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The role of flagella in Clostridium difficile pathogenicity
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Review
Microbial Translocation
The role of flagella in Clostridium difficile pathogenicity Emma Stevenson, Nigel P. Minton, and Sarah A. Kuehne Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Clostridium difficile is widely publicised as a problem in the health-care system. Disruption of the normal gut microbiota by antibiotic therapy allows C. difficile to colonise the colon. On colonisation, C. difficile produces two toxins that lead to disease, with symptoms ranging from mild-to-severe diarrhoea, to fulminant and often fatal pseudomembranous colitis (PMC). How C. difficile establishes initial colonisation of the host is an area of active investigation. Recently there has been increased research into the role of C. difficile flagella in colonisation and adherence. Novel research has also elucidated a more complex role of flagella in C. difficile virulence pertaining to the regulation of toxin gene expression. This review focuses on new insights into the specific role of C. difficile flagella in colonisation and toxin gene expression. Importance of flagella to bacteria The formation of flagella by many bacterial species provides them with the ability to be motile, to colonise abiotic or biotic surfaces, to optimise growth and survival, and to evade desiccation in a hostile host environment [1]. The flagellum comprises a molecular motor, a hook–basal-body complex, and a rotating filament and is considered to be a specialised type III secretion system derived from a common ancestral structure [2] (Figure 1). The components of the flagellum work together to drive bacterial motility. The motor proteins provide the power to the hook–basal-body structure to drive flagellum rotation. The basal body is an integral membrane protein complex [3] that also serves as a passive structure allowing the translocation of other structural flagellar proteins outside the cell [2]. Once these proteins have been assembled, the hook structure acts as a universal joint to which the filament proteins can be assembled in a helical manner. The power provided by the motor proteins can thus drive the rotation of the flagellum filament, providing the bacterium with motility [3]. Bacterial flagella have been implicated in contributing to bacterial pathogenesis by: (i) promoting adherence to host cells; (ii) providing force-driven motility to nutrients; (iii) promoting biofilm formation; (iv) facilitating translocation of virulence factors across cell membranes; and Corresponding author: Stevenson, E. ([email protected]). Keywords: flagellar gene regulation; toxin expression. 0966-842X/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2015.01.004
(v) acting as immunomodulators by triggering proinflammatory cytokines through the Toll-like receptor 5 (TLR5) signalling pathway [2]. Flagellum-driven motility is important to many gastrointestinal pathogens including Campylobacter jejuni, Vibrio cholerae, and Helicobacter pylori [4]. Mutations in flagellar genes in these bacteria have rendered strains less able to colonise the host [5,6], unable to establish persistent infections [7], and less able to adhere to small intestinal epithelial cells [8] and flagella mutants in C. jejuni are deficient in the secretion of certain virulence proteins [9]. Thus, as C. difficile occupies an environmental niche similar to that of these intestinal pathogens, the flagella of C. difficile may also play an important role in colonisation and pathogenicity. C. difficile is the causative agent of hospital-acquired antibiotic-associated diarrhoea in high-risk patients over the age of 65 years. Reports show that C. difficile infection (CDI) is 12–14 times more common than other widely publicised infections such as methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia [10]. Disruption of the normal colonic flora via antibiotics allows the establishment of CDI in the colon resulting in symptoms ranging from mild-to-severe diarrhoea to fulminant and often fatal PMC. Until recently C. difficile flagella have primarily been implicated in the colonisation of the host [11]. However, the contribution of flagella to the pathogenesis of C. difficile is complex and not yet fully understood. There is increasing scientific evidence to suggest that flagella play a more direct role in virulence, via modulation of toxin expression [4,12] rather than simply providing force-driven motility towards nutrients available in the gut. In this review the importance of flagella to the pathogenicity of C. difficile is discussed. Genetic organisation of C. difficile flagellar genes Many of the genes and proteins that govern the structural integrity and motion of flagella in other bacterial species have been identified in strains of C. difficile 630 and R20291 (BI/NAPI/027, hypervirulent ribotype) (Figure 1). Expression of the flagellar genes in all bacteria is temporal and regulated in a hierarchical manner [13]. For many bacteria, activation of flagellar gene transcription is governed by the master transcriptional regulator FlhDC [13]. However, many alternative master regulators have been found in other bacteria and include CtrA, VisNR, FleQ, FlrA, FlaK, LafK, SwrA, and MogR [14]. Interestingly, a master Trends in Microbiology xx (2015) 1–8
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Filament
F1
293002
FliD Filament cap
Flagellar assembly
CD0226 fliN CD0230
F2
304766
FlgK Hook Proximal rod FlgF
FlgG
Outer membrane
FlgH FlgI
Pepdoglycan layer
Basal body
MotB Cytoplasmic membrane
FliE
MotA
Motor/switch
FlhA FliH FliI FliO FliP
Type III secreon system
FlgN
FliJ
FliS
FlgA
FliT
FlhB
308251
CD0240
fliC F3
309272
flgC fliF
CD0241 CD0243
FliK FlgD
FlgE
CD0238
fliW fliS1 fliD
CD0242 CD0244
Hook-filament juncon
304049
csRA fliS2
CD0227 flgM flgK
FliC
FlgL
flgL
flgB fliE
fliH fliG
flil
F3 flgD
fliJ
Distal rod
flbD
motB
fliZ
fliQ
flhA
L ring
fliK
P ring FlgB
FlgC
FliF
MS ring
FliG FliM FliN
C ring
flgE
Proximal rod
motA
fliP
fliL
F3 flhG flhF
333302
CD0267 flgG1 fliA
flhB/fliR
CD0272
fliM
CD0267A flgG
fliN TRENDS in Microbiology
Bacterial chemotaxis
FliQ FliR FlhC FlhD FlgM
DNA O
Early gene products
DNA O
Late gene products
Figure 2. Genetic organisation of the flagellar operon in Clostridium difficile 630 (drawn to scale). Black arrows represent open reading frames with gene annotations above or below the respective genes. The three regions of the flagellar regulon are indicated by a solid bracket above the gene locus (F1, latestage flagellar genes; F2, flagellar glycosylation genes; F3, early-stage flagellar genes). Broken arrows below the flagellar locus mark the location of putative fliA promoter sites. White triangles represent insertion sites for ClosTron mutation and grey shading represents noncoding regions of DNA. Reprinted, with permission, from [4].
02040 6/23/10 (c) Kanehisa laboratories
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Figure 1. Representative structural organisation of a flagellum in a Gram-negative bacterium. The integral basal-body complex allows structural and filament proteins to be translocated out of the cell. The hook structure anchors the filament proteins to the basal body, allowing the formation of a mature flagellum. Rotation of the fully formed flagellum is powered by the internal motor and switch proteins attached to the basal-body complex. The flagellar genes (highlighted in green) indicate those that have been identified and/or characterised in Grampositive Clostridium difficile 630 and R20291. Reproduced from http://www. genome.jp/kegg-bin/show_pathway?org_name=cdf&mapno=02040&mapscale= &show_description=hide.
