Sporulation and Germination in Clostridial Pathogens AIMEE SHEN,1 ADRIANNE N. EDWARDS,2 MAHFUZUR R. SARKER,3,4 and DANIEL PAREDES-SABJA5 1Department of Molecular Biology and Microbiology, Tufts University Medical School, Boston, MA 2Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 3Department of Biomedical Sciences, College of Veterinary Medicine, Oregon State University, Corvallis, OR; 4Department of Microbiology, College of Science, Oregon State University, Corvallis, OR 5Department of Gut Microbiota and Clostridia Research Group, Departamento de Ciencias Biolo gicas, Facultad de Ciencias Biologicas, Universidad Andres Bello, Santiago, Chile ABSTRACT As obligate anaerobes, clostridial pathogens IMPORTANCE OF SPORES TO depend on their metabolically dormant, oxygen-tolerant spore CLOSTRIDIAL PATHOGENESIS form to transmit disease. However, the molecular mechanisms Disease transmission by clostridial pathogens depends by which those spores germinate to initiate infection and then form new spores to transmit infection remain poorly on their ability to form aerotolerant, metabolically understood. While sporulation and germination have been well dormant spores before exiting their hosts (6). Since characterized in Bacillus subtilis and Bacillus anthracis, striking spores are highly resistant to extreme temperature and differences in the regulation of these processes have been pressure changes, radiation, enzymatic digestion, and observed between the bacilli and the clostridia, with even some oxidizing agents (7), they can persist for long periods of conserved proteins exhibiting differences in their requirements time and serve as environmental reservoirs for these and functions. Here, we review our current understanding of fi organisms (8). Spores from C. perfringens, C. botuli- how clostridial pathogens, speci cally Clostridium perfringens, fi Clostridium botulinum, and Clostridioides difficile, induce num, and C. dif cile can be isolated from diverse sporulation in response to environmental cues, assemble environments, including animal gastrointestinal tracts resistant spores, and germinate metabolically dormant spores and carcasses, wastewater, lawns, hospital rooms, and in response to environmental cues. We also discuss the direct relationship between toxin production and spore formation in these pathogens. Received: 15 January 2018, Accepted: 11 April 2018, Published: 19 December 2019 Editors: Vincent A. Fischetti, The Rockefeller University, New York, NY; Richard P. Novick, Skirball Institute for Molecular Medicine, NYU Medical Center, New York, NY; Joseph J. Ferretti, Department Notably, different mechanisms exist between these of Microbiology & Immunology, University of Oklahoma Health Science Center, Oklahoma City, OK; Daniel A. Portnoy, Department organisms for forming and germinating spores. Some of Molecular and Cellular Microbiology, University of California, of these differences reflect phylogenetic differences be- Berkeley, Berkeley, CA; Miriam Braunstein, Department of Microbiology and Immunology, University of North Carolina-Chapel tween the Clostridiaceae (represented by C. perfringens Hill, Chapel Hill, NC, and Julian I. Rood, Infection and Immunity and C. botulinum) and the Peptostreptococcaceae (rep- Program, Monash Biomedicine Discovery Institute, Monash resented by C. difficile)(1). Other differences reflect University, Melbourne, Australia genetic diversity within each species (2–4). C. botulinum Citation: Shen A, Edwards AN, Sarker MR, Paredes-Sabja D. 2019. Sporulation and germination in clostridial pathogens. Microbiol is the most divergent, being divided into four metabolic Spectrum 7 (6):GPP3-0017-2018. doi:10.1128/microbiolspec.GPP3- groups (groups 1 to IV) that effectively represent dif- 0017-2018. ferent species despite their shared production of botuli- Correspondence: Aimee Shen, [email protected] num toxin (5). © 2019 American Society for Microbiology. All rights reserved. ASMscience.org/MicrobiolSpectrum 1 Downloaded from www.asmscience.org by IP: 205.193.94.40 On: Wed, 22 Jan 2020 15:45:37 Shen et al. soil (8). Infections by these pathogens typically are ini- specific bile acids sensed in the mammalian gut; the tiated upon ingestion of spores, although C. perfringens resulting vegetative cells produced secrete glucosylating can also enter the body via contaminating wounds. toxins that are the primary cause of disease symptoms Upon sensing small-molecule germinants, spores from (20). Notably, antibiotic exposure sensitizes individuals these pathogens germinate and outgrow into toxin- to antibiotic-resistant C. difficile by removing the colo- secreting vegetative cells. nization resistance conferred by our gut microflora (21). Spores are critical to this infection process