Autoinducing Peptide-Based Quorum Signaling Systems in Clostridioides difficile

Abstract

The autoinducing peptide-based Agr system in C. difficile is involved in virulence factor expression, motility, and sporulation. This review highlights several of the recent discoveries regarding C. difficile Agr. Typical Agr systems rely on the combined activities of four proteins involved in peptide expression, peptide processing, peptide sensing, and transcriptional regulation. As emphasized in this review, at least two C. difficile Agr systems (Agr1 and Agr3) lack the set of proteins associated with this regulatory network. In line with this, recent finding indicate Agr1 can function in ways that may not depend on accumulation of extracellular peptide. Also, described are the similarities and differences in Agr systems within the pathogenic Clostridia.

Introduction

Cell-to-cell communication in bacteria generally involves the production of self-secreted extracellular signals that accumulate in the local environment and correlate with cell density (1). After reaching a threshold concentration, molecules signal back to the cell to coordinate a variety of events such as expression of virulence factors, sporulation, and biofilm formation (2, 3). Broadly, quorum sensing in bacteria involves production of self-secreted extracellular signaling molecules such as acyl homoserine lactones (AHL), autoinducer-2, oligopeptides, diffusible signal factors (DSF), and autoinducing peptides (AIP) (4–10). C. difficile, the focus of this review, has been found to contain LuxS/LuxR, RNPP (Rap, Npr, PlcR, and PrgX) family of quorum sensing proteins, and Accessory gene regulator (Agr) quorum sensing systems (11–13). This review presents an overview of the C. difficile Agr systems and describes recent results that suggest at least one of these systems, Agr1, functions in ways not typically associated with AIPs in other bacteria.

The prototypical Agr System

The Gram-positive bacterial accessory gene regulator (agr) loci contains genes encoding four proteins (AgrB, AgrD, AgrC, and AgrA) important for Agr-mediated regulation of virulence factor expression and cellular processes such as sporulation (14–17). The transmembrane protease, AgrB, processes the AgrD peptide to generate a cyclic autoinducing peptide (AIP), that is secreted from the cell (18–20). The AgrC sensor histidine kinase detects extracellular AIP concentrations at a critical threshold, leading to its activation via histidine-specific autophosphorylation (21). The phosphate group of the activated AgrC is transferred to an aspartate residue on the AgrA response regulator, consequently activating AgrA (22). Activated AgrA, using its DNA-binding domain, binds to the promoter region of target genes to up-or down-regulate their expression (23). In the well-studied Staphylococcus aureus Agr system, AgrA promotes transcription of a regulatory RNA (RNAIII) that ultimately mediates many of the important transcriptional changes that occur when the Agr system is activated (Fig. 1) (1, 10, 24).

Fig. 1. The prototypical four-protein Agr system in comparison to C. difficile Agr systems.

The agr locus encodes for four genes- agrB, agrD, agrC, and agrA that express a transmembrane protease AgrB, a peptide AgrD, a histidine kinase AgrC, and a response regulator AgrA, respectively. The transmembrane protease AgrB processes the AgrD peptide into a mature AIP. Mature AIP is secreted to the extracellular milieu to bind to and activate the membrane-bound AgrC. Activated AgrC then activates AgrA, which binds to the P2 and P3 promoter to induce transcription of the agr locus genes and the small regulatory RNA, RNAIII. As shown in the image, in comparison to the typical Agr system found in S. aureus, various strains of C. difficile utilize variations of the Agr system that lack a known RNAIII and do not contain the entire suite of genes typically associated with Agr (Created with inkscape.org)

The Agr System in C. difficile

Interestingly, three different Agr systems- Agr1, Agr2, and Agr3- have been identified in C. difficile (23) and each has clear differences from the prototypical system in S. aureus. The overall sequence homology and organization of the genes substantially differ among the C. difficile Agr systems and in comparison with S. aureus Agr (Fig.2A). C. difficile Agr2, found in a subset of C. difficile strains, most closely resembles S. aureus Agr by encoding all four components of a typical Agr system. However, agr2 genes (agrACDB) are arranged in the opposite order of those found in the of S. aureus Agr system (agrBDCA) (Fig.2A). Furthermore, no RNAIII-like regulatory RNA has been identified in the C. difficile Agr2 system. How C. difficile Agr2 mediates transcriptional changes remains largely unknown, though it is possible that AgrA2 directly promotes gene expression within this system.

Fig. 2. agr loci in C. difficile and in other Clostridia.