regulator of flagellar expression in C. difficile has not yet been characterised and this raises the question of whether flagellar gene activation is governed by transcriptional regulators involved in other cellular processes. Genes that govern the assembly of C. difficile 630 flagella are organised into three distinct operons known as the F1, F2, and F3 regulons [15] (Figure 2). The hierarchy of transcription of the flagellar genes in C. difficile is proposed to start with the early-stage genes located in the F3 region (Table 1 and Figure 2). Transcriptional control of the F3 regulon in C. difficile has not yet been fully defined and remains an area for future research. However, the genes in the F3 regulon are orthologous to the class II flagellar genes found in Bacillus subtilis, which are cotranscribed by an RNA polymerase and the vegetative sigma factor 70 (s70) homolog, sA [4,16]. A putative sAlike promoter sequence has been identified upstream of flgB in C. difficile 630 [17]. Therefore, the F3 region of
C. difficile might be under similar regulation via this promoter [4,17]. In strains of C. difficile from the ribotype 078 group, the F3 region is missing altogether [15] and this results in a completely nonmotile phenotype [11,15]. The F3 region comprises early flagellar genes such as fliA (Figure 2), which is an ortholog of s28, found in many Gram-negative bacteria [4,17]. In C. difficile three fliA promoter regions have been detected in the F1 region (Figure 2) and thus FliA is thought to regulate the latestage (F1) genes [4,17]. The F3 region is separated from the F1 region by an interflagellar gene region (known as the F2 region; Figure 2), which in C. difficile 630 comprises four flagellar-biosynthetic glycan genes [18] (Figure 3). However, there is genetic diversity at this region in the ‘hypervirulent’ ribotype 027 strains (including R20291) [15,19]. Four genes found in C. difficile strain 630 have been replaced by six genes in R20291 and CD196 and in other C. difficile strains such as M120, which is a 078 ribotype (Figure 3). Recent comparative studies of the F2 region have revealed that only C. difficile 630 and strains in the ribotype 017 lineage share this four-gene F2 operon [20]. The late-stage flagellar genes are encoded in the F1 regulon and are orthologous to class III flagellar genes of other bacteria [13,21]. Transcription of these genes cannot occur until all of the protein products of the F3 region have
Table 1. Positions and genes of the flagellar operons in the Clostridium difficile 630 genome [15] Flagellar regulon F1 (late-stage genes) F2 F3 (early-stage genes) 2
Position (bp) 293002–304049 304766–308251 309272–333020
Gene region CD0226–CD0240 CD0241–CD0244 flgB–CD0272
Genes (not all) encoded in each operon fliC, fliD, and a putative glycosyltransferase Flagellar-biosynthetic glycan genes fliF, fliG, fliM flhR, fliR fliA s28 homolog
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(A) F2 region of C. difficile 630
(B) F2 region of C. difficile R20291
(C) F2 region of C. difficile CD196
(D) F2 region of C. difficile M120
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Figure 3. Organisation of the F2 flagellar region using whole-genome visualisation via Artemis [49] in (A) 630, (B) R20291, (C) CD196, and (D) M120 Clostridium difficile strains. Blue boxes represent coding regions of flagellar glycan genes. In 630, the F2 region (in red box) comprises genes CD0241–CD0244, identified as flagellar glycan genes (Table 1). In R20291 and CD196 (B,C), these genes are replaced by six genes (in red boxes) including a glycosyl transferase (family 2), two putative uncharacterised proteins, a putative carbamoyl phosphate synthetase, and a putative ornithine cyclodeaminase [15].
been assembled correctly. The F1 region is proposed to be kept inactivated by the alternative sigma factor FlgM (anti-s28) [17]. Once the proteins encoded by the F3 region have been assembled, FlgM is excreted through the cell membrane and FliA activation of the F1 region can begin [13]. Microarray data have provided evidence that there is decreased gene expression in the F1 and F2 region of a fliA mutant strain, providing evidence that FliA positively regulates the late-stage flagellar genes [17]. The F1 region of the genome encodes the genes for the flagellin FliC and the protein cap FliD (Table 1 and Figure 2), which are structural proteins essential to the formation of fully functional flagella. Divergence of the F1 region in many ‘hypervirulent’ ribotype 027 strains has been shown [15,19]. This diversity is believed to account for the differences in motility between C. difficile 630 and R20291 [15]; Stabler et al. [15] observed that C. difficile 630 was less motile than R20291. It is pertinent to note that differences in motility and number of flagella have been observed between clonal isolates of C. difficile R20291 [12,15,22]. The study of Stabler et al. [15] revealed R20291 to be more motile than 630; however, research by Baban et al. [12] has shown R20291 to be less motile than 630 and to have one polar flagellum, as opposed to Martin et al. [22], who observed multiple flagella in the R20291 strain. It is unclear what genetic changes in the R20291 isolates have contributed to the change in number of flagella and thus motility observed by different research groups. Others have observed motility differences between
isolates of the same 027 ribotype [23] and have also concluded that there is great difficulty in precisely defining the genetic differences that may contribute to differences in motility. Flagella glycosylation The glycosylation of bacterial flagella has been extensively studied in Gram-negative bacteria but only three genera of Gram-positive bacteria (Listeria spp., Clostridium spp., and Butyrvibrio spp.) have been shown to produce glycosylated flagellins [24]. In many Gram-negative bacteria, including the enteric pathogen C. jejuni, flagella glycosylation plays a role not only in flagellum assembly but also in virulence [18]. Inactivation of some of the glycosylation genes of C. jejuni suggests that lack of flagella glycosylation abolishes autoagglutination and decreases the ability of the bacterium to adhere to and invade epithelial cells, and these mutants also appear to be attenuated in the ferret model of infection [25]. In 2009, Twine et al. [18] deduced that the flagellin of C. difficile could be modified by O-linked glycan moieties. Their research demonstrated that the flagellin glycans of C. difficile were genetically distinct not only from other species of Clostridia, namely Clostridium botulinum, but also between different strains of C. difficile. While the flagellin of C. difficile 630 could be glycosylated with an N-acetyl hexosamine (HexNaAc) residue at up to seven sites, the spectrum of flagellin glycosylation in strains of 3
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Review the 027 ribotype (BI-1 and BI-7) was more complex. The flagellin from 027 ribotypes was modified via O-linkage to heterogeneous glycan moieties of up to five monosaccharide residues [18]. Further identification and characterisation of a specific glycosyltransferase gene (CD0240) from the F2 region of the flagellar operon of C. difficile 630 (Table 1) revealed it was involved in the glycosylation process. Inactivation of CD0240 led to loss of the surface-associated flagellin protein. This rendered the strain nonmotile; however, the strain still produced truncated polymerised flagella filaments [18,20]. Thus, for the first time flagellin glycosylation was shown to be important in C. difficile flagellum assembly. Whole-genome comparative studies have shown genetic differences in the F2 region (Figure 3) between C. difficile strains 630, R20291 CD196 (historic 027 ribotype) [15], and M120 (078 ribotype). Stabler et al. [15] proposed that specific differences in this region between C. difficile 630, R20291, and CD196 may translate into differences in autoagglutination. The R20291 ‘hypervirulent’ 027 ribotype showed significant differences in autoagglutination to C. difficile 630 (P < 0.05), in contrast to the historic 027 ribotype, which did not show any phenotype. Autoagglutination of bacterial flagellins is a pathogenic strategy employed by C. jejuni to facilitate virulence [25]. Autoagglutination of flagellin results in changes in antigenic specificity due to differential glycosylation of flagellin [25]. The divergence of the F2 region and differences in autoagglutination observed between the strains of C. difficile in the study of Stabler et al. [15] could be correlated with the fact that distinct spectrums of flagellin glycosylation patterns exist between C. difficile 630 and 027 strains, as observed by Twine et al. [18]. However, in C. difficile changes in autoagglutination may not directly be the result of changes in flagellin glycosylation, as it is a multilevel process and probably involves other surface proteins [15]. Further research has since characterised more of the genes from the F2 region of C. difficile 630 and a toxin Anegative epidemic ribotype 017 (RT017) strain and found them to be putative post-translational modification (PTM) genes. Individual mutants in three of the four PTM genes of the F2 region (CD0241, CD0242, and CD0244) resulted in loss of motility and showed a sedimentation phenotype in vitro [20] that was more extreme than that displayed by the flagellin ( fliC) mutant. In concordance with what was also observed by Twine et al. [18], mutants in these genes were also still found to produce flagellin; however, for each mutant differences in flagellin molecular weight were observed. These size differences were proposed to be due to changes in flagellin PTM caused by disruption of the genes [20]. Flagellin modification of the CD0241, CD0242, and CD0244 mutants was determined by mass spectrometry, which revealed that these mutants did indeed have altered glycopeptide structures leading to the differential flagellin molecular weight [20]. Thus, this evidence suggests that both the structural integrity and PTMs of flagella are important for the motility of C. difficile. However, further research outlined below has also demonstrated the importance of flagella PTMs in colonisation. 4
