Virulence Factors of Clostridioides (Clostridium) difficile Linked to Recurrent Infections

From 20 to 30% of Clostridioides (Clostridium) difficile infection (CDI), patients might develop recurrence of the infection (RCDI) and, after the first recurrence, the risk of further episodes increases up to 60%. Several bacterial virulence factors have been associated with RCDI, including the elevated production of toxins A and B, the presence of a binary toxin CDT, and mutations in the negative regulator of toxin expression, tcdC. Additional factors have shown to regulate toxin production and virulence in C. difficile in RCDI, including the accessory-gene regulator agr, which acts as a positive switch for toxin transcription. Furthermore, adhesion and motility-associated factors, such as Cwp84, SlpA, and flagella, have shown to increase the adhesion efficiency to host epithelia, cell internalization, and the formation of biofilm. Finally, biofilm confers to C. difficile protection from antibiotics and acts as a reservoir for spores that allow the persistence of the infection in the host. In this review, we describe the key virulence factors of C. difficile that have been associated with recurrent infections.


Introduction
Clostridioides (Clostridium) difficile infection (CDI) is the leading cause of healthcare-associated diarrhea worldwide. In 2011, increased CDI was reported in the United States by the Centers for Disease Control with an estimated 453 000 infections (83 000 had at least one recurrence) and 29 000 deaths [1].
depending on severity [18]. Nevertheless, 20-35% of patients may develop the recurrence of symptoms, which is defined as a recurrent infection (RCDI) [19][20][21][22][23]. After the first recurrent episode, patients are more likely to have subsequent recurrences, and by the third episode, risk of recurrence can reach 60% [24,25]. Several studies have evaluated administration of fidaxomicin versus vancomycin and metronidazole for RCDI patients, with lower recurrent episodes and fewer deaths for fidaxomicin [18,26,27].

Relapse and Reinfection
RCDI may occur due to relapse, defined as the persistence of the same strain causing the initial infection, or reinfection, defined as the acquisition of a genotypically distinct C. difficile strain from an exogenous source [28]. Furthermore, patients with ribotype 027 strains present a higher risk of relapse than those with other ribotypes [7].

Ribotypes Associated with Relapse or Reinfection
The glycosylating toxins, toxin A (TcdA) and toxin B (TcdB), are primarily responsible for the symptoms associated with CDI and are the key mediators of pathogenesis [29]. These toxins have been shown to bind to the cell surface and translocate to the cytosol of the host epithelial cells where they glycosylate and inactivate important GTPases (including Rho, Rac, and Cdc42), leading to actin cytoskeleton alternations, cell rounding, apoptosis, and cell death [30,31]. Several studies have shown elevated sporulation rates in epidemic strains, including the hypervirulent NAP1/BI/ 027 strain [32]. Also, these strains have been found to contain increased levels of toxins, which are associated with deletions in the toxin negative regulator tcdC (18 bp and 39 bp deletions for the 027 and 078 strains, respectively) in in vitro models [33,34]. However, in more complex models, the 027 strain has been shown to have a longer growth cycle, where toxin production starts slightly earlier than that of other strains, and toxins tend to accumulate [35,36].
RCDI is more frequent in patients infected with the 027 strain than in those infected with non-027 strains (P < 0.001). Besides, the clinical cure rate has been reported to be lower in 027-infected patients than in those with non- Increased pathogenicity in vivo and in vitro [37] agr1 locus (accessory-gene regulator) Positive regulation of toxin A and toxin B production, independent of tcdC Regulation of virulence, associated with increased colonization [38][39][40][41] Biofilm Survival niche of C. difficile with multispecies communities Accumulation of toxins and biomass in variant strains regulated by quorum sensing Long persistence/protection of C. difficile [36,[42][43][44][45][46] Accumulation of spores Reduced susceptibility to antibiotics [45,47] SlpA (S-layer protein A) Presence and low molecular weight subunits with sequence variability in hypervirulent strains Increased adhesion to gut mucosa [48][49][50][51][52] Cwp84 (cell wall protein 84) Cleavage of adhesins, such as SlpA, for the paracrystalline layer assembly Release and dissemination of C. difficile in the host [49,52] Degradation of several extracellular matrix proteins (fibronectin, laminin, vitronectin) Increase adhesion and colonization [48,49,53] Production of thicker biofilm in strains with high proteolytic activity associated to Cwp84 Enhanced virulence and host-pathogen adherence; maintenance of CDI [54,55] Flagella Presence of posttranscriptional modifications in flagellin and flagellar cap proteins Increased biofilm, adherence, and cell internalization, associated with efficient colonization in vivo [42,56,57] [58] Spores Development of structural morphotypes of outermost exosporium layers (thin or thick) Associated to host-spore interactions, dfferences in affinity to epithelial cells [12,45,59,60] Expression of the sporulating regulator spo0A is associated with high spore production and biofilm formation Transmission of CDI and maintenance of C. difficile in the host, despite the antibiotic treatment [42,45,59,[61][62][63] 027 infections when treated with fidaxomicin (P � 0.007) or vancomycin (P � 0.02) [3].

