Gas gangrene-associated gliding motility is regulated by the Clostridium perfringens CpAL/VirSR system

Highlights

• Clostridium perfringens strains migrates onward using gliding motility that resembles the spread of bacteria during myonecrosis.

• Gliding motility is regulated by the CpAL/VirSR system, a quorum sensing system encoded by other pathogenic clostridia.

• Transcriptions of genes encoding PFO and CPA toxins implicated in the pathogenesis of gangrene were highly expressed in gliding bacteria.

Abstract

Clostridium perfringens strains cause a wide variety of human and animal disease, including gas gangrene or myonecrosis. Production of toxins required for myonecrosis, PFO and CPA, is regulated by the C. perfringens Agr-like (CpAL) system via the VirSR two-component system. Myonecrosis begins at the site of infection from where bacteria migrate deep into the host tissue likely using a previously described gliding motility phenotype. We therefore assessed whether gliding motility was under the control of the CpAL/VirSR regulon. The migration rate of myonecrosis-causing C. perfringens strain 13 (S13) was investigated during a 96 h period, including an adaptation phase with bacterial migration (∼1.4 mm/day) followed by a gliding phase allowing bacteria faster migration (∼8.6 mm/day). Gliding required both an intact CpAL system, and signaling through VirSR. Mutants lacking ΔagrB, or ΔvirR, were impaired for onward gliding while a complemented strain S13ΔagrB/pTS1303 had the gliding phenotype restored. Gene expression studies revealed upregulated transcription of pili genes (pilA1, pilA2 and pilT) whose encoded proteins were previously found to be required for gliding motility and CpAL/VirSR-regulated pfoA and cpa toxin genes. Compared to S13, transcription of cpa and pfoA significantly decreased in S13ΔagrB, or S13ΔvirR, strains but not that of pili genes. Further experiments demonstrated that mutants S13ΔpfoA and S13Δcpa migrated at the same rate as S13 wt. We demonstrated that CpAL/VirSR regulates C. perfringens gliding motility and that gliding bacteria have an increased transcription of toxin genes involved in myonecrosis.

Introduction

The Gram-positive, spore-forming bacterium Clostridium perfringens affects both humans and animals, by producing severe diseases such as food poisoning [[1], [2], [3], [4]], gastrointestinal syndromes, and enterotoxemias [3,5,6], gas gangrene, and numerous histotoxic infections [[7], [8], [9]]. With the exception of food poisoning caused by C. perfringens enterotoxin (CPE), a common characteristic in most toxigenic C. perfringens vegetative infections is the rapid progression of the disease to an often fatal outcome. Whereas the virulence of C. perfringens is directly related to the production of toxins, other virulence traits such as adhesion, biofilm formation and gliding motility should also contribute to an integrated, and highly successful, virulence mechanism. C. perfringens isolates are classified into seven pathogenic types (A through G) based on the production of typing toxins alpha (CPA), beta (CPB), enterotoxin (CPE), epsilon (Etx), iota (Itx) and/or NetB [2,[10], [11], [12], [13], [14]]. C. perfringens type A, for example, produces CPA whereas type C strains produce CPA and CPB. All toxinotypes make other biomedically important toxins such as perfringolysin O (PFO) or CPB2 [10].

C. perfringens type A causes near ∼90% of all gas gangrene cases [3,9,13]. Gas gangrene/myonecrosis is considered one of the most fulminant Gram-positive infections in humans and animals [8,9]. Infection starts at the site of a recent surgical wound or trauma. C. perfringens type A strains first attach to the disrupted epidermal epithelium, proliferate and produce toxins that necrotize the tissue [8,[15], [16], [17]]. Tissue destruction associated with C. perfringens infection progresses rapidly to involve an entire extremity [8,15,[18], [19], [20]]. Amputation remains the single best life-saving treatment, though mortality still remains high [8,9,16,17].

Production of C. perfringens alpha toxin (CPA), and to some extent PFO, has largely been implicated in clostridial myonecrosis [3,9,13,21,22]. For example, a C. perfringens type A strain [cpa⁺ and pfoA⁺] was highly virulent in a mouse model of clostridial myonecrosis, while isogenic cpa or pfoA mutants showed reduced virulence (including reduced tissue destruction) [18,20,23,24]. Elimination of both CPA and PFO production (a double cpa-pfoA toxin gene mutant) removed most of the histopathological features typical of clostridial myonecrosis [25].