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The role of flagella in host colonisation Studies before the publication of the C. difficile 630 genome highlighted that the flagellin and flagella cap genes ( fliC and fliD) were involved in adherence to murine mucus. Tasteyre et al. showed that there was a tenfold decrease (P < 0.01) in adherence of nonflagellated strains versus flagellated strains to mouse caeca in vivo [11], indicating that flagella may be important for the colonisation of C. difficile in the host. While the work of Tasteyre et al. [11] has elucidated a small part of the role of flagella in C. difficile colonisation, research relied on the use of naturally occurring nonmotile strains. More recently, the development of targeted mutation technology such as ClosTron [26] and allele-coupled exchange (ACE) [27,28] has enabled researchers to target specific genes of the flagellar gene operon and gain a greater depth of understanding of the contribution that the flagella make to C. difficile pathogenicity. The use of this technology has recently implicated the importance of flagella in the later stages of biofilm formation [29], when a C. difficile R20291 fliC mutant showed decreased biofilm formation (P < 0.05) at day five of the experiment. Recent studies [4,12,30] have found that fliC and fliD mutants of 630Derm displayed increased adherence to Caco2 cells compared with the parental strain and this is contradictory to previous evidence that flagella do play a role in C. difficile colonisation [11]. However, a study by Baban et al. [12] showed that fliC and fliD mutants of strain R20291 exhibited decreased adherence to Caco2 cells (P < 0.05) and thus, for some strains, intact flagella may be necessary for adherence. The study of Baban et al. [12] also included a mutant with paralysed flagella (the motB mutant) to further define whether the observed differences were due to loss of intact flagellar structure or specific to the inactivation of certain flagellar genes. Co-challenge experiments in a diaxenic mouse model showed that both the motB and the parental C. difficile R20291 strain outcompeted the fliC mutant in adherence, with the fliC mutant showing significantly less adherence (P < 0.05) to mouse caeca than the motB mutant. They further concluded that, for C. difficile R20291, flagella may play a more significant role in bacterial adherence than initial motility-driven colonisation. The study of Baban et al. also suggested that the inverse relationship of flagellar gene mutation and adherence is true for strain 630Derm fliC and fliD mutants. Challenge of 630Derm fliC and fliD mutants in monoxenic mice revealed no difference in colonisation. Co-challenge experiments revealed that the parental strain outcompeted the fliC mutant at some time points and the 630Derm parental strain adhered less well to mouse caeca than the fliC mutant. Taking these findings together, they proposed that, in the 630Derm strain, flagella and motility are not essential to colonise mice but may provide a motility fitness advantage over nonflagellated strains in environments where the two coexist. Publication of the C. difficile 630 genome sequence [Reference Sequence (RefSeq) NC_009089)] and the progression of next-generation sequencing platforms have led to the publication of many more C. difficile genomes, facilitating genome-comparison studies. These studies
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Review have provided useful insights into genetic changes between strains, which have led to the recognition of altered motility, toxicity, and antibiotic-resistance phenotypes [15]. Whole-genome comparison studies of C. difficile isolates have been used to identify differences in the flagellar gene regions that might affect motility [15] and virulence [18]. These whole-genome comparison studies have again highlighted that flagella, although important, may not be essential for C. difficile colonisation and adherence. The publication of the genome of C. difficile M120 (ribotype 078; GenBank FN665653) has confirmed that this strain has complete loss of the F3 flagellar region while retaining the F1 region, and this has been corroborated in microarray data from phylogenetic studies [19]. Studies as early as 2000 have indicated that nonflagellated C. difficile serotypes retain transcription of fliC and fliD genes but the protein products have remained undetected [31,32]. Therefore, whole-genome sequencing and characterisation on a wider panel of 078 ribotypes would reveal whether loss of the F3 and conservation of the F1 region is a common phenomenon among these strains. The PCR ribotype 078 strains have recently been recognised by Public Health England (PHE) and the Clostridium difficile Ribotyping Network (CDRN) as one of the emerging C. difficile strains in England, with a significant (P < 0.05) [33] increase in prevalence of 3.9% from 2007 to 2010 (http://www.gov.uk/government/uploads/system/uploads/ attachment_data/file/347168/CDRN_2009_10_Report. pdf). The 078 strain has also been found in both humans [34,35] and farm animals such as calves [36] and pigs [34] and is thought to contribute to a more community-based source of infection. The emergence of this strain as a common circulating ribotype in health-care facilities therefore suggests that nonflagellated strains are as capable of causing infection as other, flagellated strains. More recently, mutations in PTM genes of the F2 region in C. difficile 630 were shown to display alterations in adherence to abiotic surfaces. More specifically, a mutation in the PTM gene CD0241 led to attenuation of colonisation and relapse of infection in a murine model [20]. This observation combined with other work suggests that both the structural integrity of FliC and its PTM status play a role in the adherence and colonisation of C. difficile during the infection process [4,12,20]. The evidence above suggests that flagellum-driven motility may not be essential to the survival and colonisation of C. difficile, but the role of the flagellum in pathogenicity cannot be overlooked, especially when recent evidence suggests there may be a link between flagellar regulation and toxin production. Flagellar and toxin gene regulation An initial indication of a link between flagellar gene regulation and toxin expression was shown when hamsters infected with C. difficile 630 fliC and fliD mutants succumbed to infection faster than those with the parental 630Derm strain [30]. The observations of faster time to death of hamsters infected with the fliC and fliD mutants also correlated with increased cytotoxicity of culture supernatants at 24 h in these strains. However, in vivo there were no observable differences in caecal toxin titres
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between hamsters infected with any of the strains. Therefore, while a direct link between toxin and flagellar expression could not explain in vivo outcomes [30], it left an intriguing area of research open to investigation by others into whether there is a direct link between toxin and flagellar gene expression. In recent years there has been strong evidence to suggest that the expression of flagellar genes is coupled to toxin gene regulation, as well as other genes involved in the early stages of bacterial colonisation and virulence [4,12,30,37]. Studies of flagellar and toxin gene expression in C. difficile 630Derm In 2012, Aubry et al. [4] examined the disruption of flagellar genes in C. difficile strain 630Derm. Disruption of some F3 genes, such as fliF, fliG, and fliM, decreased the expression of some of the genes of the C. difficile pathogenicity locus (PaLoc), including the gene responsible for positive regulation of toxins (tcdR), the toxin genes (tcdB and tcdA), and the accessory holin gene (tcdE). This also correlated with decreased toxin in supernatants of cultures. Similarly to Aubry et al., Baban et al. [12] reported that disruption of an F3 gene, flgE, resulted in a tenfold decrease in tcdA expression. Interestingly, Aubry et al. did not find any change in levels of expression of tcdC (debated as the negative regulator of toxins [38,39]) with mutants in any of the flagellar genes. They surmised that toxin gene regulation at the early- to mid-log phase of cellular growth is a more dynamic process than previously thought and may not be solely controlled by repression of tcdR by TcdC during vegetative growth. Recent transcriptomic and quantitative real-time (qRT)-PCR data published by ElMeouche et al. [17] revealed decreased tcdR, tcdA, and tcdB expression in a C. difficile 630 fliA (sigD) mutant. Bioinformatic analysis of the PaLoc revealed a fliA-like consensus sequence upstream of the tcdR promoter site and further characterisation of both the mutant and parental strains revealed that FliA directly activates tcdR expression by binding and directing the core RNA polymerase tcdR promoter. Thus, FliA mediates the regulation of toxin gene expression via TcdR [17]. Inversely, disruption of the F1 genes such as fliC increased the expression of toxin genes and the toxin found in culture supernatant [4]. Baban et al. [12] corroborated these findings and reported that expression of tcdA (deduced by qRT-PCR) was 44.4 times greater in a fliC mutant and 7.4 times greater in a fliD mutant than in the 630Derm control strain (P < 0.05). Aubry et al. also investigated a C. difficile 630 mutant in a glycosylation gene (CD0240) in the F2 region. Data indicated that this strain clearly had the same nonmotile phenotype as the 630 fliC mutant. However, nonglycosylated FliC was found to be secreted, which suggested the formation of a nonfunctional flagellum and supports the hypothesis of Twine et al. [18]. Toxin gene expression of the C. difficile 630 CD0240 mutant remained unchanged during their experiments, and perhaps direct changes or loss of FliC rather than loss of a functional flagellum is responsible for changes in toxin expression. If this were true, it would be interesting to see whether disruption of fliC in the 5