Antibody Response to Toxin A
In several studies, the immune response to toxins A and B has been described, with higher titers of immunoglobulins IgG antitoxin A in asymptomatic carriers than noncarriers, and higher titers of IgG and IgM against toxin A and toxin B, but regression analysis showed significance for recurrent infection and low antitoxin A for 027 and primarily for all types with patients with little antitoxin B (P � 0.02) [65,66].
Patients with a single episode of CDI had higher concentrations of serum IgM against toxin A on day 3 of initial CDI than those with RCDI (n � 22; P � 0.004). On day 12, patients who had a single episode of diarrhea (n � 7) had higher serum IgG values against toxin A than those with RCDI (n � 9; p � 0.009). IgG response to toxin A (12 days after onset of CDI) during an initial episode confers protection against recurrence (OR, 48.0; 95% CI, 3.5-663) [25]. Nevertheless, CDI patients who received neutralizing antibodies against toxin A showed no difference in the frequency of recurrence in comparison with CDI patients receiving placebo (17% and 18%, respectively, P � NS) [67].

Biofilm Production
Bacterial biofilms are associated with antimicrobial resistance, act as a survival niche, and protect bacteria, which can be in a dormant form with prolonged growth rates deep within the biofilm structure. Biofilms have been reported for several Clostridium species, including C. perfringens, C. thermocellum, and C. acetobutylicum [68,69]. Similarly, C. difficile growth has shown well-organized communities on abiotic surfaces and well-structured biofilms in vitro and in vivo [42,61,70], with differences in the level of biofilm production between some strains in monoculture biofilms [42,71]. In addition, a range of studies has characterized the supernatant and the polymeric composition and architecture of the biofilm matrix in in vivo and in vitro models, which is composed of extracellular DNA, polysaccharides, and proteins similar to B. subtilis biofilm [42,54,61,72].
Notably, in a chemostat gut model, C. difficile (vegetative and spore forms) has been shown to participate in multispecies communities forming a robust biofilm that accumulates toxins. In addition, this biofilm is a potential reservoir for the reestablishment of C. difficile after primary antimicrobial therapy has finished, when gut levels of antimicrobials are at subminimal inhibitory concentration [36,43,44]. Furthermore, the biofilm matrix showed a preferential localization of spores that have a higher resistance to some antibiotics (metronidazole and vancomycin) ( Table 1). Taken together, these observations may explain the long-term persistence of strains involved in primary and/or recurrent CDI [42,45]

Regulation by Quorum Sensing
Quorum sensing (QS) is the regulation of gene expression of virulence factors (biofilm production, attachment, motility, toxin production, and sporulation) in response to environmental changes due to cell-to-cell communication. It is mediated by small diffuse molecules known as autoinducers produced by individual bacteria. The level of autoinducers is cell-density dependent: when the density is high, autoinducers are detected by other bacteria, enabling them to coordinate physiological activities [38,73,74].
Orthologues of the accessory-gene regulator (Agr) ACDB, the global regulatory locus that encodes AgrA, have been found within the genome of most C. difficile isolates, including the hypervirulent strain 027/BI/NAP1. AgrA has critical roles in controlling gene expression and enhancing the production of colonization factors and exoproteins essential for the pathogenic process [38,39]. Furthermore, it is the transcriptional regulator of the best-understood QS system in Gram-positive bacteria, including Staphylococcus aureus [39].
C. difficile production of toxins A and B are controlled by an Agr-quorum signaling system mediated through a small thiolactone that can be detected in stools of CDI patients [40]. Some strains encode two Agr loci in their genomes (ag1 and agr2), with the first being present in all strains and the second being present in a few strains. It has been shown that the agr1 mutant cannot produce both toxins and that toxin production can be restored with the wild type agr1. Furthermore, it has been demonstrated that the agr1 mutant can colonize but cannot cause disease in a murine CDI model (Table 1) [39,75].

