Sporulation and Germination in Clostridial Pathogens · 2020-03-13 · Sporulation and Germination...

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Sporulation and Germination inClostridial 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, MA2Department 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 pathogensdepend on their metabolically dormant, oxygen-tolerant sporeform to transmit disease. However, the molecular mechanismsby which those spores germinate to initiate infection and thenform new spores to transmit infection remain poorlyunderstood. While sporulation and germination have been wellcharacterized in Bacillus subtilis and Bacillus anthracis, strikingdifferences in the regulation of these processes have beenobserved between the bacilli and the clostridia, with even someconserved proteins exhibiting differences in their requirementsand functions. Here, we review our current understanding ofhow clostridial pathogens, specifically Clostridium perfringens,Clostridium botulinum, and Clostridioides difficile, inducesporulation in response to environmental cues, assembleresistant spores, and germinate metabolically dormant sporesin response to environmental cues. We also discuss the directrelationship between toxin production and spore formationin these pathogens.

Notably, different mechanisms exist between theseorganisms for forming and germinating spores. Someof these differences reflect phylogenetic differences be-tween the Clostridiaceae (represented by C. perfringensand C. botulinum) and the Peptostreptococcaceae (rep-resented by C. difficile) (1). Other differences reflectgenetic diversity within each species (2–4). C. botulinumis the most divergent, being divided into four metabolicgroups (groups 1 to IV) that effectively represent dif-ferent species despite their shared production of botuli-num toxin (5).

IMPORTANCE OF SPORES TOCLOSTRIDIAL PATHOGENESISDisease transmission by clostridial pathogens dependson their ability to form aerotolerant, metabolicallydormant spores before exiting their hosts (6). Sincespores are highly resistant to extreme temperature andpressure changes, radiation, enzymatic digestion, andoxidizing agents (7), they can persist for long periods oftime and serve as environmental reservoirs for theseorganisms (8). Spores from C. perfringens, C. botuli-num, and C. difficile can be isolated from diverseenvironments, including animal gastrointestinal tractsand carcasses, wastewater, lawns, hospital rooms, and

Received: 15 January 2018, Accepted: 11 April 2018,

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, Departmentof Microbiology & Immunology, University of Oklahoma HealthScience Center, Oklahoma City, OK; Daniel A. Portnoy, Departmentof Molecular and Cellular Microbiology, University of California,Berkeley, Berkeley, CA; Miriam Braunstein, Department ofMicrobiology and Immunology, University of North Carolina-ChapelHill, Chapel Hill, NC, and Julian I. Rood, Infection and ImmunityProgram, Monash Biomedicine Discovery Institute, MonashUniversity, Melbourne, Australia

Citation: Shen A, Edwards AN, Sarker MR, Paredes-Sabja D. Sporulation and germination in clostridial pathogens. Microbiol

Spectrum (6):GPP3-0017-2018. doi:10.1128/microbiolspec.GPP3- 0017-2018.

Correspondence: Aimee Shen, [email protected]

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Published: 19 December 2019

2019.

© 2019 American Society for Microbiology. All rights reserved.

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soil (8). Infections by these pathogens typically are ini-tiated upon ingestion of spores, although C. perfringenscan also enter the body via contaminating wounds.Upon sensing small-molecule germinants, spores fromthese pathogens germinate and outgrow into toxin-secreting vegetative cells.

C. perfringensC. perfringens causes two major human diseases: foodpoisoning and gas gangrene (also known as clostridialmyonecrosis [9]). Clostridial myonecrosis occurs whenspores from the soil enter muscle tissue, typically througha wound, while food poisoning arises when spores orvegetative cells in contaminated food are ingested. Un-like most clostridial pathogens, C. perfringens’ vegetativeform can initiate infection when present at sufficientlyhigh levels to survive passage through the stomach.C. perfringens causes disease by secreting a number oftoxins; C. perfringens strains are subdivided into toxi-genic types A to E based on the combination of thealpha-, beta-, epsilon-, and iota-toxins they produce.

