Regulation of Neurotoxin Production and Sporulation by a Putative agrBD Signaling System in Proteolytic Clostridium botulinum

A significant number of genome sequences of Clostridium botulinum and related species have now been determined. In silico analysis of these data revealed the presence of two distinct agr loci (agr-1 and agr-2) in all group I strains, each encoding putative proteins with similarity to AgrB and AgrD of the well-studied Staphylococcus aureus agr quorum sensing system. In S. aureus, a small diffusible autoinducing peptide is generated from AgrD in a membrane-located processing event that requires AgrB. Here the characterization of both agr loci in the group I strain C. botulinum ATCC 3502 and of their homologues in a close relative, Clostridium sporogenes NCIMB 10696, is reported. In C. sporogenes NCIMB 10696, agr-1 and agr-2 appear to form transcriptional units that consist of agrB, agrD, and flanking genes of unknown function. Several of these flanking genes are conserved in Clostridium perfringens. In agreement with their proposed role in quorum sensing, both loci were maximally expressed during late-exponential-phase growth. Modulation of agrB expression in C. sporogenes was achieved using antisense RNA, whereas in C. botulinum, insertional agrD mutants were generated using ClosTron technology. In comparison to the wild-type strains, these strains exhibited drastically reduced sporulation and, for C. botulinum, also reduced production of neurotoxin, suggesting that both phenotypes are controlled by quorum sensing. Interestingly, while agr-1 appeared to control sporulation, agr-2 appeared to regulate neurotoxin formation.Clostridium botulinum neurotoxins are made up of seven structurally related but antigenically distinct (serogroups A to G) proteins which form heterocomplexes with other, nontoxic proteins and are the most potent toxins known to humans (29). The Gram-positive, spore-forming organism C. botulinum is a heterogeneous species consisting of four physiologically and phylogenetically distinct groups (11). In humans and various animal species, the toxin induces a potentially fatal condition known as botulism. Botulism is characterized by a progressive, descending, symmetrical paralysis, which initially affects the musculature innervated by cranial nerves before spreading through the rest of the body (29).Naturally occurring forms of botulism include food-borne, intestinal, and wound botulism. Food-borne botulism is the most common form and occurs when humans or animals ingest food or drink that contains the preformed neurotoxin. In humans, members of group I (proteolytic) and group II (nonproteolytic) C. botulinum are responsible for most of the cases observed. In contrast, intestinal and wound forms of botulism are a consequence of C. botulinum infection followed by toxin production in vivo. In humans, these forms of botulism are mostly caused by group I C. botulinum. Intestinal botulism, which is commonly seen in infants, occurs after ingestion of spores and subsequent colonization of the intestinal tract, whereas wound botulism develops as a consequence of localized tissue infection (13).The extremely high potency of the botulinum neurotoxin has triggered fears that it might be an attractive weapon for bioterrorists (38). C. botulinum has therefore developed renewed strategic importance post-9/11, particularly in the United States.In addition to toxin production, perhaps one of the most important virulence factors of C. botulinum is its ability to form heat-stable endospores. These spores are present in the environment, i.e., in soil, water, and dust, and are responsible for the survival of C. botulinum in cooked foods. However, in contrast to the well-studied organism Bacillus subtilis, little is known about initiation of sporulation or the factors that influence germination of the resulting C. botulinum endospores (22).To prevent botulism, it is imperative that we understand the environmental factors that affect the ability of the organism to grow and/or elaborate toxin, either in food or in the gastrointestinal tract. Various potential regulators of neurotoxin expression have been proposed. These include the availability of exogenous carbon and nitrogen sources and regulation by the botR gene, which shows characteristics of a DNA-binding protein (16).In several bacteria, toxins are known to be controlled by cell-cell communication (quorum sensing) and are thus often expressed in a cell-density-dependent fashion. This is achieved via accumulation of a secreted signal molecule in the environment. The concentration of this signal molecule, often termed an autoinducer, is thought to serve as a measure of the population density that has been achieved. Once a critical threshold concentration of autoinducer has been reached, a response is triggered, leading to concerted, population-wide changes in gene expression.Intriguingly, all recently completed genomes of C. botulinum contain putative homologues of the staphylococcal agr quorum sensing system. The staphylococcal system comprises four genes, agrC, agrA, agrB, and agrD, which, in Staphylococcus aureus, mediate the global regulation of a battery of virulence factors (see the review by Novick [19]). The secreted signal molecule involved (the autoinducing peptide [AIP]) is derived from an internal fragment of AgrD through the action of the AgrB transmembrane protein. In silico analysis of the completed C. botulinum genomes revealed the presence of two sets of genes encoding homologues of the staphylococcal AgrB and AgrD proteins. Equivalent genes are also present in other clostridial genomes (30, 31, 37) and have recently been shown to regulate toxin production in Clostridium perfringens (21, 37).Here we report the characterization of these systems in C. botulinum ATCC 3502 (group I) and C. sporogenes NCIMB 10696. The latter bacterium was used as a model system for initial experiments, as it is considered a nontoxigenic version of group I (proteolytic) C. botulinum (5).