bacteria in which natural transformation was reported in 1928 by Frederick Griffith.³⁷ Natural transformation ensures genomic plasticity of pneumococcus, which is associated with better adaptation to different environmental stressors and spread crucial features, such as antibiotic resistance. This process takes place only in pneumococcal competent cells, which can acquire DNA from the surrounding environment.^38–40 The competence regulon ComCDE QS circuitry plays a decisive role in regulating competence, biofilm formation and virulence factor production in S. pneumoniae.^39,41,42

In the S. pneumoniae ComCDE QS system, the signaling molecule is a 17-residue oligopeptide CSP (competence stimulating peptide, Table 1), which is formed from the precursor peptide, called ComC. The CSP is exported out of the bacterial cell by an ATP-binding cassette (ABC) transporter, termed ComAB. Once accumulation of extracellular mature CSP reaches a threshold concentration, it binds to its cognate ComD histidine kinase receptor, which triggers the phosphorylation and activation of a response regulator protein, ComE. Activated ComE then acts as a transcription factor for the comAB and comCDE genes. ComE also activates the transcription of the comX gene, an alternative sigma factor, also known as sigX, which is involved in the expression of a large regulon of effector genes involved in the acquisition of competence within the cells (Figure 5).^43–45 Previous studies have shown that deletion of the comA, comB, comC, comD, comE, as well as comX genes reduces competence induction and the severity of pneumococcal infections.^39,42,46

Figure 5: S. pneumoniae ComCDE QS system.

The precursor peptide ComC is processed to the mature peptide signal, CSP, and transported outside the cell by the ComAB trasporter system. When the CSP concentration reaches a certain threshold, it binds and activates the transmembrane histidine kinase receptor, ComD. Upon activation, ComD undergoes autophosphorylation, leading to phosphorylation of the response regulator, ComE. Phosphorylated ComE then acts a transcription factor to upregulate the expression of the QS genes, comABCDE, as well as genes involved in group phenotypes, such as comX.

Different strains of pneumococcus can produce different CSP signals and the two major forms of CSP, namely CSP1 and CSP2 (Table 1), are used by the majority of pneumococcus strains. Although these two CSP signals share 50% homology in their sequence, they are highly specific toward their respective cognate receptors, ComD1 and ComD2, respectively, and only at higher signal concentration can activate their non-cognate receptor.^45,47 Streptococci belonging to the mitis and anginosus groups use this ComCDE system to regulate different phenotypes associated with infectivity, making this QS circuit a prime target for the attenuation of pathogenicity in multiple pathogens.

Peptide-based QS systems as therapeutic targets

QS interference is an attractive strategy to limit both the spread of infectious organisms and selective growth pressure that results in the proliferation of resistant organisms. Targeting virulence factors, rather than bacterial growth, has been shown to be effective in controlling bacterial infections without engendering resistance development.^9,48,49 The close connection between QS and pathogenicity has led to the development of novel antimicrobial therapeutic approaches, such as quorum quenching and other QS-blocking methodologies, that focus on controlling virulence factor production.^49,50 Targeting the ability of the microbe to recognize and respond to the AIP signaling molecules has proven to be an effective approach for the treatment of several human pathogens.^9,48 Current technologies, such as solid-phase peptide synthesis (SPPS) and high-performance liquid chromatography (HPLC), facilitate the design and synthesis of numerous peptides or modified peptide analogs that can modulate QS-mediated phenotypic responses in vitro.^51,52 By having superior properties like excellent selectivity, remarkable potency, improved stability and low toxicity, peptides, the biological mediators, have gained significant attention to consider them as promising therapeutic candidates.⁵¹ Several reports demonstrated the importance of developing synthetic peptide analogs capable of inhibiting QS circuits in many of the human pathogens, including S. aureus, S. pneumoniae, and E. faecalis.^{8,9,14,48–50}

Although the development of potent synthetic peptide analogs capable of inhibiting QS communication systems has been reported, many of these compounds suffer from poor affinity and low pharmacokinetic profiles. To overcome these barriers, the stability of lead peptide sequences can be improved through the utilization of several chemical alterations to the peptide, such as, incorporation of non-proteinogenic amino acids, N-methylated residues, or D-amino acids.⁵³ These modifications could lead to improved protease resistance and enhanced biological activity of the parent compound.^51,52 Peptide cyclization has also been shown to be an effective approach to increase peptide stability, binding affinity, and specificity to the receptor.^54–57