C. perfringens spores can be remarkably heat resis-tant, with some type A strains surviving boiling for >1 h(10, 11). Spores from food-poisoning isolates exhibitgreater resistance to heat (∼60-fold), cold, and oxidizingagents (12, 13) than nonfoodborne isolates, suggestingthat their resistance properties facilitate their survival inundercooked or improperly held food (10).

C. botulinumC. botulinum causes a flaccid paralysis known as botu-lism through the production of the potent neurotoxin,botulinum toxin (BoNT). C. botulinum strains are sub-divided based on their production of one or more ofseven BoNT types (A to G) (14, 15). Botulism typicallyresults from the ingestion of preformed BoNT in con-taminated foods, but ingestion of C. botulinum sporesin contaminated foods such as unpasteurized honey cancause infant paralysis, particularly in those <1 year old(16). Even in cases of botulism intoxication, the sporeform is critical for contaminating food, where incom-plete sterilization or processing, such as during homecanning, can create anaerobic environments that allowC. botulinum spores to germinate and form toxin-producing vegetative cells that subsequently intoxicatethe food (17).

C. difficileC. difficile is a leading cause of antibiotic-associateddiarrhea and pseudomembranous colitis worldwide (18,19). C. difficile-associated disease occurs when spores,ingested by susceptible hosts, germinate in response to

specific bile acids sensed in the mammalian gut; theresulting vegetative cells produced secrete glucosylatingtoxins that are the primary cause of disease symptoms(20). Notably, antibiotic exposure sensitizes individualsto antibiotic-resistant C. difficile by removing the colo-nization resistance conferred by our gut microflora (21).Spores are critical to this infection process not onlybecause they are essential for transmitting C. difficileinfections (6) but also because they are inert to anti-biotics and resist many commonly used disinfectants inhealth care settings (22, 23). Accordingly, spores areeasily detected in health care-associated environments(24) in addition to numerous other sites (8).

OVERVIEW OF SPORE FORMATIONThe first morphological stage of sporulation is the for-mation of a polar septum, which generates two mor-phologically distinct, but genetically identical, cells (Fig. 1)(25). The larger mother cell engulfs the smaller foresporecell, leaving the forespore within the mother cell cytosolsurrounded by two membranes. A thick layer of modifiedpeptidoglycan known as the cortex forms between thetwomembranes, conferring spores with heat and ethanolresistance (26, 27). A series of proteinaceous layers knownas the coat assembles around the outer forespore mem-brane and protects the spore against enzymatic and oxi-dative insults. In C. difficile, an additional layer known asthe exposporium assembles on the coat, but this layer isnot present in all spore-forming organisms.

The mother cell and forespore also coordinately pre-pare the forespore for dormancy. The mother cell pro-duces large amounts of dipicolinic acid (pyridine-2,6-dicarboxylic acid) complexed with calcium (Ca-DPA),which it pumps into the developing forespore in ex-change for water (28). The resulting partially dehydratedforespore cytosol prevents metabolism. The foresporeproduces large amounts of small acid-soluble proteins(SASPs), which coat the chromosome, prevent tran-scription, and protect against DNA damage (26, 28).Once the forespore completes its maturation, the mothercell induces lysis and releases the metabolically dormantspore into the environment.

SPORULATION INITIATION INCLOSTRIDIAL PATHOGENSThe decision to initiate sporulation requires that vege-tative cells recognize specific environmental and nu-tritional signals and assimilate these cues into a robustresponse. All sporulating Firmicutes use the conservedtranscriptional regulator, Spo0A, as a key checkpoint