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Review 078 ribotypes had any significant impact on toxin production, since these strains have lost the entire F3 region but retained the F1 region. Studies of flagellar and toxin gene expression in C. difficile R20291 Data from studies of other virulence factors governing C. difficile pathogenicity, such as sporulation, have proved that different C. difficile ribotypes and even strains of the same type behave differently [40] and that interpretation of data for one strain should not be used to generalise about the characteristics of others. Thus, although there is good evidence for the coregulation of toxin and flagellar genes in C. difficile 630Derm [4,12], the link between the two in other strains, such as C. difficile R20291, is not so clear. Baban et al. [12] confirmed the findings of Aubry et al. [4], deducing that toxin gene expression was altered in mutants of flagellar genes in C. difficile 630. Baban et al. also sought to confirm whether this was true for other strains of C. difficile, namely the ‘hypervirulent’ 027 ribotype called R20291. Results from their experiments showed no difference in cytotoxicity between the parental strain of R20291 and any of the flagellar gene mutants. However, 70% of mice infected with a R20291 fliC mutant had succumbed to infection within 4 days but those challenged with the parental strain did not succumb to infection at all [12]. The findings of Baban et al. have prompted others to investigate the pleiotropic effects of a fliC mutation in R20291 during colonisation in mice. Using in vivo transcriptomic data gathered 14 h post-infection of germ-free mice with either a R20291 fliC mutant or the parental R20291 strain, Barketi et al. [37] showed that there was no difference in toxin gene expression between the fliC mutant and the parental strain of R20291. Furthermore, both parental and mutant strains of R20291 showed no differences in cytotoxicity. They also found that 310 genes were differentially regulated in the fliC mutant compared with the parental strain during this time period. Most of the genes differentially regulated were those involved in motility, membrane transport [including phosphotransferase systems (PTSs) and ATP-binding cassette (ABC) transporters], sporulation, and carbon metabolism. This suggests coregulation between the expression of flagellar genes and those involved in the early stages of colonisation by C. difficile and also that, in the R20291 fliC mutant, there may be a global increase in genes associated with virulence that is responsible for the greater fatality in mice, as observed by Baban et al. [12]. The interaction of flagella and the host immune system Recent publications have implicated the flagellar proteins in the regulation of toxin gene expression, culminating in the release of toxins, which leads to the pathology of C. difficile infection. The toxins disrupt the tight junctions of epithelial cells, altering their morphology and increasing cell permeability [41]. The effect of toxins on gut epithelia evokes a host inflammatory response, which facilitates intestinal damage, and results in the clinical presentation of disease, such as diarrhoea and PMC [42]. 6
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Bacterial flagellins are highly immunogenic [43] and in many Gram-negative and Gram-positive bacteria have been shown to activate an innate immune response via the TLR5 pathway [2,44]. Thus, the immune response elicited by C. difficile flagellin could also contribute to the pathology of infection. In 2005, two publications by Pechine et al. [45,46] demonstrated that antibody levels against FliC and FliD were increased in a patient control cohort compared with a cohort with active C. difficile infection. They concluded that these proteins were able to induce an immune response that could play a role in host defence. More recently, Jarchum et al. [47] deduced that exogenous C. difficile flagellin protects mice from lethal C. difficile infection by mechanisms that maintain the integrity of the intestinal epithelial barrier. They proposed that the response requires host TLR5 activation. However, in vivo the response is unlikely to occur until epithelial damage (presumably via the toxins) has occurred, due to the location of TLR5 expression on the basolateral surface of intestinal epithelial cells, and thus flagellin may not provide a protective immune response on active infection [47]. Yoshino et al. [48] expanded on the flagellin immune activation repertoire by deducing that epithelial cells could indeed be stimulated, via TLR5, by flagellin and that this stimulation induced activation of nuclear factor kappa B (NF-kB) and p38 and the production of interleukin-8 (IL-8) and chemokine (C–C motif) ligand 20 (CCL20). The implication of flagellin in the immune response could have importance in the development of novel vaccine candidates against flagellin. Protective antibodies to flagellin may not only delay or prevent colonisation but may also help reduce the inflammatory response and might contribute to a less severe outcome of infection. Concluding remarks Here we have highlighted the increasing importance of flagella in the pathogenicity of C. difficile. The dawn of genetic tools to manipulate the genome has facilitated a greater understanding of the role of the flagellum in colonisation, adherence, and the regulation of toxin production. However, the important questions and avenues of investigation that remain will facilitate greater understanding (Box 1). Insights into the evolution of nonmotile strains have strengthened the hypothesis that flagellum-driven motility is not necessary for in vivo colonisation. However, they may play a greater role in adherence to the host. Box 1. Outstanding questions Is there a difference in virulence between strains that are peritrichous or monoflagellated? What is the link between flagella and toxin expression in a wider panel of emerging Clostridium difficile strains? Do other flagellar genes or gene products directly regulate toxin expression? Are there any other virulence genes coregulated with flagellar genes in other C. difficile flagellar mutants or naturally occurring nonmotile strains? How do parental C. difficile strains and flagellar gene mutants effect the complex immune host–pathogen interactions that occur during infection and determine infection outcomes? What are the roles of distinct flagellar glycan structures in C. difficile pathogenicity?
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Review Clearly, there is mounting evidence to suggest an indistinct relationship between toxin production and flagellar gene regulation in C. difficile. Although the relationship is not yet fully understood, it is most certainly strain specific. Furthermore, this is yet another way in which C. difficile governs transcription of virulence factors. This is another example of how intricate and convoluted the transcriptional regulatory network of C. difficile is and how deciphering the precise mechanisms of regulation will continue to be a challenge. This presents another avenue of investigation for researchers who are already working on transcriptomic data to unravel the intricate control of toxin gene regulation in C. difficile. Understanding novel regulatory pathways of toxin production could in the future lead to novel therapies against this persistent hospital pathogen, disease due to which has potentially dire consequences for an elderly population of patients. Acknowledgements This work was supported by the National Institute for Health Research (NIHR) Digestive Disease Biomedical Research Unit Nottingham (NIHR 2012-2017). E.S. wrote the manuscript, E.S. and S.K. evaluated the manuscript, and N.M. supported the manuscript.
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17 El Meouche, I. et al. (2013) Characterization of the sigD regulon of C. difficile and its positive control of toxin production through the regulation of tcdR. PLoS ONE 8, e83748 18 Twine, S. et al. (2009) Motility and flagellar glycosylation in Clostridium difficile. J. Bacteriol. 191, 7050–7062 19 Stabler, R.A. et al. (2006) Comparative phylogenomics of Clostridium difficile reveals clade specificity and microevolution of hypervirulent strains. J. Bacteriol. 188, 7297–7305 20 Faulds-Pain, A. et al. (2014) The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence. Mol. Microbiol. 94, 272–289 21 Aldridge, P. and Hughes, K.T. (2002) Regulation of flagellar assembly. Curr. Opin. Microbiol. 5, 160–165 22 Martin, M.J. et al. (2013) The agr locus regulates virulence and colonization genes in Clostridium difficile 027. J. Bacteriol. 195, 3672–3681 23 Valiente, E. et al. (2012) Emergence of new PCR ribotypes from the hypervirulent Clostridium difficile 027 lineage. J. Med. Microbiol. 61, 49–56 24 Logan, S.M. (2006) Flagellar glycosylation – a new component of the motility repertoire? Microbiology 152, 1249–1262 25 Guerry, P. et al. (2006) Changes in flagellin glycosylation affect Campylobacter autoagglutination and virulence. Mol. Microbiol. 60, 299–311 26 Heap, J.T. et al. (2007) The ClosTron: a universal gene knock-out system for the genus Clostridium. J. Microbiol. Methods 70, 452–464 27 Heap, J.T. et al. (2012) Integration of DNA into bacterial chromosomes from plasmids without a counter-selection marker. Nucleic Acids Res. 40, e59 28 Ng, Y.K. et al. (2013) Expanding the repertoire of gene tools for precise manipulation of the Clostridium difficile genome: allelic exchange using pyrE alleles. PLoS ONE 8, e56051 29 Ðapa, T. et al. (2013) Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile. J. Bacteriol. 195, 545–555 30 Dingle, T.C. et al. (2011) Mutagenic analysis of the Clostridium difficile flagellar proteins, FliC and FliD, and their contribution to virulence in hamsters. Infect. Immun. 