In vivo studies have been reported determining the efficacy of the peptide modulators in preventing or reducing diseases. For example, our group, together with the Lau lab, reported the development of potent synthetic pneumococcal QS modulators that can attenuate pneumococcal infections in vivo.^55,56,58 Blocking the Agr QS system to control virulence in S. aureus could be a fruitful target as the pathology of an Agr group IV strain can be attenuated through vaccination with hapten-linked AIP-4.⁵⁹ Recently, Blackwell and co-workers reported a stable and potent synthetic peptide-based inhibitor of the S. aureus agr system attenuating S. aureus (MRSA) infection in an in vivo mouse model of skin infection.⁶⁰ This work present a new, useful, and modular anti-virulence approach to controlling bacterial skin infections in vivo. The following section will detail the use of peptide-based probes in modulating QS circuitries and their related phenotypes in important Gram-positive human pathogenic bacteria.

Agr QS system as therapeutic target

As the expression of many virulence genes is regulated by the Agr QS system, targeting Agr could result in the reduction in S. aureus pathogenicity.^61,62 Various methods have been reported to block Agr function and attracted significant attention as potential anti-infective therapies to prevent S. aureus infections.^9,63–72 Among them, interference of AIP:receptor interactions represents a direct strategy to block QS, and may have the potential to prevent virulence in S. aureus.^66,73 Development of non-native synthetic ligands, such as, small peptides and macromolecules capable of inhibition of the AgrC receptor have been promoted by many researchers.^{59,71,73–77} Initial studies have shown that each of the four native staphylococcal AIPs (Table 1) can antagonize the other three, noncognate AgrC receptors to prevent activation of the Agr regulon.^30,71,74,78 Previously, an in vivo study conducted by Wright et al. showed that S. aureus pathologies can be reduced by blocking the agr function.⁷⁹ The authors showed that by injecting inhibitory concentrations of the AIP-II peptide in a mouse dermonecrosis model, ulcers and abscesses caused by an Agr-I strain could be impeded.

There are numerous examples of developing AIP:AgrC modulators based on the AIP-I and AIP-II signals.^{69,71,74,78,80–83} Detailed Structure-Activity Relationship (SAR) studies of AIPs -I and -II were conducted by Muir, Novick, Williams, and co-workers and several potent inhibitors capable of inhibiting both cognate and noncognate AgrC receptors were developed.^30,84 The SAR studies revealed that the AIP macrocycle is important for initial receptor recognition/binding, and the involvement of the AIP exocyclic tail in interactions is responsible for receptor activation. Hydrophobic residues at the C-terminal region (residues 6−8 for AIP-I and residues 8 and 9 for AIP-II) were found to be important for both AgrC cognate and noncognate receptor binding, and based on this analysis, the most potent inhibitor, tAIP-I D2A, was developed, which is a truncated version of AIP-I lacking an exocyclic tail and having an aspartic acid to alanine mutation in the macrocyclic core.

Studies by Blackwell and co-workers closely examined the SAR of AIP-III (Figure 6a) and through the design, synthesis, and biological testing of a series of first- and second- generation AIP-III mimetics, the authors identified a set of non-native AIP-III analogs that can inhibit all four staphylococcal AgrC receptors with picomolar IC₅₀ values.⁸⁵ The systematic SAR analysis of the AIP-III analogs revealed several important structural features that are responsible for the ability of AIP-III to modulate the four AgrC receptors. The AIP-III SAR analysis was consistent with the previous AIPs -I and -II SAR analyses, emphasizing the role of the endocyclic hydrophobic residues (residues 5–7 in AIP-III) in receptor binding and the exocyclic tail in receptor activation. Moreover, the most potent global AgrC inhibitor, AIP-III D4A (Figure 6b), was developed through the inclusion of a previously reported identical mutation (replacement of Asp4 with alanine), emphasizing the previous findings regarding the importance of the Asp4 side chain in AgrC receptor activation. This global inhibitor was able to block QS-regulated hemolysis in all four S. aureus specificity groups (Agr-I−IV) at sub-nanomolar concentrations. The production of toxic shock syndrome toxin-1 (TSST-1) is a hallmark QS phenotype in group-III S. aureus, and this analog was also able to attenuate TSST-1 production in a S. aureus group-III strain by over 80% at nanomolar concentrations. However, the presence of a hydrolytically unstable thioester linkage in AIP-III D4A makes this most potent inhibitor less stable and soluble in aqueous media. To address these issues, a new AIP-III analog containing a non-native amide bridge, AIP-III D4A Amide (Figure 6c), was developed, and exhibited improved stability and solubility in aqueous media.⁷⁵

