A Sporulation Factor Is Involved in the Morphological Change of Clostridium perfringens Biofilms in Response to Temperature

Abstract

Biofilm formation has been associated with bacterial pathogenesis, such as nosocomial and chronic infections, as the resistance of biofilms to environmental stresses has increased. Clostridium perfringens is a Gram-positive spore-forming anaerobic pathogen. This organism survives antibiotic treatment through the formation of biofilms or spores, but the environmental and regulatory factors involved in the biofilm formation remain unclear. Here, we observed that temperature regulates C. perfringens biofilm morphology. At 37°C, C. perfringens adhered to the substrate surface and formed a flat, thin biofilm, herein referred to as adhered biofilm. However, at 25°C, this bacterium did not adhere and produced a threadlike extracellular matrix, forming a viscous, thick biofilm, herein referred to as pellicle biofilm. Pellicle biofilm formation requires the sporulation master regulator, Spo0A, and the toxin regulator, CtrAB, and is enhanced in the absence of the global repressor, AbrB. These transcriptional regulator genes are regulated by each other and temperature. Adhered-biofilm formation requires AbrB and pilA2, which encodes a component of type IV pili (TFP). TFP expression was activated at 37°C and regulated through Spo0A, AbrB, and CtrAB. These results indicate that the morphology of C. perfringens biofilm is dependent on temperature through the differential production of extracellular matrix and the activity of TFP. Moreover, pellicle biofilm formation is involved in sporulation and toxin production. Here, we demonstrated that clostridial biofilm formation is closely associated with sporulation and that the morphological change of the biofilms could play an important role in the pathogenesis of this organism.

The CpAL Quorum Sensing System Regulates Production of Hemolysins CPA and PFO To Build Clostridium perfringens Biofilms

“…Two different research groups recently showed that treatment of preformed biofilms with proteinase K, cellulose, or DNase I dispersed C. perfringens biofilms, suggesting the presence of proteins, ␤-1,4-linked polysaccharides, and eDNA (49,50). Our studies further demonstrated the presence of eDNA in the supernatant of biofilm cultures acting as a source of DNA to be incorporated into the matrix.…”

“…Furthermore, incubation at 37°C, but not at 25°C, upregulated levels of pilA2 mRNA, which encodes the main pilin subunit of the matrix-associated C. perfringens TFP (50). These data suggest that C. perfringens biofilms are differentially regulated.…”

“…Whereas the VirR/S system is the logical downstream candidate, since the VirS transmembrane protein appears to be the receptor for the AgrD pheromone (37), another transcriptional regulator called CtrAB has been recently described to indirectly (i.e., via an unknown factor) control the transcript levels of cpa and pfoA (74). A ctrAB-null mutant, however, produced wt biofilm biomass when incubated at 37°C but demonstrated some effects on biofilms formed at 25°C (50). Our data showing upregulated transcription in biofilm cells of CpAL-regulated cpa and pfoA genes, but not cbp, further reinforce our hypothesis that biofilms activate specific downstream signals in response to the CpAL-released AgrD pheromone.…”

“…The biofilm matrix of other pathogens, such as Streptococcus pneumoniae (61,62) or Enterococcus faecalis (63), is partially composed of DNA that has been excreted into the medium (i.e., eDNA). It has been recently reported that eDNA is present in the C. perfringens biofilm matrix (49,50). To assess whether C. perfringens cells release DNA to the extracellular milieu, DNA was purified from supernatants of cultures of C. perfringens type C strain CN3685.…”

“…Initial attempts to characterize C. perfringens biofilms demonstrated that biofilms are produced in vitro by some strains (48,49). The matrix has been recently studied and found to be made of some proteins, ␤-1,4-linked polysaccharides and extracellular DNA (eDNA) (49,50). A type IV pilus (TFP) is also required for biofilm formation; these pili were located on the biofilm matrix (48).…”