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for integrating these signals. The response regulatorSpo0A initiates spore formation by activating or re-pressing the expression of genes encoding early spor-ulation regulators (6, 29–34), and its DNA-bindingactivity is directly controlled by phosphorylation. Inthe model organism B. subtilis, Spo0A phosphorylationis orchestrated by a complex regulatory network, knownas a phosphorelay, which consists of several orphansensor histidine kinases (KinA-E; orphan refers to his-tidine kinases that are not encoded beside a responseregulator) and two phosphotransfer proteins (Spo0Fand Spo0B) (35). The KinA to KinE kinases directlyphosphorylate Spo0F, which subsequently transfers thephosphate to Spo0B and finally to Spo0A (Fig. 2) (36).Antikinases and two classes of phosphatases inhibitSpo0A phosphorylation levels by either blocking kinaseactivity or stripping Spo0A or Spo0F of its phosphate(37). The complexity of this regulatory pathway func-

tions as a noise generator, creating heterogeneous levelsof Spo0A phosphorylation within a population such thatonly a portion of its population commits to sporulation(38–40).

Notably, the B. subtilis phosphorelay is absent inclostridial pathogens, since clostridia lack orthologs ofthe Spo0F and Spo0B phosphotransfer proteins (41, 42).Thus, Spo0A appears to be directly phosphorylated byhistidine kinases in clostridial organisms (32, 33, 43–46), although the kinases, regulatory pathway, and en-vironmental signals used to control clostridial Spo0Aphosphorylation remain largely uncharacterized.

C. difficileSporulation initiation has been most extensively studiedin C. difficile, where key Spo0A regulatory proteins havebeen identified (Fig. 3). An initial study identified threeorphan histidine kinases with significant homology to

FIGURE 1 Lifecycle of endospore formers. (A) Sporulation. Upon sensing certain envi-ronmental conditions, endospore formers activate Spo0A and initiate sporulation. The firstmorphological event is the formation of a polar septum, which creates a larger mother celland smaller forespore. The mother cell engulfs the forespore, and the two cells worktogether to assemble the dormant spore. Calcium dipicolinic acid (Ca-DPA) is synthesizedin the mother cell and transported into the forespore in exchange for water. The cortex isformed between the two membranes, and coat proteins polymerize on the surface of themother cell-derived membrane. Once the spore is mature, the mother cell lyses andreleases the dormant spore into the environment. (B) Germination. Upon sensing theappropriate small molecule germinants, the spore initiates a signaling cascade that leadsto activation of cortex hydrolases and core hydration, which is necessary for metabolismto resume in the germinating spore.


Sporulation and Germination in Clostridial Pathogens



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the B. subtilis family of sporulation-associated phos-photransfer histidine kinases (32). Loss of one of theseputative histidine kinases, CD2492, decreased sporeformation ∼3-fold in rich liquid media, while anotherkinase, CD1579, directly phosphorylated Spo0A in vitro(32). These results suggest that both CD2492 andCD1579 promote Spo0A activation. Loss of the thirdputative histidine kinase, CD1492, increases sporulation∼4-fold on solid sporulation media in a manner de-pendent on its conserved histidine residue (47), suggest-ing that CD1492 functions as a phosphatase rather thanas a kinase.

Interestingly, CD2492 does not necessarily alwayspromote sporulation, since a CD2492 mutant exhibitsincreased sporulation on solid sporulation media in-dependent of its conserved histidine residue (A. Edwardsand S. McBride, unpublished data). The discrepancy inCD2492 mutant sporulation phenotypes could be dueto differences in growth conditions and/or methodsfor measuring spore formation. The signals that con-trol kinase versus phosphatase activity of sporulation-associated histidine kinases are largely unknown, evenin B. subtilis; thus, the activity observed may depend onthe presence or absence of unidentified signals. Indeed,

one B. subtilis kinase mutant exhibits varying sporula-tion phenotypes depending on the growth conditionsused (48, 49). This contradiction highlights the impor-tance of assessing sporulation using multiple conditionsand verifying spore frequencies with at least two differ-ent methods (50).

Notably, sporulation-associated histidine kinasesdirectly control Spo0A phosphorylation through com-peting kinase and phosphatase activities in C. ace-tobutylicum and C. thermocellum, where at least onesporulation-associated histidine kinase in each speciesinhibits sporulation in vivo (44, 45), and a C. aceto-butylicum histidine kinase has been shown to dephos-phorylate Spo0A in vitro (45). Altogether, clostridialsporulation-associated histidine kinases appear to re-versibly regulate Spo0A phosphorylation and thus theonset of sporulation.