Figure 6. Lead Inhibitors of the S. aureus Agr QS circuitry.

a) Structure of the native AIP-III; b) Structure of the lead inhibitor, AIP-III D4A; and c) Structure of the inhibitor AIP-III D4A Amide, which was found to inhibit all four S. aureus specificity groups (Agr-I-IV) while exhibiting improved pharmacological properties. Red color indicates the modifications at specific amino acids in AIP-III. Here, D4A represents that Asp at position 4 was replaced by Ala.

As studies suggested that AIP-III might provide a superior scaffold for the development of potent AgrC inhibitors, Blackwell and co-workers further conducted in depth structural analysis of all four native S. aureus AIPs, along with several AIP-III analogs, using 2D NMR to obtain valuable insight as to the structural features that drive AIP-mediated AgrC activation and inhibition.^73,76,86 The NMR data revealed two important structural motifs within AIP-type ligands that are required for AgrC receptors modulation: (i) the presence of a triangular, hydrophobic knob motif on the macrocycle that is needed for receptor binding, and (ii) a fourth hydrophobic contact or anchor on the N-terminal tail that is essential for receptor activation. These structural findings can be utilized for the design of new staphylococcal modulators with better potency and selectivity.

Com QS system as therapeutic target

The streptococcal Com QS system controls bacterial competence and biofilm formation in numerous streptococci. While competence is not directly related to bacterial pathogenesis in most of the streptococcal species, S. pneumoniae ability to utilize exogenous genetic material has been recognized as an important factor for genetic variability and subsequent evolution. For instance, previous work has shown that non-opsonizing antibodies against pneumococcal polysaccharide capsular antigens alter the expression of QS genes and thus improve bacterial viability and pathogenesis.⁸⁷ Another significant QS-regulated phenotype, biofilm formation, enhances pneumococcal survival by facilitating evasion of host immune responses during pneumococcal colonization of the nasopharynx.^41,88 A novel biofilm regulating peptide, BriC, which is upregulated by the ComE transcriptional regulator, has been shown to act as a molecular linker of pneumococcal competence, biofilm formation, and colonization.⁸⁸ Lastly, S. pneumoniae produces a wide range of toxins and virulence factors, including Ply, autolysin, choline-binding proteins, lipoproteins, LPXTG cell wall bound proteins, capsule polysaccharide, cell wall polysaccharide, and Immunoglobulin A1 (IgA1) protease, and the production of many of these virulence factors is influenced by the competence regulon (ComCDE system).^40,89 Thus, to control pneumococcus infections, targeting this nonessential communication system could be considered as a potential anti-virulence approach.

Extensive work has been done by our lab focusing on the development of CSP-based QS-modulators in S. pneumoniae. To do so, systematic alanine and epimer scans were conducted on both pneumococcal CSPs (CSP1 and CSP2, Table 1) to evaluate the structural and functional properties of each residue, eventually leading to the development of several potent QS-modulators of both S. pneumoniae serotype groups.^47,90 It has been found that N-terminal amino acids play critical roles in both CSPs activity, whereas the last three C-terminal amino acids are unnecessary for activity. Evaluation of the CSP1 central region revealed that the hydrophobic residues (L4, F7, F8, F11, I12) are critical for receptor binding (Figure 7). Follow-up studies have focused on optimizing the degree of occupancy of the CSP1 binding pockets within the ComD1 receptor by utilizing conservative substitutions at these key hydrophobic residues of CSP1.^91,92,93 Through the incorporation of bulkier, hydrophobic nonproteogenic amino acids, Milly et al. were able to develop pneumococcal QS modulators with high potency and superior metabolic stability, while remaining nontoxic against mammalian cells.⁹³

Figure 7. Key structural features required for CSP:ComD binding.

a) The structures of CSP1 and CSP2 highlighting the hydrophobic residues that constitute the hydrophobic patches (in red) required for effective ComD1 and ComD2 binding, respectively. The Glu1 residue that is critical for receptor activation is marked in blue; b) 3D structures of CSP1 (PDB 6COW) and CSP2-d10 (PDB 6COT) exhibiting the two distinct hydrophobic patches. The two structures were obtained through structural NMR studies.⁹⁴