A. sequence homology and organization of the agr genes vary among C. difficile agr loci. B. The C. acetobutylicum agr and C. difficile agr2 encode for a complete Agr system. The agr loci in C. perfringens, C. botulinum, C. difficile agr1 encode for only AgrB (transmembrane protease) and AgrD (peptide), and C. difficile agr3 locus lacks gene encoding the response regulator. HG, hypothetical gene. (Created with geneious.com)

The Agr2 system is present in the epidemic R20291 strain of C. difficile (25). By examining an agrA2 mutant in C. difficile R20291, investigators found decreased expression in fliC (flagellin), fliA/SigD (alternative sigma factor), and fliM (anti-sigma factor) (26). Likewise, this mutant lost detectable flagella production and did not produce TcdA, one of the major toxins encoded by C. difficile. Finally, mice infected with the C. difficile agrA2 mutant showed significantly lower fecal shedding than mice infected with the parental strain. Thus, the Agr2 system in C. difficile R20291 is an important regulator of flagella formation, TcdA production, and colonization.

The C. difficile Agr3 system consists of agrB3, agrD3, and agrC3, but lacks a gene encoding agrA3 (Fig.2) (27, 28). Several C. difficile strains such as NAP07 and NAP08 contain genes for Agr3, but little is known about the its function and regulatory mechanisms (27). Interestingly, Agr3 appears to be encoded by a C. difficile bacteriophage phiCDHM1, suggesting this Agr system could be highly mobile and transmitted among various C. difficile strains (27).

The C. difficile Agr1 system is found in all sequenced C. difficile strains (25, 28). In contrast to the C. difficile Agr2 system, the C. difficile agr1 locus includes only the genes for the AgrB1 protease and the AgrD1 peptide. A two-gene agr loci resembling C. difficile agr1 has also been identified in other pathogenic clostridia such as C. perfringens, C. botulinum, and C. sporogenes (Fig.2B) (26, 29, 30). In the case of C. perfringens a histidine kinase/response regulator system, virS/virR has been shown to directly bind to the extracellular signals generated by the C. perfringens agr locus and likely substitutes for the AgrC1 and AgrA1 (31). Yet, similar genes encoding AgrC1 and AgrA1 surrogates have not been discovered in C. difficile, C. botulinum, or C. sporogenes. The absence of agrC1 and agrA1 within the agr1 locus suggests that the receptor histidine kinase and response regulator are encoded elsewhere in the genome or Agr1 functions in ways that do not entirely depend on these two other proteins.

A study by Darkoh et. al. found that the Agr1 system regulates the production of two major virulence factors of C. difficile, TcdA and TcdB (25). Additionally, the AIP signaling molecule generated from the Agr1 system is detected in the stool of CDI patients (28), suggesting a potential role of this system in C. difficile pathogenesis.

Results from more recent work supports the notion that C. difficile Agr1 acts as both a typical AIP signaling system in the case of sporulation, but may have other novel activities involving regulation of toxin expression (13). Deleting either agrB1 or agrD1, or the complete agr1 locus, downregulates the expression of sporulation-associated regulators and significantly reduces spore production, suggesting that both AgrB1 and AgrD1 are important for the regulation of sporulation in C. difficile. This is similar to what is known about Agr-mediated regulation of sporulation in C. perfringens, C. botulinum, and C. acetobutylicum (Summarized in Table 1) (32–34). Culture supernatant from C. difficile wild type strains, but not from the agrD1 mutant, induced sporulation in both single and double deletion mutants, suggesting that Agr1 functions similarly to a typical Agr system and generates an extracellular AIP signaling molecule that signals to regulate sporulation in C. difficile (13). These findings suggest Agr1 activity influences sporulation and does so in a way consistent with the known mechanisms of AIP signaling.

Table 1.

Regulatory role of the Agr system in Clostridia

Clostridia	Discovered Roles of the Quorum Signaling Systems in Clostridia
C. difficile Agr1	Regulate toxin production, sporulation, and motility
C. difficile Agr2	Positive regulator of TcdA production and flagella formation
C. difficile Agr3	Unknown
C. acetobutylicum Agr	Positively regulates sporulation
C. perfringens Agr	Positive regulator of PFO, PLC, ColA, CPA, CPB, CPE, ETX, NETB toxins, and of sporulation.
C. botulinum Agr1	Positively regulates sporulation
C. botulinum Agr2	Positively regulates sporulation