Spo0A activity in C. difficile is further modulated byan RRNPP family ortholog, RstA (regulator of sporu-lation and toxins) (51), which shares homology with theRap phosphatases that directly dephosphorylate Spo0Fin B. subtilis. RRNPP family members have multipleC-terminal tetratricopeptide repeat domains that bindquorum-sensing peptides and regulate the cognate

FIGURE 2 Sporulation initiation via Spo0A phosphorylation. Sigma factors are shown ascircles, histidine kinases and phosphatases as hexagons (adapted from Al-Hinai et al. [85]).Positive regulators are shown in green (with the exception of σK, which is shown in purple),and negative regulators are shown in red. In B. subtilis, the KinA-E orphan histidine kinasesphosphorylate the phosphotransfer protein Spo0F, the first component in the phos-phorelay (25). The Rap phosphatases remove phosphates from phosphorylated Spo0F.The phosphate is transferred from Spo0F to Spo0B to Spo0A. In C. difficile, the orphanhistidine kinases CD1579 and CD2492 appear to phosphorylate Spo0A (32), while CD1492likely dephosphorylates Spo0A (47). A more detailed description of C. difficile Spo0Aregulation is shown in Fig. 3. Although all the putative orphan histidine kinases with thepotential to phosphorylate Spo0A in C. perfringens and C. botulinum are shown, whetherthey act as positive or negative regulators remains unstudied. The stationary factor σH

activates expression of spo0A in B. subtilis and C. difficile (101), while σK activates spo0Atranscription in C. botulinum (74) and possibly C. perfringens, the latter of which inducessporulation during log-phase growth (65).


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N-terminal helix-turn-helix DNA-binding domain and/or Spo0A/Spo0F-binding domain (52, 53). RstA con-tains these three conserved regulatory domains, and anrstA mutant produces ∼20-fold fewer spores than thewild type, indicating that RstA promotes early sporula-tion events in C. difficile (29). Although the helix-turn-helix domain appears to be dispensable for RstA tomodulate sporulation (29), the putative Spo0A/Spo0F-binding domain may directly bind and control Spo0Aphosphorylation and/or dephosphorylation (A. Edwardsand S. McBride, unpublished data). Interestingly, in ad-dition to regulating sporulation, RstA represses motilityand toxin production (see below) (51). Homologs ofRstA are observed in both pathogenic and nonpathogenicclostridial organisms, including C. sordellii, C. perfri-ngens, C. botulinum, and C. acetobutylicum (51).

RRNPP systems in other spore formers use quorum-sensing peptides to control activity of the regulatorand, thus, sporulation initiation. For example, B. subtilisRap phosphatase activity is inhibited by quorum-sensing

peptides imported by the conserved oligopeptide per-meases Opp and App, promoting sporulation (32, 34–36). Further, C. acetobutylicum sporulation is affectedby the deletion or overexpression of genes encodingRRNPP-type regulators, which are located adjacent toputative quorum-sensing peptide genes (54). However, itis unclear whether C. difficile RstA activity is regulatedby quorum-sensing peptides. Regardless, peptide trans-port coordinates the onset of sporulation in C. difficile,since the loss of Opp and App increases C. difficilesporulation ∼20-fold (55). These results suggest thatthe peptides imported by C. difficile Opp and App serveas nutrients rather than quorum-sensing molecules suchthat C. difficile opp and app mutants may initiate sporu-lation earlier due to decreased nutrient acquisition.