The first position on the pherotype 1 AIP (CSP1, Table 1; with its unique primary structure, each CSP represents a separate pheromone type – pherotype) was found to be necessary for competence development, as Glu1 to Ala mutation (to afford CSP1-E1A) was sufficient to convert the signaling peptide into a competitive inhibitor capable of inhibiting competence induction and regulation of down-stream genes in S. pneumoniae.⁴² The N-terminal first residue (Glu1) was further investigated through the implication of amino acids (AAs) with different side-chain length, polarity, and chirality.⁹⁵ Structural analysis of both native CSPs, along with several synthetic abiotic analogs, revealed that an α-helical character is important for receptor binding.⁹⁴ Results from these studies directed the design of cyclic CSP1 AIPs with enhanced binding characteristics. To this end, Yang et al. first applied ring position scan of the CSP1 scaffold, followed by ring size conformational scan to afford two potent pan-group activators with low nanomolar potency, (CSP1-cyc(Dab6E10) and CSP1-cyc(Dap6E10)).⁵⁷ Then, by incorporating the E1A modification, the lead pan-group inhibitor, CSP1-E1A-cyc(Dap6E10) (Figure 8), was developed, which can inhibit the ComD1 receptor with an IC₅₀ value of 75.8 nM and ComD2 receptor with an IC₅₀ value of 182 nM. Structural analysis revealed that this peptide also contains the previously identified important hydrophobic “patches” that are crucial for both ComD1 and ComD2 receptor binding.

Figure 8. Pan-group inhibitor of the S. pneumoniae competence regulon QS circuitry.

The most potent inhibitor, CSP1-E1A-cyc(Dap6E10), was found to effectively inhibit both group 1 and group 2 pneumococcal competence regulon. The modifications at specific amino acids in CSP1 are highlighted as red color. Here, E1A represents that Glu at position 1 was replaced by Ala, Dap represents 2,3-diaminopropionic acid, and cyclization was introduced by connecting the 6^th and 10^th amino acid side chains.

The use of dominant-negative competence-stimulating peptide (dnCSP) analogs is considered to be an attractive therapeutic strategy, as apart from their high receptor interference capability, these agents also exhibited reduced antibiotic resistance and capsule biosynthesis genes acquisition, decreased allolytic factors LytA and CbpD expression and PLY release, and mouse mortality attenuation in in vivo studies.^42,57,58 Furthermore, the pharmacological properties and safety profiles of CSP1-E1A-cyc(Dap6E10) were recently evaluated, demonstrating the superior safety and pharmacokinetics profiles of this lead analog.⁵⁶ Biostability of this dnCSP, along with the native CSP1 and CSP2 signals, was examined using an IVIS Spectrum in vivo imaging system. To this end, the Cy7.5 fluorophore was attached to the N-terminus of the peptide to construct Cyanine7.5 (Cy7.5)-labeled dnCSP and both native CSP analogs. Comparison between the biostability of the dnCSP and native signals revealed the superior biostability property of the cyclized dnCSP, demonstrating the importance of macrocyclization of the peptide. In vitro cytotoxicity and in vivo toxicity assays further supported the strong safety and pharmacological profiles of this promising peptide-based drug candidate.

As previous studies evidenced that nonproteogenic amino acid substitutions and cyclization resulted in improved modulators of the pneumococcal competence regulon,⁵⁷ Lella et al. intended to design and develop pharmacologically enhanced cyclic peptidomimetic scaffolds by utilizing the side chain-to-side chain urea-bridge cyclization chemistry.⁵⁵ This strategy led to the development of the first pneumococcus dual-action CSP modulator that can block group 1 while activating group 2 pneumococcus competence regulon. This is an interesting finding showing that analogs bearing the E1A modification, a reported key modification in the conversion of CSPs into competitive ComD inhibitors, can activate noncognate pneumococcal ComD receptors. The lead dual-action urea-bridged cyclic peptide, CSP1-E1A-cyc(Dab6Dab10) (Figure 9), can attenuate group 1 pneumococcal infections without affecting the bacterial burden, as was shown by in vivo studies using a mouse model of infection. These findings further highlighted the therapeutic potential of utilizing peptide-based competitive inhibitors to block the competence regulon without exposing the bacteria to selective pressure for resistance development.

Figure 9. The dual-action urea-bridged cyclic CSP.

