Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens

Abstract

Clostridium perfringens enterotoxin (CPE) is an important virulence factor for food poisoning and non-food borne gastrointestinal (GI) diseases. Although CPE production is strongly regulated by sporulation, the nature of the signal(s) triggering sporulation remains unknown. Here, we demonstrated that inorganic phosphate (P i ), and not pH, constitutes an environmental signal inducing sporulation and CPE synthesis. In the absence of P i -supplementation, C. perfringens displayed a spo0A phenotype, i.e., absence of polar septation and DNA partitioning in cells that reached the stationary phase of growth. These results received support from our Northern blot analyses which demonstrated that P i was able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0A expression, indicating that P i acts as a key signal triggering spore morphogenesis. In addition to being the first study reporting the nature of a physiological signal triggering sporulation in clostridia, these findings have relevance for the development of antisporulation drugs to prevent or treat CPE-mediated GI diseases in humans.Clostridium perfringens is a gram-positive, anaerobic, endospore-forming bacterium causing gastrointestinal and histotoxic infections in humans and animals (2, 6, 9, 17). The virulence of this bacterium largely results from its prolific ability to produce at least 15 different toxins (18). In addition, enterotoxigenic C. perfringens isolates produce a 35-kDa enterotoxin (C. perfringens enterotoxin [CPE]), whose synthesis is under a strict positive control of sporulation (3,5,6,9,17). In C. perfringens, the production of CPE is confined to the large compartment (mother cell) of the sporangium where cpe transcription is believed to be driven from the mother cell-specific forms of the RNA polymerase, RNA-E and RNA-K (30). The copious amount of CPE (as much as 10% or more of the total protein of the developing sporangium) is accumulated probably only in the cytoplasm of the mother cell compartment until its release when the mother cell lyses at the completion of sporulation to liberate the mature spore (17). The released CPE rapidly binds to protein receptors present on the apical surface of enterocytes and induces cell permeabilization with the concomitant appearance of the symptoms of enterotoxaemia, intestinal cramping, and diarrhea (2,17,18).Despite the key role of spores in CPE synthesis and in the dissemination and developing of clostridial diseases, very little is known at the molecular level about the regulatory mechanisms governing the formation of spores in clostridia (6,9,11,13,20,23). Although from genome sequence analyses it can be assumed that the mechanism of spore formation in Bacillus and Clostridium is conserved (21,24,25), the main differences reside at the level of the initiation of the sporulation process (24, 25). While orthologs for spo0A and the genes activated by Spo0AϳP, along with most of the spo genes that are subsequently expressed during the morphogenesis of the spore,...

Germination of spores of Clostridium difficile strains, including isolates from a hospital outbreak of Clostridium difficile-associated disease (CDAD)

“…To our knowledge, P i has not been reported to induce germination of bacterial spores, although P i at pH 5.5-6.7 induces sporulation of C. perfringens (Philippe et al, 2006). The precise mechanism of initiation of C. difficile spore germination by P i is not known.…”

Characterization of the Sporulation Initiation Pathway of Clostridium difficile and Its Role in Toxin Production

“…The precise nature of sporulation-inducing signal ligands has generally remained elusive. In C. perfringens, inorganic phosphate has been shown to be a signal capable of promoting spo0A expression, sporulation, and toxin synthesis (25), but any involvement of sporulation-associated sensor kinases in this process has yet to be demonstrated.…”

Carbon Catabolite Repression of Type IV Pilus-Dependent Gliding Motility in the Anaerobic Pathogen Clostridium perfringens

“…At various time points, 1 ml of each culture was centrifuged for 2 min at 9,000 rpm, and the pellet was stored frozen at Ϫ20°C until used. The cell pellets were resuspended with 1 ml of buffer Z (8.54 g of Na 2 HPO 4 , 5.5 g of NaH 2 PO 4 ⅐ H 2 O, 0.75 g of KCl, 0.25 g of MgSO 4 , and 1.4 ml of ␤-mercaptoethanol ⅐ 7H 2 O, pH 7, per liter) (13,28). Adequate aliquots of the resuspended cells were brought to a final volume of 730 l with buffer Z.…”

“…In this respect, diverse bacterial behaviors, such as biofilm development, sporulation, fruiting body formation, and surface-associated motility, are regulated by environmental, metabolic, and quorum-sensing signals (9,10,16,28,46). In particular, carbon catabolite regulation is a widespread phenomenon in bacteria where the expressions of a number of genes are regulated by the presence of a preferred carbon source, such as glucose or fructose (27,39,43).…”

“…Clostridium perfringens is a gram-positive, anaerobic, sporeforming bacterium that causes severe gastrointestinal and histotoxic infections in humans and animals (28,32,33). This pathogen has been traditionally described as a nonmotile bacterium, as no genes that encode flagellar proteins or genes involved in chemotaxis were identified in the complete genomic sequences of the three human-pathogenic C. perfringens strains 13, SM101, and ATCC13124 (26,36).…”