Sporulation and Germination
Another key virulence factor involved in C. difficile pathogenesis and colonization is spore production, with differences in germination rates being lower in spores from biofilm than those from a vegetative culture [45]. Spore production is mediated by the master regulator of the sporulation pathway (spo0A). In mice infected with sporulating and nonsporulating C. difficile strains (spo0A mutants), no recurrence of CDI was found after vancomycin treatment, and the spo0A mutant infection was not transmissible between hosts [59]. Spo0A mutants are associated with defective biofilm formation and low sporulation in biofilms (0.0001%), suggesting an essential link between Spo0A and biofilm production, such as that seen in Bacillus subtilis [42,61,62].
The production and accumulation of spores within C. difficile biofilms are likely to be significantly associated with RCDI, with further germination of vegetative toxinproducing cells after cessation of antibiotic therapy. Metabolically dormant spore forms can protect C. difficile from adverse conditions, such as nutrient starvation, antimicrobial agents, disinfectants, heat, and desiccation, and help the bacteria survive attacks of phagocytic cells [76]. Furthermore, antibiotic treatment triggers the excretion of higher sporulation of C. difficile in mice. Therefore, in most cases, when antibiotic therapy is stopped, a recovery occurs and the super-shedder state of C. difficile is suppressed [77].
A novel exosporial layer has been found in spores from biofilms, composed of fine fibers and darkly staining granules. This layer is surrounded by a thin layer and is acquired after mother cell lysis; it has been found in 027 strains associated with multiple recurrent episodes of CDI (Table 1) [45,59,60]. The specific role of these structural differences of the exosporium in spores is not clear. A previous study showed that the C. difficile R20291 strain (RT-027) showed higher affinity to the host cell membrane and microvilli of intestinal epithelial cells [78], suggesting that the differences in composition of the exosporium of C. difficile spores might regulate the adherence to intestinal cells of the host (Table 1) [78][79][80]. Additional studies are needed to determine the role of the structural properties of the C. difficile layer and spore exosporium for the development of recurrent infection.

Adhesion Factors
Several nontoxin factors involved in the virulence and infection processes have been described, including surface proteins (cell wall and surface layer (S-layer) proteins), pili, flagellin, flagellar cap, and fibronectin-binding proteins [42,53].
The S-layer protein A (SlpA) is the predominant outer surface and has shown to be the major contributor of C. difficile adherence to epithelial cells in vitro. SlpA is cleaved after translation in high and low molecular weight (HMW and LMW) subunits for the assembly of the paracrystalline layer. Interstrain sequence variability of LMW subunits has been associated with higher adhesion efficiency in hypervirulent strains [48][49][50][51].
The cell wall protein 84 (Cwp84) is one of the primary proteases that is exported by the cell and cleave several adhesins such as SlpA for the assembly of the paracrystalline layer and the degradation of extracellular matrix proteins (fibronectin, laminin, and vitronectin). This degradation triggers the release and dissemination of C. difficile in the host, which are related to the recurrence of infection (Table 1) [49,[81][82][83].
Cwp84 is present in all C. difficile strains, and those with the highest proteolytic activity are associated with stronger adhesion and production of thicker biofilm, planktonic growth defect, and virulence in vivo [49,72]. In addition, a recent study found overexpression of cwp84 in a biofilm model from recurrence causing strains, this phenomenon was not observed in the biofilm produced by nonrecurrent strains [84], suggesting an association with recurrent infection.
SlpA cleavage could be accomplished by other proteases in the absence of cwp84, such as cwpV, cwp66, and cwp13 [49,52]. These findings suggest an essential role of some surface proteins associated with increased host-pathogen adherence, which may be related to the maintenance of CDI.
In addition to propulsion, motility components provide bacteria with other advantages, such as adherence and cell internalization. C. difficile possesses peritrichous flagella, which induce the adhesion and establishment of the bacteria including the strains without complete and functional flagella as a result of mutations [56,57,[85][86][87]. The filament from C. difficile flagella is mostly composed of single flagellin subunits and flagellar cap proteins, both of which are modified posttranslationally. Therefore, as a consequence of a noncomplete functional flagella, these components do not confer motility but enhance binding of C. difficile to abiotic surfaces, as well as reduced biofilm formation, leading to the attenuation of colonization and relapse in vivo, suggesting a role of flagella in the process of adherence and biofilm formation independent to motility (Table 1) [42,56,57].

Concluding Remarks
RCDI development is associated with hypervirulent strains and may be attributed to a high rate of sporulation and the maintenance of spores encased in a C. difficile biofilm, which is resistant to antibiotic therapies. Besides, several adhesionrelated proteins are involved in RCDI development and the establishment of the infection.
Antibiotics have been demonstrated to disrupt colonic microbiota, placing the patient at a high risk of further recurrent episodes. Further studies on RCDI development are needed to assess the correlation of these potential virulence traits and the persistence of C. difficile infection.