In contrast to sporulation, other C. difficile activities appear to be influenced in a manner that does not follow the typical models for Agr-mediated regulation (13). For example, because AgrB processes AgrD to generate the extracellular AIP (35) it is assumed that deletion of either agrB1 or agrD1 will result in no extracellular AIP and result in phenotypes similar to those found in a strain lacking both genes. Yet, expression of the alternative sigma factor tcdR is substantially increased only in the agrB1 deletion mutant and remains unchanged when only agrD1 or agrB1D1 are deleted (13). TcdR positively regulates TcdA and TcdB production (36), and both toxins are expressed at higher levels in the agrB1 mutant. Deletion of agrB1 also results in an increase in the activity of the motility-associated sigma factor SigD (13). SigD directly binds to the promoter region to upregulate the expression of tcdR, and thus tcdA and tcdB toxins (12, 37). It is possible that SigD may be under direct regulation of the Agr1 system, which in turn regulates TcdR. Alternatively, it is possible that tcdR is directly and indirectly (through sigD) regulated by the Agr1 system, leading to the regulation of toxin production.

A C. difficile agrB1D1 mutant does not exhibit changes in expression of tcdR, or SigD activity, or toxin production (13), indicating that the observed phenotypes in the agrB1 mutant are not a direct effect of AgrB1. Instead, it might be a consequence of the intracellular AgrD1 peptide is present in the agrB1 mutant. In line with this, the AgrD1 peptide has been shown to accumulate in an agrB1 deletion mutant of C. difficile (13).

Previous studies have also reported that the C. perfringens Agr system-mediated regulation of target genes does not entirely depend on the VirS/VirR histidine kinase/response regulator pair (38). For example, Agr-mediated regulation of epsilon toxin (ETX) production in C. perfringens type D strain CN3718 does not require virS/virR (39), suggesting the presence of an additional regulatory mechanism employed by this system. Future studies investigating whether intracellular AgrD1 directly regulates the expression of the toxin genes or interacts with regulators of toxin production will be important in understanding the AgrB1-independent activities in this system.

Agr system as a therapeutic target

The most common predisposing factor in C. difficile infections (CDI) is antibiotic treatment (40, 41). Nevertheless, antibiotics remain the most available treatment option for CDI patients (42, 43). Additionally, antibiotics are often ineffective in preventing recurrent CDI (44). In the cases where antibiotics are not sufficient, many times the only remaining option to treat CDI is the surgical removal of the colon (45–47), emphasizing the necessity for development of non-antibiotic therapies to treat CDI. The Agr quorum sensing system is an attractive non-antibiotic therapeutic target, as this system coordinates the regulation of multiple virulence factors in many Gram-positive bacteria (48). While detailed regulatory mechanisms of the Clostridial Agr system are just beginning to be understood, we know that the Agr system is essential for toxin production and sporulation in many Clostridia family members. Spores are critical for the transmission of C. difficile from host to host and to initiate CDI, while the toxins directly mediate disease symptoms (49). Therefore, inhibition of the Agr system, and thus of Agr mediated induction of spore formation and toxin production could limit the disease’s spread and severity. Two inhibitory peptides designed to target VirS, the receptor histidine kinase that detects AIP in C. perfringens, were found to attenuate Agr-mediated toxin production in this pathogen (50). Using peptide analogs that would transform AgrB to an inactive form, irreversibly antagonizing AgrB-mediated processing of AgrD, or use of antibodies to sequester intracellular AgrD1 and extracellular AIP are only a few of the options that could provide a basis for an Agr-based treatment. Furthermore, identifying the Agr1 responsive histidine kinase-response regulator pair and the downstream effector proteins of the intracellular AgrD1 might provide additional therapeutic targets.

Conclusion and Future Directions

Exciting progress is taking place in understanding Agr quorum sensing in Clostridia. The curious absence of any definitive two-component system genes in most Clostridial agr loci suggest a unique mechanism of action employed by this system in these pathogens. Indeed, a distinct toxin phenotype observed only in the C. difficile agrB1 mutant cannot be explained by the currently known mechanisms of Agr systems, where defective sporulation in the C. difficile agr1 mutant appears to be mediated by a typical quorum sensing mechanism. Furthermore, how possessing more than one Agr system affects C. difficile R20291 function and whether Agr1 and Agr2 synergize or antagonize is currently not known. A further in-depth understanding of the Agr system in C. difficile may reveal a novel regulatory mechanism utilized by this system and provide non-antibiotic therapeutic targets to treat C. difficile infections.

Highlights.

• Agr typically function through the extracellular accumulation of an autoinducing peptide.

• Three variants of the Agr system have been identified in C. difficile.

• Agr1, the most common Agr system in C. difficile, lacks a sensor kinase and response regulator.

• Agr systems regulate C. difficile virulence factor expression and sporulation.

• Agr1 may function in ways that do not require extracellular peptide.