We recently demonstrated that C. perfringens strains encode a functional agr-like (accessory gene regulator) quorum sensing (QS) system that controls production of most, but not all, toxins [26,27]. The C. perfringens Agr-like system (referred to as CpAL for simplicity) is related to the Agr system from S. aureus and other Gram positives [26,[28], [29], [30], [31]]. Signaling through CpAL requires a secreted pheromone encoded by the agrD gene which is processed into a functional cyclic peptide by a transmembrane protein encoded by agrB [26,27]. The CpAL system regulates in vitro production of CPA, and PFO in all studied toxin types including type A strains [26,[32], [33], [34]]. CpAL also regulates production of beta toxin (CPB) in type B and type C strains [32,33], and it is required to produce necrotizing enteritis in rabbit ileal loops by regulating intestinal levels of CPB [32].

C. perfringens strains form biofilm-like structures on host tissues and epithelia [[35], [36], [37], [38]]. Biofilms are bacterial communities surrounded by a matrix providing the mechanical stability, mediating their adhesion to surfaces, and forming a cohesive, three-dimensional polymer network that protect them from the host response and antibiotics [39]. In C. perfringens, the matrix is made of proteins, hemolysins, a type IV pilus (TFP), β-1,4-linked polysaccharides, and extracellular DNA (eDNA) [35,[40], [41], [42]]. Altough C. perfringens is an ubiquitous bacterium, studies by Obana et al. (2014) demonstrated that the biofilm density (i.e., biomass) increased at 37 °C compared to 25 °C with a phenotypically different composition of the biofilm matrix, indicating that there is a potential mechanism regulating the increased biomass [41]. Accordingly, we recently demonstrated that biofilms were regulated by the CpAL system, and more importantly, that transcription levels of toxin genes, cpa and pfoA, are increased in biofilm structures made by C. perfringens strain 13, when compared to planktonic cultures.

As mentioned earlier, gas gangrene caused by type A strains advances rapidly on the infected tissue. Whereas gangrene, and other histotoxic infections appear to be biofilm-related diseases. The rapid progression of bacteria on the infected tissue resembles a type of motility known as gliding motility [[35], [36], [37], [38]]. Studies by Varga et al. (2006) showed that gliding in C. perfringens strains was linked to the production of type IV pili (T4P) [38]. Subsequent studies demonstrated that C. perfringens strains isolated from several mammals, including humans, exhibit gliding motility [43]. In silico analysis found that C. perfringens strain 13 encodes genes of the T4P in a major cluster in the chromosome, including pilA1 and pilA2, which encode for major pilin subunits. There are two other “small” secondary gene clusters containing putative T4P genes [38] and genes encoding a putative sortase (SrtC)-dependent pili [44]. A similar arrangement of T4P genes has been observed in C. difficile [45]. Evidence of the potential role of the T4P in gliding motility includes studies of strains with mutations in genes encoded in secondary clusters, such as pilC (encoding a putative inner membrane core protein) and pilT (encoding a putative retraction ATPase). Both mutants showed reduced gliding motility [38].

Mendez et al. (2008) published evidence of a regulatory network controlling gliding motility. The authors demonstrated that gliding was repressed by incubating strains in agar plates containing glucose and other sugars [43]. A similar repression of virulence traits, including production of toxins, has been documented in planktonic cultures grown in the presence of glucose, indicating a common regulatory network controlling both gliding motility and toxin production [46]. In this study, we demonstrated that gliding motility was regulated by the quorum sensing CpAL system. Whereas CpAL controls production of toxins, the gliding motility phenotype did not require gangrene-associated toxins CPA and PFO as individual mutants showed a similar gliding rate as that of the wild-type (wt) strain. Moreover, we demonstrated that gliding bacteria produce hundreds times higher levels of the pfoA gene transcript linking PFO production and gliding motility to the central regulatory network mediated by CpAL.