79, 4061–4067 31 Tasteyre, A. et al. (2000) Phenotypic and genotypic diversity of the flagellin gene ( fliC) among Clostridium difficile isolates from different serogroups. J. Clin. Microbiol. 38, 3179–3186 32 Tasteyre, A. et al. (2001) Molecular characterization of fliD gene encoding flagellar cap and its expression among Clostridium difficile isolates from different serogroups. J. Clin. Microbiol. 39, 1178–1183 33 Wilcox, M.H. et al. (2012) Changing epidemiology of Clostridium difficile infection following the introduction of a national ribotyping-based surveillance scheme in England. Clin. Infect. Dis. 55, 1056–1063 34 Goorhuis, A. et al. (2008) Clostridium difficile PCR ribotype 078: an emerging strain in humans and in pigs? J. Clin. Microbiol. 46, 1157– 1158 35 Cairns, M.D. et al. (2012) The continually evolving Clostridium difficile species. Future Microbiol. 7, 945–957 36 Schneeberg, A. et al. (2013) Presence of Clostridium difficile PCR ribotype clusters related to 033, 078 and 045 in diarrhoeic calves in Germany. J. Med. Microbiol. 62, 1190–1198 37 Barketi-Klai, A. et al. (2014) The flagellin FliC of Clostridium difficile is responsible for pleiotropic gene regulation during in vivo infection. PLoS ONE 9, e96876 38 Cartman, S.T. et al. (2012) Precise manipulation of the Clostridium difficile chromosome reveals a lack of association between the tcdC genotype and toxin production. Appl. Environ. Microbiol. 78, 4683–4690 39 Dupuy, B. et al. (2008) Clostridium difficile toxin synthesis is negatively regulated by TcdC. J. Med. Microbiol. 57, 685689 40 Burns, D.A. et al. (2010) The diverse sporulation characteristics of Clostridium difficile clinical isolates are not associated with type. Anaerobe 16, 618–622 41 Solomon, K. (2013) The host immune response to Clostridium difficile infection. Ther. Adv. Infect. Dis. 1, 19–35 42 Kelly, C.P. and Kyne, L. (2011) The host immune response to Clostridium difficile. J. Med. Microbiol. 60, 1070–1079 7
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Review 43 Ghose, C. (2013) Clostridium difficile infection in the twenty-first century. Emerg. Microbes Infect. 2, e62 44 Hayashi, F. et al. (2001) The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature 410, 1099–1103 45 Pe´chine´, S. et al. (2005) Variability of Clostridium difficile surface proteins and specific serum antibody response in patients with Clostridium difficile-associated disease. J. Clin. Microbiol. 43, 5018–5025 46 Pe´chine´, S. et al. (2007) Diminished intestinal colonization by Clostridium difficile and immune response in mice after mucosal
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immunization with surface proteins of Clostridium difficile. Vaccine 25, 3946–3954 47 Jarchum, I. et al. (2011) Toll-like receptor 5 stimulation protects mice from acute Clostridium difficile colitis. Infect. Immun. 79, 1498–1503 48 Yoshino, Y. et al. (2013) Clostridium difficile flagellin stimulates Tolllike receptor 5, and toxin B promotes flagellin-induced chemokine production via TLR5. Life Sci. 92, 211–217 49 Rutherford, K. et al. (2000) Artemis: sequence visualization and annotation. Bioinformatics 16, 944–945
Review
Microbial Translocation
The role of flagella in Clostridium difficile pathogenicity Emma Stevenson, Nigel P. Minton, and Sarah A. Kuehne Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK
Clostridium difficile is widely publicised as a problem in the health-care system. Disruption of the normal gut microbiota by antibiotic therapy allows C. difficile to colonise the colon. On colonisation, C. difficile produces two toxins that lead to disease, with symptoms ranging from mild-to-severe diarrhoea, to fulminant and often fatal pseudomembranous colitis (PMC). How C. difficile establishes initial colonisation of the host is an area of active investigation. Recently there has been increased research into the role of C. difficile flagella in colonisation and adherence. Novel research has also elucidated a more complex role of flagella in C. difficile virulence pertaining to the regulation of toxin gene expression. This review focuses on new insights into the specific role of C. difficile flagella in colonisation and toxin gene expression. Importance of flagella to bacteria The formation of flagella by many bacterial species provides them with the ability to be motile, to colonise abiotic or biotic surfaces, to optimise growth and survival, and to evade desiccation in a hostile host environment [1]. The flagellum comprises a molecular motor, a hook–basal-body complex, and a rotating filament and is considered to be a specialised type III secretion system derived from a common ancestral structure [2] (Figure 1). The components of the flagellum work together to drive bacterial motility. The motor proteins provide the power to the hook–basal-body structure to drive flagellum rotation. The basal body is an integral membrane protein complex [3] that also serves as a passive structure allowing the translocation of other structural flagellar proteins outside the cell [2]. Once these proteins have been assembled, the hook structure acts as a universal joint to which the filament proteins can be assembled in a helical manner. The power provided by the motor proteins can thus drive the rotation of the flagellum filament, providing the bacterium with motility [3]. Bacterial flagella have been implicated in contributing to bacterial pathogenesis by: (i) promoting adherence to host cells; (ii) providing force-driven motility to nutrients; (iii) promoting biofilm formation; (iv) facilitating translocation of virulence factors across cell membranes; and Corresponding author: Stevenson, E. ([email protected]). Keywords: flagellar gene regulation; toxin expression. 0966-842X/ ß 2015 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2015.01.004
(v) acting as immunomodulators by triggering proinflammatory cytokines through the Toll-like receptor 5 (TLR5) signalling pathway [2]. Flagellum-driven motility is important to many gastrointestinal pathogens including Campylobacter jejuni, Vibrio cholerae, and Helicobacter pylori [4]. Mutations in flagellar genes in these bacteria have rendered strains less able to colonise the host [5,6], unable to establish persistent infections [7], and less able to adhere to small intestinal epithelial cells [8] and flagella mutants in C. jejuni are deficient in the secretion of certain virulence proteins [9]. Thus, as C. difficile occupies an environmental niche similar to that of these intestinal pathogens, the flagella of C. difficile may also play an important role in colonisation and pathogenicity. C. difficile is the causative agent of hospital-acquired antibiotic-associated diarrhoea in high-risk patients over the age of 65 years. Reports show that C. difficile infection (CDI) is 12–14 times more common than other widely publicised infections such as methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia [10]. Disruption of the normal colonic flora via antibiotics allows the establishment of CDI in the colon resulting in symptoms ranging from mild-to-severe diarrhoea to fulminant and often fatal PMC. Until recently C. difficile flagella have primarily been implicated in the colonisation of the host [11]. However, the contribution of flagella to the pathogenesis of C. difficile is complex and not yet fully understood. There is increasing scientific evidence to suggest that flagella play a more direct role in virulence, via modulation of toxin expression [4,12] rather than simply providing force-driven motility towards nutrients available in the gut. In this review the importance of flagella to the pathogenicity of C. difficile is discussed. Genetic organisation of C. difficile flagellar genes Many of the genes and proteins that govern the structural integrity and motion of flagella in other bacterial species have been identified in strains of C. difficile 630 and R20291 (BI/NAPI/027, hypervirulent ribotype) (Figure 1). Expression of the flagellar genes in all bacteria is temporal and regulated in a hierarchical manner [13]. For many bacteria, activation of flagellar gene transcription is governed by the master transcriptional regulator FlhDC [13]. However, many alternative master regulators have been found in other bacteria and include CtrA, VisNR, FleQ, FlrA, FlaK, LafK, SwrA, and MogR [14]. Interestingly, a master Trends in Microbiology xx (2015) 1–8
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Filament
F1
293002
FliD Filament cap
Flagellar assembly
CD0226 fliN CD0230
F2
304766
FlgK Hook Proximal rod FlgF
FlgG
Outer membrane
FlgH FlgI
Pepdoglycan layer
Basal body
MotB Cytoplasmic membrane
FliE
MotA
Motor/switch
FlhA FliH FliI FliO FliP
Type III secreon system
FlgN
FliJ
FliS
FlgA
FliT
FlhB
308251
CD0240
fliC F3
309272
flgC fliF
CD0241 CD0243
FliK FlgD
FlgE
CD0238
fliW fliS1 fliD
CD0242 CD0244
Hook-filament juncon
304049
csRA fliS2
CD0227 flgM flgK
FliC
FlgL
flgL
flgB fliE
fliH fliG
flil
F3 flgD
fliJ
Distal rod
flbD
motB
fliZ
fliQ
flhA
L ring
fliK
P ring FlgB
FlgC
FliF
MS ring
FliG FliM FliN
C ring
flgE
Proximal rod
motA
fliP
fliL
F3 flhG flhF
333302
CD0267 flgG1 fliA
flhB/fliR
CD0272
fliM
CD0267A flgG
fliN TRENDS in Microbiology
Bacterial chemotaxis
FliQ FliR FlhC FlhD FlgM
DNA O
Early gene products
DNA O
Late gene products
Figure 2. Genetic organisation of the flagellar operon in Clostridium difficile 630 (drawn to scale). Black arrows represent open reading frames with gene annotations above or below the respective genes. The three regions of the flagellar regulon are indicated by a solid bracket above the gene locus (F1, latestage flagellar genes; F2, flagellar glycosylation genes; F3, early-stage flagellar genes). Broken arrows below the flagellar locus mark the location of putative fliA promoter sites. White triangles represent insertion sites for ClosTron mutation and grey shading represents noncoding regions of DNA. Reprinted, with permission, from [4].