The dual-action analog, CSP1-E1A-cyc(Dab6Dab10), was found to inhibit group 1 while activating group 2 pneumococcus competence regulon. The modifications at specific amino acids in CSP1 are highlighted as red color. Here, E1A represents that Glu at position 1 was replaced by Ala, Dab represents 2,4-diaminobutyric acid, and the urea bridge cyclization was introduced by connecting the 6^th and 10^th amino acid side chains.

RRNPP QS system as therapeutic target

The RRNPP protein family has been found in several members of the Bacillus genus and is a critical factor in regulating the expression of several genes responsible for biofilm formation, sporulation, and pathogenic responses.¹⁴ The transcription factor PlcR and its associated AIP regulate the secretion of several virulence factors, including hemolysin, in B. cereus. Similar to the four Agr groups in S. aureus, five PapR signal peptides (Table 1) have been reported so far, and cross-reactivity has been shown in four of these pherotypes.⁹⁶ Binding of the signaling heptapeptide PapR₇ (ADLPFEF) triggers PlcR activity.^97,98 Alanine and epimer scans of PapR₇ provided valuable SAR insights, including the importance of stereochemistry of the side chains in positions 3–7 for PlcR activation. This analysis eventually led to the development of one activator and five inhibitors of the PlcR-PapR QS circuitry in B. cereus.

B. thuringiensis produces an insecticidal toxin and deletion of PapR resulted in a reduction of toxin production and the associated killing of susceptible insects.⁹⁸ The NprR transcriptional regulator activity was analyzed in B. thuringiensis and findings revealed that it is important for biofilm formation and sporulation in insect cadavers.⁹⁹ Analysis of NprR and its associated AIPs, termed NprRB (six known pherotypes), in B. thuringiensis revealed that the native peptide SKPDI (Table 1), along with another pherotype, SKPDT, and its K2A substituted synthetic analog, SAPDT, can induce the expression of the insecticidal toxin, cry1Aa in this species.¹⁰⁰ Among these three peptides, only SKPDT was able to induce a response above background levels at 100 nM. Further analysis with the heptapeptide, SKPDIVG, and octapeptide, SSKPDIVG, revealed that both could upregulate bacterial sporulation (efficiency was increased by 2.1- and 1.6-fold, respectively). Three heptapeptide analogs were developed based on SKPDIVG: YSSKPDI, SSKPDIV, and SKPDIVG, and all exhibited mild inhibitory effects. The octapeptide, SSKPDIVG (Figure 10) was found to be the best inhibitor (35% drop in signal relative to the untreated control) whereas the nonapeptide YSSKPDIVG had no effect.¹⁰⁰ These virulence-regulating QS systems in Bacillus species could be targeted as an anti-virulence strategy.

Figure 10. Inhibitor of the B. thuringiensis NprR QS circuitry.

The octapeptide SSKPDIVG was found to be the most potent inhibitor of B. thuringiensis cry1Aa expression.

Highly homologous Rgg/SHP pairs, which act as an activator and a repressor, respectively, have been reported in several species of streptococci and it has been established that cross-communication could be taking place between these different streptococcal Rgg/SHP systems.^5,18,26–28 A bidirectional signaling associated with SHP pheromones was documented between human pathogens, including Group A streptococcus (GAS, S. pyogenes), group B streptococcus (GBS, S. agalactiae), and S. dysgalactiae subsp. equisimilis.²⁷ GAS can cause a verity of illnesses and life-threatening infections in humans.¹⁰¹ In GAS, two competing transcriptional regulator pairs, Rgg2/SHP2 and Rgg3/SHP3, are required to regulate the expression of a wide range of genes, including genes involved in biofilm formation.²³ GAS is often found to colonize with GBS at the same sites within the human host, and in GBS, virulence genes are regulated by an ortholog of the Rgg2 regulator, termed RovS, which is associated with a peptide pheromone signal nearly identical to SHP2.²⁷ Spent culture supernatant and co-culture experiments demonstrated that Rgg2/3-regulated gene expression, specifically genes involved in biofilm formation in GAS could be modulated by the production and secretion of GBS-produced SHPs, and on the other hand, GAS-produced SHP2/3 signals (Table 1) can stimulate RovS-mediated gene regulation in GBS. The results also revealed that both GAS and GBS Rgg/SHP QS circuits can be induced by using an orthologous Rgg2/SHP2 pair of S. dysgalactiae subsp. equisimilis and S. porcinus. Rgg/SHP systems of S. mutans, and S. thermophilus were also shown to be triggered by using slightly different and non-cognate synthetic SHPs from similar Rgg classes of streptococci.²⁸ These Rgg/SHP mediated cross-talks among streptococci provide a means to influence the regulation of a variety of pathogenic behaviors of Rgg/SHP-carrying pathogens by deceiving, diverting, or dissuading the Rgg/SHP QS systems of competing bacteria.