Metabolic cues regulate the initiation of C. difficilesporulation because the global regulators CcpA andCodY directly control the expression of genes encodingearly sporulation regulators. CcpA senses carbon avail-ability (56, 57), and CodY senses GTP and branched-

FIGURE 3 Regulatory pathway controlling Spo0A activation in C. difficile. Early sporula-tion factors experimentally determined to function as positive regulators of Spo0A arehighlighted in green, and those that inhibit Spo0A are highlighted in red (32, 47, 51, 55, 56,59, 61, 62, 101). Hexagons indicate histidine kinase/phosphatases, rounded rectanglesdemarcate transcription factors, and circles highlight sigma factors. Red lines indicatenegative regulation, and black lines indicate positive regulation. SinR and SinR′, C. difficileorthologs for regulatory proteins characterized in B. subtilis (gray), were recently shown topromote sporulation (60). Solid lines indicate defined regulatory interactions, and dashedlines suggest proposed, and potentially indirect, regulatory effects. Branched-chain aminoacids are a CodY cofactor (59), and their precursors are likely imported primarily throughthe Opp and App oligopeptide transporters (55, 61). CcpA-independent carbon-specificregulation is not shown (56). The reciprocal transcriptional regulation of early sporulationfactors by Spo0A has also been omitted for simplicity (100).





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chain amino acid levels (58, 59). CcpA directly repressesthe expression of spo0A and the opp and sigF operons(sigF encodes the first sporulation-specific sigma factorto be activated). CcpA also indirectly downregulatestranscription of sinR (56), which encodes an earlysporulation regulator that enhances sporulation (60).Not surprisingly, sporulation is increased ∼10-fold ina ccpA mutant (56), providing further evidence thatC. difficile initiates sporulation in response to nutrientdeprivation. Similarly, CodY downregulates transcrip-tion of sinR and the opp operon, and a codY mutantexhibits increased sporulation (61). However, CodY’seffect on sporulation is strain specific, given that anR20291 codY mutant produces ∼100-fold more sporesthan the parent strain, but the equivalent mutant in630Δerm produces only ∼2-fold more spores (61). In-terestingly, the addition of glucose reduces sporulationfrequency in a CcpA-independent manner (56), reveal-ing that additional regulatory pathways impact the tim-ing of sporulation in response to nutritional cues.

C. difficile also uses alternative sigma factors tocontrol sporulation initiation. SigH, which controls thetransition to stationary phase, is essential for sporula-tion and directly drives the expression of spo0A, simi-lar to B. subtilis (35), and CD2492, which modulatesSpo0A phosphorylation. In contrast, SigB, the gen-eral stress response sigma factor, inhibits predivisionalsporulation-specific gene expression and decreases sporeformation (62). SigB likely decreases Spo0A phosphor-ylation by reducing transcription of CD1579, whichencodes a Spo0A kinase (12), and increasing trans-cription of CD1492, which encodes a putative Spo0Aphosphatase (47).

The C. difficile genome encodes orthologs of addi-tional B. subtilis early sporulation factors, such as SinRI,Spo0J-Soj, Spo0E, and KipI/KipA (see reference 63 formore detail). However, if the trend from recent researchholds true, these uncharacterized C. difficile orthologslikely function differently from those in B. subtilis. In-deed, recent analyses indicate that the C. difficile sinRlocus encodes two sinR homologs, sinR and sinR′, withSinR′ antagonizing SinR function, analogous to B. sub-ilis SinI’s negative effect on SinR activity. In C. difficile,SinR functions as a DNA-binding transcriptional regu-lator and enhances sporulation, although the mechanismis unclear. There is some evidence that SinR regulatesearly sporulation events, because Spo0A-dependent geneexpression is significantly decreased in a sinRR′ mu-tant and is not rescued by spo0A overexpression (60).Furthermore, additional novel C. difficile regulators ofsporulation initiation are likely to be discovered given

that C. difficile encounters a diverse array of environ-mental conditions during growth in the gut, whichstrongly induces sporulation gene expression relative tobroth culture growth to promote survival outside of thehost (64).

C. perfringensIn contrast to most spore-forming organisms, C. per-fringens induces sporulation during the exponentialphase (65). Sporulation induction depends in part onsensing cell density through the Agr-like quorum-sensingsystem because an agrB mutant, which no longer makesa mature quorum-sensing peptide, has reduced Spo0Aprotein levels and produces ∼1,000-fold fewer sporesthan the wild type (66). Like C. difficile, C. perfringensCcpA and CodY modulate sporulation (67, 68), al-though unlike C. difficile, C. perfringens CcpA activatesrather than represses sporulation (68), with a C. per-fringens ccpA mutant making ∼60-fold fewer sporesthan the wild type. Nevertheless, similar to C. difficile,glucose strongly reduces C. perfringens sporulation fre-quency (∼2,000-fold) in a CcpA-independent manner.