02040 6/23/10 (c) Kanehisa laboratories
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Figure 1. Representative structural organisation of a flagellum in a Gram-negative bacterium. The integral basal-body complex allows structural and filament proteins to be translocated out of the cell. The hook structure anchors the filament proteins to the basal body, allowing the formation of a mature flagellum. Rotation of the fully formed flagellum is powered by the internal motor and switch proteins attached to the basal-body complex. The flagellar genes (highlighted in green) indicate those that have been identified and/or characterised in Grampositive Clostridium difficile 630 and R20291. Reproduced from http://www. genome.jp/kegg-bin/show_pathway?org_name=cdf&mapno=02040&mapscale= &show_description=hide.
regulator of flagellar expression in C. difficile has not yet been characterised and this raises the question of whether flagellar gene activation is governed by transcriptional regulators involved in other cellular processes. Genes that govern the assembly of C. difficile 630 flagella are organised into three distinct operons known as the F1, F2, and F3 regulons [15] (Figure 2). The hierarchy of transcription of the flagellar genes in C. difficile is proposed to start with the early-stage genes located in the F3 region (Table 1 and Figure 2). Transcriptional control of the F3 regulon in C. difficile has not yet been fully defined and remains an area for future research. However, the genes in the F3 regulon are orthologous to the class II flagellar genes found in Bacillus subtilis, which are cotranscribed by an RNA polymerase and the vegetative sigma factor 70 (s70) homolog, sA [4,16]. A putative sAlike promoter sequence has been identified upstream of flgB in C. difficile 630 [17]. Therefore, the F3 region of
C. difficile might be under similar regulation via this promoter [4,17]. In strains of C. difficile from the ribotype 078 group, the F3 region is missing altogether [15] and this results in a completely nonmotile phenotype [11,15]. The F3 region comprises early flagellar genes such as fliA (Figure 2), which is an ortholog of s28, found in many Gram-negative bacteria [4,17]. In C. difficile three fliA promoter regions have been detected in the F1 region (Figure 2) and thus FliA is thought to regulate the latestage (F1) genes [4,17]. The F3 region is separated from the F1 region by an interflagellar gene region (known as the F2 region; Figure 2), which in C. difficile 630 comprises four flagellar-biosynthetic glycan genes [18] (Figure 3). However, there is genetic diversity at this region in the ‘hypervirulent’ ribotype 027 strains (including R20291) [15,19]. Four genes found in C. difficile strain 630 have been replaced by six genes in R20291 and CD196 and in other C. difficile strains such as M120, which is a 078 ribotype (Figure 3). Recent comparative studies of the F2 region have revealed that only C. difficile 630 and strains in the ribotype 017 lineage share this four-gene F2 operon [20]. The late-stage flagellar genes are encoded in the F1 regulon and are orthologous to class III flagellar genes of other bacteria [13,21]. Transcription of these genes cannot occur until all of the protein products of the F3 region have
Table 1. Positions and genes of the flagellar operons in the Clostridium difficile 630 genome [15] Flagellar regulon F1 (late-stage genes) F2 F3 (early-stage genes) 2
Position (bp) 293002–304049 304766–308251 309272–333020
Gene region CD0226–CD0240 CD0241–CD0244 flgB–CD0272
Genes (not all) encoded in each operon fliC, fliD, and a putative glycosyltransferase Flagellar-biosynthetic glycan genes fliF, fliG, fliM flhR, fliR fliA s28 homolog
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(A) F2 region of C. difficile 630
(B) F2 region of C. difficile R20291
(C) F2 region of C. difficile CD196
(D) F2 region of C. difficile M120
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Figure 3. Organisation of the F2 flagellar region using whole-genome visualisation via Artemis [49] in (A) 630, (B) R20291, (C) CD196, and (D) M120 Clostridium difficile strains. Blue boxes represent coding regions of flagellar glycan genes. In 630, the F2 region (in red box) comprises genes CD0241–CD0244, identified as flagellar glycan genes (Table 1). In R20291 and CD196 (B,C), these genes are replaced by six genes (in red boxes) including a glycosyl transferase (family 2), two putative uncharacterised proteins, a putative carbamoyl phosphate synthetase, and a putative ornithine cyclodeaminase [15].
been assembled correctly. The F1 region is proposed to be kept inactivated by the alternative sigma factor FlgM (anti-s28) [17]. Once the proteins encoded by the F3 region have been assembled, FlgM is excreted through the cell membrane and FliA activation of the F1 region can begin [13]. Microarray data have provided evidence that there is decreased gene expression in the F1 and F2 region of a fliA mutant strain, providing evidence that FliA positively regulates the late-stage flagellar genes [17]. The F1 region of the genome encodes the genes for the flagellin FliC and the protein cap FliD (Table 1 and Figure 2), which are structural proteins essential to the formation of fully functional flagella. Divergence of the F1 region in many ‘hypervirulent’ ribotype 027 strains has been shown [15,19]. This diversity is believed to account for the differences in motility between C. difficile 630 and R20291 [15]; Stabler et al. [15] observed that C. difficile 630 was less motile than R20291. It is pertinent to note that differences in motility and number of flagella have been observed between clonal isolates of C. difficile R20291 [12,15,22]. The study of Stabler et al. [15] revealed R20291 to be more motile than 630; however, research by Baban et al. [12] has shown R20291 to be less motile than 630 and to have one polar flagellum, as opposed to Martin et al. [22], who observed multiple flagella in the R20291 strain. It is unclear what genetic changes in the R20291 isolates have contributed to the change in number of flagella and thus motility observed by different research groups. Others have observed motility differences between
isolates of the same 027 ribotype [23] and have also concluded that there is great difficulty in precisely defining the genetic differences that may contribute to differences in motility. Flagella glycosylation The glycosylation of bacterial flagella has been extensively studied in Gram-negative bacteria but only three genera of Gram-positive bacteria (Listeria spp., Clostridium spp., and Butyrvibrio spp.) have been shown to produce glycosylated flagellins [24]. In many Gram-negative bacteria, including the enteric pathogen C. jejuni, flagella glycosylation plays a role not only in flagellum assembly but also in virulence [18]. Inactivation of some of the glycosylation genes of C. jejuni suggests that lack of flagella glycosylation abolishes autoagglutination and decreases the ability of the bacterium to adhere to and invade epithelial cells, and these mutants also appear to be attenuated in the ferret model of infection [25]. In 2009, Twine et al. [18] deduced that the flagellin of C. difficile could be modified by O-linked glycan moieties. Their research demonstrated that the flagellin glycans of C. difficile were genetically distinct not only from other species of Clostridia, namely Clostridium botulinum, but also between different strains of C. difficile. While the flagellin of C. difficile 630 could be glycosylated with an N-acetyl hexosamine (HexNaAc) residue at up to seven sites, the spectrum of flagellin glycosylation in strains of 3
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Review the 027 ribotype (BI-1 and BI-7) was more complex. The flagellin from 027 ribotypes was modified via O-linkage to heterogeneous glycan moieties of up to five monosaccharide residues [18]. Further identification and characterisation of a specific glycosyltransferase gene (CD0240) from the F2 region of the flagellar operon of C. difficile 630 (Table 1) revealed it was involved in the glycosylation process. Inactivation of CD0240 led to loss of the surface-associated flagellin protein. This rendered the strain nonmotile; however, the strain still produced truncated polymerised flagella filaments [18,20]. Thus, for the first time flagellin glycosylation was shown to be important in C. difficile flagellum assembly. Whole-genome comparative studies have shown genetic differences in the F2 region (Figure 3) between C. difficile strains 630, R20291 CD196 (historic 027 ribotype) [15], and M120 (078 ribotype). Stabler et al. [15] proposed that specific differences in this region between C. difficile 630, R20291, and CD196 may translate into differences in autoagglutination. The R20291 ‘hypervirulent’ 027 ribotype showed significant differences in autoagglutination to C. difficile 630 (P < 0.05), in contrast to the historic 027 ribotype, which did not show any phenotype. Autoagglutination of bacterial flagellins is a pathogenic strategy employed by C. jejuni to facilitate virulence [25]. Autoagglutination of flagellin results in changes in antigenic specificity due to differential glycosylation of flagellin [25]. The divergence of the F2 region and differences in autoagglutination observed between the strains of C. difficile in the study of Stabler et al. [15] could be correlated with the fact that distinct spectrums of flagellin glycosylation patterns exist between C. difficile 630 and 027 strains, as observed by Twine et al. [18]. However, in C. difficile changes in autoagglutination may not directly be the result of changes in flagellin glycosylation, as it is a multilevel process and probably involves other surface proteins [15]. Further research has since characterised more of the genes from the F2 region of C. difficile 630 and a toxin Anegative epidemic ribotype 017 (RT017) strain and found them to be putative post-translational modification (PTM) genes. Individual mutants in three of the four PTM genes of the F2 region (CD0241, CD0242, and CD0244) resulted in loss of motility and showed a sedimentation phenotype in vitro [20] that was more extreme than that displayed by the flagellin ( fliC) mutant. In concordance with what was also observed by Twine et al. [18], mutants in these genes were also still found to produce flagellin; however, for each mutant differences in flagellin molecular weight were observed. These size differences were proposed to be due to changes in flagellin PTM caused by disruption of the genes [20]. Flagellin modification of the CD0241, CD0242, and CD0244 mutants was determined by mass spectrometry, which revealed that these mutants did indeed have altered glycopeptide structures leading to the differential flagellin molecular weight [20]. Thus, this evidence suggests that both the structural integrity and PTMs of flagella are important for the motility of C. difficile. However, further research outlined below has also demonstrated the importance of flagella PTMs in colonisation. 4