Crosstalk between pathogenic and non-pathogenic streptococci has been evidenced by a recent study conducted by Junges et.al.²⁶ In silico analyses revealed the presence of a Rgg/SHP cell-to-cell communication system in a commensal species from the mitis group, S. mitis. This S. mitis-Rgg regulator exhibited greater similarity to a S. pyogenes-Rgg repressor, however, the S. mitis-Rgg regulator functions as an activator, as confirmed by genetic mutation analysis and autoinducing assays. The S. mitis-Rgg regulator exhibited 74% identity and 87% similarity to the Rgg transcriptional regulator of the closely related human pathogen, S. pneumoniae. The predicted mature SHP sequence in S. mitis is DIIIVGG (Table 1), having a unique feature of containing one less isoleucine residue than the other streptococcal SHPs (DIIIIVGG or DILIIVGG). The S. mitis-Rgg regulator can respond to other streptococcal SHPs and the similar noncognate peptides (DIIIIVGG and DILIIVGG) exhibited the highest activity potential of S. mitis shp induction. Not only the S. pneumoniae SHP (DIIIIVGG) can activate the QS system in S. mitis, but S. mitis SHP can also trigger the activation of the Rgg/SHP system in S. pneumoniae, which regulates pneumococcal surface polysaccharide production. Cross-communication of Rgg/SHP systems can provide important insights regarding the host-microbiome relationships, and interference with these signaling pathways may have therapeutic potential by controlling bacterial pathological behavior.

Conclusions and Outlook

With the increasingly recognized importance of tackling antimicrobial resistance, the interference and alteration of QS systems have the potential to promote the development of novel anti-virulence therapeutic strategies for treating harmful diseases. The attenuation of these non-vital communication systems with the use of peptide-based modulators in Gram-positive bacteria may aid in the fight to treat bacterial infections by preventing the spread of antimicrobial resistance while inhibiting bacterial pathogenesis. Therapeutically relevant peptide-based QS modulators that can be utilized as promising alternatives to current antibiotic therapy have been developed for the treatment of several Gram-positive bacterial pathogens, including S. pneumoniae and S. aureus. Such QS modulators can both act as drug leads and as probes to delineate the molecular mechanisms that drive QS activation as well as interspecies interference.

Although several promising preliminary in vivo studies have been reported in recent years showcasing the potential of QS modulation as a therapeutic approach; before these lead compounds could be brought to clinical trials extensive systematic in vivo studies as well as comprehensive ADME pharmacokinetic studies must be completed. To the best of our knowledge, such studies have not been completed yet. Furthermore, it is still not clear how QS modulators could best be utilized, as a standalone therapeutic or as an adjuvant as part of a combination therapy with traditional antibiotics. Both strategies have their advantages, yet failure in the pursue of either one may discourage future attempts to pursue the other approach. Therefore, the specific system and therapeutic strategy selected for advancement may determine the fate of QS modulation as a therapeutic approach in the near future.

Biographies

Tahmina Ahmed Milly grew up in Bangladesh and received her B.S. (2012) and M.S. (2014) degrees in Applied Chemistry and Chemical Engineering, respectively, from the University of Dhaka, Bangladesh. She is currently working in the Tal-Gan group at University of Nevada, Reno, U.S.A, completing her final year at the Chemistry Ph.D. program. Her Ph.D. work is focused on investigating bacterial quorum sensing and interspecies interactions in streptococci.

Yftah Tal-Gan was born in Jerusalem, Israel and received his B.S. (2001), M.S. (2006) and Ph.D. (2011) degrees from The Hebrew University of Jerusalem, working with Chaim Gilon and Alexander Levitzki. He then joined Helen Blackwell’s group at the University of Wisconsin–Madison, U.S.A., as a Postdoctoral Research Associate. Yftah was appointed Assistant Professor in the Department of Chemistry at University of Nevada, Reno, U.S.A. in 2014, was promoted to Associate Professor with tenure in 2020, and to Professor in 2022. Yftah is the recipient of the 2020 American Peptide Society Early Career Lectureship Award.

Data Availability Statement

Data sharing not applicable – no new data generated.