Type IV Pili and the CcpA Protein Are Needed for Maximal Biofilm Formation by the Gram-Positive Anaerobic Pathogen Clostridium perfringens

Abstract

The predominant organizational state of bacteria in nature is biofilms. Biofilms have been shown to increase bacterial resistance to a variety of stresses. We demonstrate for the first time that the anaerobic gram-positive pathogen Clostridium perfringens forms biofilms. At the same concentration of glucose in the medium, optimal biofilm formation depended on a functional CcpA protein. While the ratio of biofilm to planktonic growth was higher in the wild type than in a ccpA mutant strain in middle to late stages of biofilm development, the bacteria shifted from a predominantly biofilm state to planktonic growth as the concentration of glucose in the medium increased in a CcpA-independent manner. As is the case in some gram-negative bacteria, type IV pilus (TFP)-dependent gliding motility was necessary for efficient biofilm formation, as demonstrated by laser confocal and electron microscopy. However, TFP were not associated with the bacteria in the biofilm but with the extracellular matrix. Biofilms afforded C. perfringens protection from environmental stress, including exposure to atmospheric oxygen for 6 h and 24 h and to 10 mM H 2 O 2 for 5 min. Biofilm cells also showed 5-to 15-fold-increased survival over planktonic cells after exposure to 20 g/ml (27 times the MIC) of penicillin G for 6 h and 24 h, respectively. These results indicate C. perfringens biofilms play an important role in the persistence of the bacteria in response to environmental stress and that they may be a factor in diseases, such as antibiotic-associated diarrhea and gas gangrene, that are caused by C. perfringens.

Type IV Pilin Proteins: Versatile Molecular Modules

“…O'Toole and Kolter (304) first reported that P. aeruginosa mutants lacking T4P are deficient in biofilm formation. Since then, similar findings have been reported for a number of bacteria and archaea, including (but not limited to) V. cholerae, A. actinomycetemcomitans, Aeromonas caviae, Shewanella oneidensis, Legionella pneumophila, Caulobacter crescentus, Deinococcus geothermalis, C. perfringens, and Sulfolobus acidocaldarius (40,44,102,133,178,342,389,398,408,431).…”

“…All classes of T4P, and even the T2S hyperpseudopili produced upon overexpression of the major pseudopilin, can participate in biofilm development in a wide variety of bacterial and archaeal species by promoting initial (and often highly tenacious) adherence to surfaces, as well as cell-cell interactions (122,128,208,209,328,398,400). O'Toole and Kolter (304) first reported that P. aeruginosa mutants lacking T4P are deficient in biofilm formation.…”

Enhancement of Biofilm Formation on Pyrite by Sulfobacillus thermosulfidooxidans

“…Cell detachment might occur, which leads to the poor biofilm formation. In addition, many publications emphasized the importance of motility for the biofilm formation [21,22]. When cells were incubated with pyrite slices, large flat surfaces and reduced shearing forces provide a chance to facilitate their attachment on the surface.…”

NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

“…There is accumulating evidence that CcpA can also be involved in the control of virulence gene expression by several Gram-positive pathogens, including C. difficile and S. aureus (25)(26)(27)(28)(29). In C. perfringens, CcpA has been shown to control expression of the enterotoxin gene (cpe) and genes involved in type IV pilus formation and function (30,31). In addition to toxins, C. perfringens produces many enzymes, including three sialidases named NanJ, NanI, and NanH.…”