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The role of flagella in host colonisation Studies before the publication of the C. difficile 630 genome highlighted that the flagellin and flagella cap genes ( fliC and fliD) were involved in adherence to murine mucus. Tasteyre et al. showed that there was a tenfold decrease (P < 0.01) in adherence of nonflagellated strains versus flagellated strains to mouse caeca in vivo [11], indicating that flagella may be important for the colonisation of C. difficile in the host. While the work of Tasteyre et al. [11] has elucidated a small part of the role of flagella in C. difficile colonisation, research relied on the use of naturally occurring nonmotile strains. More recently, the development of targeted mutation technology such as ClosTron [26] and allele-coupled exchange (ACE) [27,28] has enabled researchers to target specific genes of the flagellar gene operon and gain a greater depth of understanding of the contribution that the flagella make to C. difficile pathogenicity. The use of this technology has recently implicated the importance of flagella in the later stages of biofilm formation [29], when a C. difficile R20291 fliC mutant showed decreased biofilm formation (P < 0.05) at day five of the experiment. Recent studies [4,12,30] have found that fliC and fliD mutants of 630Derm displayed increased adherence to Caco2 cells compared with the parental strain and this is contradictory to previous evidence that flagella do play a role in C. difficile colonisation [11]. However, a study by Baban et al. [12] showed that fliC and fliD mutants of strain R20291 exhibited decreased adherence to Caco2 cells (P < 0.05) and thus, for some strains, intact flagella may be necessary for adherence. The study of Baban et al. [12] also included a mutant with paralysed flagella (the motB mutant) to further define whether the observed differences were due to loss of intact flagellar structure or specific to the inactivation of certain flagellar genes. Co-challenge experiments in a diaxenic mouse model showed that both the motB and the parental C. difficile R20291 strain outcompeted the fliC mutant in adherence, with the fliC mutant showing significantly less adherence (P < 0.05) to mouse caeca than the motB mutant. They further concluded that, for C. difficile R20291, flagella may play a more significant role in bacterial adherence than initial motility-driven colonisation. The study of Baban et al. also suggested that the inverse relationship of flagellar gene mutation and adherence is true for strain 630Derm fliC and fliD mutants. Challenge of 630Derm fliC and fliD mutants in monoxenic mice revealed no difference in colonisation. Co-challenge experiments revealed that the parental strain outcompeted the fliC mutant at some time points and the 630Derm parental strain adhered less well to mouse caeca than the fliC mutant. Taking these findings together, they proposed that, in the 630Derm strain, flagella and motility are not essential to colonise mice but may provide a motility fitness advantage over nonflagellated strains in environments where the two coexist. Publication of the C. difficile 630 genome sequence [Reference Sequence (RefSeq) NC_009089)] and the progression of next-generation sequencing platforms have led to the publication of many more C. difficile genomes, facilitating genome-comparison studies. These studies
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Review have provided useful insights into genetic changes between strains, which have led to the recognition of altered motility, toxicity, and antibiotic-resistance phenotypes [15]. Whole-genome comparison studies of C. difficile isolates have been used to identify differences in the flagellar gene regions that might affect motility [15] and virulence [18]. These whole-genome comparison studies have again highlighted that flagella, although important, may not be essential for C. difficile colonisation and adherence. The publication of the genome of C. difficile M120 (ribotype 078; GenBank FN665653) has confirmed that this strain has complete loss of the F3 flagellar region while retaining the F1 region, and this has been corroborated in microarray data from phylogenetic studies [19]. Studies as early as 2000 have indicated that nonflagellated C. difficile serotypes retain transcription of fliC and fliD genes but the protein products have remained undetected [31,32]. Therefore, whole-genome sequencing and characterisation on a wider panel of 078 ribotypes would reveal whether loss of the F3 and conservation of the F1 region is a common phenomenon among these strains. The PCR ribotype 078 strains have recently been recognised by Public Health England (PHE) and the Clostridium difficile Ribotyping Network (CDRN) as one of the emerging C. difficile strains in England, with a significant (P < 0.05) [33] increase in prevalence of 3.9% from 2007 to 2010 (http://www.gov.uk/government/uploads/system/uploads/ attachment_data/file/347168/CDRN_2009_10_Report. pdf). The 078 strain has also been found in both humans [34,35] and farm animals such as calves [36] and pigs [34] and is thought to contribute to a more community-based source of infection. The emergence of this strain as a common circulating ribotype in health-care facilities therefore suggests that nonflagellated strains are as capable of causing infection as other, flagellated strains. More recently, mutations in PTM genes of the F2 region in C. difficile 630 were shown to display alterations in adherence to abiotic surfaces. More specifically, a mutation in the PTM gene CD0241 led to attenuation of colonisation and relapse of infection in a murine model [20]. This observation combined with other work suggests that both the structural integrity of FliC and its PTM status play a role in the adherence and colonisation of C. difficile during the infection process [4,12,20]. The evidence above suggests that flagellum-driven motility may not be essential to the survival and colonisation of C. difficile, but the role of the flagellum in pathogenicity cannot be overlooked, especially when recent evidence suggests there may be a link between flagellar regulation and toxin production. Flagellar and toxin gene regulation An initial indication of a link between flagellar gene regulation and toxin expression was shown when hamsters infected with C. difficile 630 fliC and fliD mutants succumbed to infection faster than those with the parental 630Derm strain [30]. The observations of faster time to death of hamsters infected with the fliC and fliD mutants also correlated with increased cytotoxicity of culture supernatants at 24 h in these strains. However, in vivo there were no observable differences in caecal toxin titres
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between hamsters infected with any of the strains. Therefore, while a direct link between toxin and flagellar expression could not explain in vivo outcomes [30], it left an intriguing area of research open to investigation by others into whether there is a direct link between toxin and flagellar gene expression. In recent years there has been strong evidence to suggest that the expression of flagellar genes is coupled to toxin gene regulation, as well as other genes involved in the early stages of bacterial colonisation and virulence [4,12,30,37]. Studies of flagellar and toxin gene expression in C. difficile 630Derm In 2012, Aubry et al. [4] examined the disruption of flagellar genes in C. difficile strain 630Derm. Disruption of some F3 genes, such as fliF, fliG, and fliM, decreased the expression of some of the genes of the C. difficile pathogenicity locus (PaLoc), including the gene responsible for positive regulation of toxins (tcdR), the toxin genes (tcdB and tcdA), and the accessory holin gene (tcdE). This also correlated with decreased toxin in supernatants of cultures. Similarly to Aubry et al., Baban et al. [12] reported that disruption of an F3 gene, flgE, resulted in a tenfold decrease in tcdA expression. Interestingly, Aubry et al. did not find any change in levels of expression of tcdC (debated as the negative regulator of toxins [38,39]) with mutants in any of the flagellar genes. They surmised that toxin gene regulation at