not only C. perfringens because they are essential for transmitting C. difficile C. perfringens causes two major human diseases: food infections (6) but also because they are inert to anti- poisoning and gas gangrene (also known as clostridial biotics and resist many commonly used disinfectants in myonecrosis [9]). Clostridial myonecrosis occurs when health care settings (22, 23). Accordingly, spores are spores from the soil enter muscle tissue, typically through easily detected in health care-associated environments a wound, while food poisoning arises when spores or (24) in addition to numerous other sites (8). vegetative cells in contaminated food are ingested. Un- like most clostridial pathogens, C. perfringens’ vegetative OVERVIEW OF SPORE FORMATION form can initiate infection when present at sufficiently high levels to survive passage through the stomach. The first morphological stage of sporulation is the for- C. perfringens causes disease by secreting a number of mation of a polar septum, which generates two mor- toxins; C. perfringens strains are subdivided into toxi- phologically distinct, but genetically identical, cells (Fig. 1) genic types A to E based on the combination of the (25). The larger mother cell engulfs the smaller forespore alpha-, beta-, epsilon-, and iota-toxins they produce. cell, leaving the forespore within the mother cell cytosol C. perfringens spores can be remarkably heat resis- surrounded by two membranes. A thick layer of modified tant, with some type A strains surviving boiling for >1 h peptidoglycan known as the cortex forms between the (10, 11). Spores from food-poisoning isolates exhibit two membranes, conferring spores with heat and ethanol greater resistance to heat (∼60-fold), cold, and oxidizing resistance (26, 27). A series of proteinaceous layers known agents (12, 13) than nonfoodborne isolates, suggesting as the coat assembles around the outer forespore mem- that their resistance properties facilitate their survival in brane and protects the spore against enzymatic and oxi- undercooked or improperly held food (10). dative insults. In C. difficile, an additional layer known as the exposporium assembles on the coat, but this layer is C. botulinum not present in all spore-forming organisms. C. botulinum causes a flaccid paralysis known as botu- The mother cell and forespore also coordinately pre- lism through the production of the potent neurotoxin, pare the forespore for dormancy. The mother cell pro- botulinum toxin (BoNT). C. botulinum strains are sub- duces large amounts of dipicolinic acid (pyridine-2,6- divided based on their production of one or more of dicarboxylic acid) complexed with calcium (Ca-DPA), seven BoNT types (A to G) (14, 15). Botulism typically which it pumps into the developing forespore in ex- results from the ingestion of preformed BoNT in con- change for water (28). The resulting partially dehydrated taminated foods, but ingestion of C. botulinum spores forespore cytosol prevents metabolism. The forespore in contaminated foods such as unpasteurized honey can produces large amounts of small acid-soluble proteins cause infant paralysis, particularly in those <1 year old (SASPs), which coat the chromosome, prevent tran- (16). Even in cases of botulism intoxication, the spore scription, and protect against DNA damage (26, 28). form is critical for contaminating food, where incom- Once the forespore completes its maturation, the mother plete sterilization or processing, such as during home cell induces lysis and releases the metabolically dormant canning, can create anaerobic environments that allow spore into the environment. C. botulinum spores to germinate and form toxin- producing vegetative cells that subsequently intoxicate SPORULATION INITIATION IN the food (17). CLOSTRIDIAL PATHOGENS C. difficile The decision to initiate sporulation requires that vege- C. difficile is a leading cause of antibiotic-associated tative cells recognize specific environmental and nu- diarrhea and pseudomembranous colitis worldwide (18, tritional signals and assimilate these cues into a robust 19). C. difficile-associated disease occurs when spores, response. All sporulating Firmicutes use the conserved ingested by susceptible hosts, germinate in response to transcriptional regulator, Spo0A, as a key checkpoint 2 ASMscience.org/MicrobiolSpectrum Downloaded from www.asmscience.org by IP: 205.193.94.40 On: Wed, 22 Jan 2020 15:45:37 Sporulation and Germination in Clostridial Pathogens FIGURE 1 Lifecycle of endospore formers. (A) Sporulation. Upon sensing certain envi- ronmental conditions, endospore formers activate Spo0A and initiate sporulation. The first morphological event is the formation of a polar septum, which creates a larger mother cell and smaller