CodY regulates C. perfringens sporulation in a strain-specific manner, again analogous to C. difficile. Loss ofCodY in the type D strain CN3178 increases sporeformation ∼10-fold (67, 68), whereas loss of CodY inthe food-poisoning strain SM101 reduces sporulation∼1,000-fold relative to the wild type (69). CodY likelyregulates sporulation by altering expression of abrB,which encodes a negative regulator of sporulation thatfunctions in a strain-specific manner (67). C. perfringensalso employs a regulatory RNA, virX, to repress spor-ulation by decreasing the expression of genes encodingsporulation-specific factors (70).

While the kinases that phosphorylate C. perfringensSpo0A remain unknown, six putative orphan histidinekinases have been identified in BLAST searches (71).Given that the bile acid deoxycholate induces C. per-fringens Spo0A phosphorylation and thus sporulation(72), these kinases may specifically respond to this bileacid. Similarly, inorganic phosphate induces C. per-fringens sporulation (73) and could stimulate kinaseactivity.

C. botulinumUnlike C. perfringens, C. botulinum sporulates duringthe transition from exponential to stationary phase (74).Like C. perfringens, the Agr-like quorum-sensing sys-tem is important for sporulation initiation, with agrBand agrD C. botulinummutants producing∼1,000-foldfewer spores (75). While the impact of CcpA and CodY


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on C. botulinum sporulation remains to be studied,five putative orphan kinases have been identified aspossible regulators of Spo0A (33). The CB01120 histi-dine kinase appears to phosphorylate Spo0A based onthe observation that production of CB01120 with wild-type Spo0A, but not with a nonphosphorylatable formof Spo0A, causes lethality when heterologously pro-duced in B. subtilis (33).

THE LINK BETWEEN SPORULATION ANDTOXIN GENE EXPRESSION IN C. DIFFICILEAlthough most Clostridium species initiate sporulationto survive unfavorable conditions, C. perfringens andC. botulinum directly couple toxin production (10, 30)to sporulation, while C. difficile coordinates these pro-cesses in a strain-specific manner (76).

C. perfringens Enterotoxin (CPE)C. perfringens type A causes food-poisoning and non-foodborne gastrointestinal disease (3). These diseasesare caused by the CPE toxin (13), which is encodedeither (cpe) chromosomally or on a large plasmid: mostfood-poisoning isolates carry chromosomal cpe, whilemost nonfoodborne isolates carry a plasmid-borne cpe(13, 77, 78). Food poisoning typically occurs whenchromosomal cpe isolates are ingested with food, whilenonfoodborne disease is primarily acquired by ingestingspores. Regardless, growth of C. perfringens in the smallintestine leads to CPE production and thus disease (3).

Numerous studies have demonstrated a strong cor-relation between spore formation and CPE production(79–82). The first genetic evidence linkingC. perfringenssporulation with CPE synthesis came from studiesshowing that mutants blocked at asymmetric divisionduring sporulation failed to produce CPE, while mutantsblocked at later stages of sporulation produced CPE(81). For example, a C. perfringens strain SM101 spo0Amutant cannot produce CPE (29), while inorganicphosphate (Pi) induces both sporulation and CPE pro-duction in the wild type (73).