the early- to mid-log phase of cellular growth is a more dynamic process than previously thought and may not be solely controlled by repression of tcdR by TcdC during vegetative growth. Recent transcriptomic and quantitative real-time (qRT)-PCR data published by ElMeouche et al. [17] revealed decreased tcdR, tcdA, and tcdB expression in a C. difficile 630 fliA (sigD) mutant. Bioinformatic analysis of the PaLoc revealed a fliA-like consensus sequence upstream of the tcdR promoter site and further characterisation of both the mutant and parental strains revealed that FliA directly activates tcdR expression by binding and directing the core RNA polymerase tcdR promoter. Thus, FliA mediates the regulation of toxin gene expression via TcdR [17]. Inversely, disruption of the F1 genes such as fliC increased the expression of toxin genes and the toxin found in culture supernatant [4]. Baban et al. [12] corroborated these findings and reported that expression of tcdA (deduced by qRT-PCR) was 44.4 times greater in a fliC mutant and 7.4 times greater in a fliD mutant than in the 630Derm control strain (P < 0.05). Aubry et al. also investigated a C. difficile 630 mutant in a glycosylation gene (CD0240) in the F2 region. Data indicated that this strain clearly had the same nonmotile phenotype as the 630 fliC mutant. However, nonglycosylated FliC was found to be secreted, which suggested the formation of a nonfunctional flagellum and supports the hypothesis of Twine et al. [18]. Toxin gene expression of the C. difficile 630 CD0240 mutant remained unchanged during their experiments, and perhaps direct changes or loss of FliC rather than loss of a functional flagellum is responsible for changes in toxin expression. If this were true, it would be interesting to see whether disruption of fliC in the 5
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Review 078 ribotypes had any significant impact on toxin production, since these strains have lost the entire F3 region but retained the F1 region. Studies of flagellar and toxin gene expression in C. difficile R20291 Data from studies of other virulence factors governing C. difficile pathogenicity, such as sporulation, have proved that different C. difficile ribotypes and even strains of the same type behave differently [40] and that interpretation of data for one strain should not be used to generalise about the characteristics of others. Thus, although there is good evidence for the coregulation of toxin and flagellar genes in C. difficile 630Derm [4,12], the link between the two in other strains, such as C. difficile R20291, is not so clear. Baban et al. [12] confirmed the findings of Aubry et al. [4], deducing that toxin gene expression was altered in mutants of flagellar genes in C. difficile 630. Baban et al. also sought to confirm whether this was true for other strains of C. difficile, namely the ‘hypervirulent’ 027 ribotype called R20291. Results from their experiments showed no difference in cytotoxicity between the parental strain of R20291 and any of the flagellar gene mutants. However, 70% of mice infected with a R20291 fliC mutant had succumbed to infection within 4 days but those challenged with the parental strain did not succumb to infection at all [12]. The findings of Baban et al. have prompted others to investigate the pleiotropic effects of a fliC mutation in R20291 during colonisation in mice. Using in vivo transcriptomic data gathered 14 h post-infection of germ-free mice with either a R20291 fliC mutant or the parental R20291 strain, Barketi et al. [37] showed that there was no difference in toxin gene expression between the fliC mutant and the parental strain of R20291. Furthermore, both parental and mutant strains of R20291 showed no differences in cytotoxicity. They also found that 310 genes were differentially regulated in the fliC mutant compared with the parental strain during this time period. Most of the genes differentially regulated were those involved in motility, membrane transport [including phosphotransferase systems (PTSs) and ATP-binding cassette (ABC) transporters], sporulation, and carbon metabolism. This suggests coregulation between the expression of flagellar genes and those involved in the early stages of colonisation by C. difficile and also that, in the R20291 fliC mutant, there may be a global increase in genes associated with virulence that is responsible for the greater fatality in mice, as observed by Baban et al. [12]. The interaction of flagella and the host immune system Recent publications have implicated the flagellar proteins in the regulation of toxin gene expression, culminating in the release of toxins, which leads to the pathology of C. difficile infection. The toxins disrupt the tight junctions of epithelial cells, altering their morphology and increasing cell permeability [41]. The effect of toxins on gut epithelia evokes a host inflammatory response, which facilitates intestinal damage, and results in the clinical presentation of disease, such as diarrhoea and PMC [42]. 6
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Bacterial flagellins are highly immunogenic [43] and in many Gram-negative and Gram-positive bacteria have been shown to activate an innate immune response via the TLR5 pathway [2,44]. Thus, the immune response elicited by C. difficile flagellin could also contribute to the pathology of infection. In 2005, two publications by Pechine et al. [45,46] demonstrated that antibody levels against FliC and FliD were increased in a patient control cohort compared with a cohort with active C. difficile infection. They concluded that these proteins were able to induce an immune response that could play a role in host defence. More recently, Jarchum et al. [47] deduced that exogenous C. difficile flagellin protects mice from lethal C. difficile infection by mechanisms that maintain the integrity of the intestinal epithelial barrier. They proposed that the response requires host TLR5 activation. However, in vivo the response is unlikely to occur until epithelial damage (presumably via the toxins) has occurred, due to the location of TLR5 expression on the basolateral surface of intestinal epithelial cells, and thus flagellin may not provide a protective immune response on active infection [47]. Yoshino et al. [48] expanded on the flagellin immune activation repertoire by deducing that epithelial cells could indeed be stimulated, via TLR5, by flagellin and that this stimulation induced activation of nuclear factor kappa B (NF-kB) and p38 and the production of interleukin-8 (IL-8) and chemokine (C–C motif) ligand 20 (CCL20). The implication of flagellin in the immune response could have importance in the development of novel vaccine candidates against flagellin. Protective antibodies to flagellin may not only delay or prevent colonisation but may also help reduce the inflammatory response and might contribute to a less severe outcome of infection. Concluding remarks Here we have highlighted the increasing importance of flagella in the pathogenicity of C. difficile. The dawn of genetic tools to manipulate the genome has facilitated a greater understanding of the role of the flagellum in colonisation, adherence, and the regulation of toxin production. However, the important questions and avenues of investigation that remain will facilitate greater understanding (Box 1). Insights into the evolution of nonmotile strains have strengthened the hypothesis that flagellum-driven motility is not necessary for in vivo colonisation. However, they may play a greater role in adherence to the host. Box 1. Outstanding questions Is there a difference in virulence between strains that are peritrichous or monoflagellated? What is the link between flagella and toxin expression in a wider panel of emerging Clostridium difficile strains? Do other flagellar genes or gene products directly regulate toxin expression? Are there any other virulence genes coregulated with flagellar genes in other C. difficile flagellar mutants or naturally occurring nonmotile strains? How do parental C. difficile strains and flagellar gene mutants effect the complex immune host–pathogen interactions that occur during infection and determine infection outcomes? What are the roles of distinct flagellar glycan structures in C. difficile pathogenicity?
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Review Clearly, there is mounting evidence to suggest an indistinct relationship between toxin production and flagellar gene regulation in C. difficile. Although the relationship is not yet fully understood, it is most certainly strain specific. Furthermore, this is yet another way in which C. difficile governs transcription of virulence factors. This is another example of how intricate and convoluted the transcriptional regulatory network of C. difficile is and how deciphering the precise mechanisms of regulation will continue to be a challenge. This presents another avenue of investigation for researchers who are already working on transcriptomic data to unravel the intricate control of toxin gene regulation in C. difficile. Understanding novel regulatory pathways of toxin production could in the future lead to novel therapies against this persistent hospital pathogen, disease due to which has potentially dire consequences for an elderly population of patients. Acknowledgements This work was supported by the National Institute for Health Research (NIHR) Digestive Disease Biomedical Research Unit Nottingham (NIHR 2012-2017). E.S. wrote the manuscript, E.S. and S.K. evaluated the manuscript, and N.M. supported the manuscript.
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