Expression of the cpe gene is induced during spor-ulation, with the cpe transcript being detected only insporulating, but not vegetative, C. perfringens cultures(80, 83). cpe is transcribed from three promoters,named P1, P2, and P3, that are either σE- or σK-dependent (83). These promoters induce high levels ofCPE production during sporulation, with CPE consti-tuting up to 20% of the total protein present (84). σE

and σK are likely mother cell-specific (85), so CPEproduction appears to be restricted to the mother

cell cytoplasm, where it reaches sufficiently highconcentrations to induce paracrystalline inclusionbody formation (84). However, rather than being se-creted from sporulating cells, CPE is released uponmother cell lysis in the late stage of sporulation (84).Accordingly, sigK and sigE mutants of the food-poi-soning strain SM101 fail to express cpe (65), while asigF, but not a sigG, mutant exhibit defects in cpe ex-pression and CPE production based on reverse tran-scription PCR (RT-PCR) and Western blot analyses(86). Thus, only σF, σE, and σK are necessary for cpetranscription and CPE production (65), even though allfour sporulation-specific sigma factors are required forC. perfringens sporulation (10, 65).

Since CPE production is strictly sporulation depen-dent, factors that regulate early events of C. perfringenssporulation also regulate CPE production. Accordingly,the Agr-like quorum-sensing system in the nonfood-borne strain F5603 and CodY in the food-poisoningstrain SM101 both activate CPE production by acti-vating Spo0A and inducing σF production (66, 67). virX,a regulatory RNA that represses sporulation, accord-ingly reduces cpe expression and CPE production (70).Notably, while sporulation is crucial for CPE produc-tion, CPE is not required for sporulation, because cpemutants sporulate at wild-type levels (13, 87).

C. perfringens TpeL ToxinMany C. perfringens isolates encode a novel toxinnamed TpeL, which belongs to the family of large clos-tridial toxins (88) and is encoded both chromosomallyand on plasmids (89–91). While the contribution ofTpeL to C. perfringens pathogenesis is unknown, tpeLexpression is specifically induced during sporulationbased on transcriptional reporter studies (92). The tpeLpromoter region contains σE- and σK-dependent se-quences, and loss of σE strongly reduces tpeL expression(∼100-fold) (92), indicating that tpeL expression alsodepends on σE, similar to cpe. More recent analyses in-dicate that tpeL expression is induced by TpeR, a tran-scriptional regulator encoded in the same pathogenicitylocus (PaLoc) as tpeL; in these analyses, TpeL produc-tion was observed under conditions that promote vege-tative cell growth (93).

C. botulinum Type Neurotoxin (BoNT)Toxin production and sporulation coincide during thetransition from exponential to stationary phase, sug-gesting that these processes may be coregulated. TheAgr-like quorum-sensing system may coordinate theseprocesses as in C. perfringens, since toxin and spore





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formation are reduced in agrB and agrD mutants (75).In aquatic C. botulinum type E strains (94, 95), sporu-lation and BoNT production are directly linked, becauseloss of Spo0A prevents spore formation and reducesBoNT production >10-fold relative to the wild type (30).Spo0A likely directly regulates toxin production, be-cause Spo0A directly binds the botE promoter in vitro,which contains a Spo0A box (30). Interestingly, Spo0Ais the first neurotoxin regulator identified in C. botuli-num type E, which does not encode the alternative sigmafactor, BotR, which activates botulinum gene expressionin other strains (96).

C. difficile Glucosylating ToxinsC. difficile produces two large exotoxins, toxin A (TcdA)and toxin B (TcdB), which are critical for virulence(97–99). The regulatory links between C. difficile toxinproduction and sporulation are complex and appear tobe strain dependent. Spo0A represses toxin expressionin epidemic 027 ribotypes (6, 76), does not impact toxinexpression in the emerging 078 ribotype (76), and var-iably impacts toxin expression in the historic 012 ribo-type (6, 31, 32, 76, 100). In the 630 background (012ribotype), the stationary phase sigma factor, SigH, down-regulates toxin gene expression (101), and the phos-photransfer protein, CD1492, positively affects toxinproduction (47), presumably through indirect means.However, as with sporulation, nutrient availabilitystrongly influences toxin gene expression since aminoacids and glucose repress toxin gene expression throughthe global regulators CodY and CcpA, respectively (57,59, 102).

The most direct link between toxin gene express-ion and sporulation is RstA, the RRNPP family mem-ber discussed above, which inversely regulates toxinproduction and sporulation (51). RstA inhibits tran-scription of tcdA and tcdB by directly binding to thepromoters and inhibiting the transcription of tcdR andsigD (29; A. Edwards and S. McBride, unpublisheddata). tcdR encodes the sigma factor that directly ac-tivates toxin gene expression (103), while sigD encodesthe flagellar-specific sigma factor that also directs tcdRtranscription (104, 105). This multitiered regulationsuggests that RstA tightly controls toxin production.While RstA-dependent repression of toxin gene ex-pression requires its DNA-binding domain, its regula-tion of sporulation does not. Thus, RstA regulatessporulation and toxin gene expression through inde-pendent mechanisms (51). However, it remains unclearif this bifunctional protein links sporulation and toxinregulation in the same cell: single-cell analyses would

reveal whether toxin-producing cells also sporulate orwhether these important processes are asynchronous.Interestingly, the regulatory pathways between spor-ulation and toxin gene expression may be reciprocal insome C. difficile strains, because TcdR enhances sporeformation in R20291 (027 ribotype) but not 630(106).

Overall, several C. difficile regulators control bothsporulation and toxin gene expression, suggesting thatthe coordinate regulation of both of these processes isimportant for C. difficile survival.

THE SPORULATION TRANSCRIPTIONALPROGRAMOnce Spo0A is phosphorylated, it induces asymmetricdivision, which eventually leads to the activation of foursporulation-specific sigma factors: σF, σE, σG, and σK.These sigma factors are essential for sporulation (25, 85,107) because they coordinate the activation of distincttranscriptional programs within the mother cell andforespore, respectively, that culminate in the formationof a metabolically dormant spore. While the regulationof sporulation-specific sigma factors has been exten-sively analyzed in B. subtilis, the conserved sporulationsigma factors exhibit notable differences in their regu-lation and function in clostridial organisms relative toB. subtilis as well as between clostridial organisms. Wefirst describe the activation and functions of sporulation-specific sigma factors in B. subtilis and then comparethese properties with those in C. difficile, C. perfringens,and C. botulinum.

B. subtilis sporulation-specific sigma factors are post-translationally activated in a compartment-specific andsequential manner. σF is activated in the forespore,followed by σE in the mother cell; σE then activates σG

in the forespore, which subsequently activates σK inthe mother cell (Fig. 4) (25). Intercompartmental sig-naling regulates sporulation sigma factor activationand couples it to specific morphological changes (108).Spo0A induces the transcription of sigF and sigE inthe predivisional cell such that σF and σE are presentin both the mother cell and forespore, although bothsigma factors remain inactive until asymmetric divisionis complete. σF is first activated in the forespore when thepreferential activation of the SpoIIE phosphatase in theforespore leads to dephosphorylation of the anti-anti-sigma factor, SpoIIAA (109–111), which antagonizesthe anti-sigma factor, SpoIIAB. Inhibition of SpoIIABfrees σF to bind its target promoters, which include sigGand spoIIR. The resulting production of SpoIIR leads


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FIGURE 4 Diversity in the regulation of the transcriptional programs controlling sporu-lation in the Firmicutes. The temporal progression of sporulation is shown from top tobottom. Transcription factors and sigma factors are shown in bold, and proteins enclosedin boxes directly participate in signaling between the mother cell and forespore (dashedboxes indicate that trans-septum signaling has not been tested yet). Text color denoteswhether the factor has been detected at both the transcript and protein level (black), ateither the transcript or protein level (purple), or has not been tested yet at the transcript orprotein level (blue). Black arrows delineate transcriptional control of gene expression, redarrows indicate signaling pathways, dashed lines indicate that the regulatory relationshipremains unknown, and thick arrows demarcate notable points of divergence from thepathway defined in B. subtilis. AND gates are indicated. The figure is adapted from Fimlaidet al. (137) under Creative Commons BY 4.0.





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to σE activation because SpoIIR activates SpoIIGA, theprotease that removes σE’s inhibitory propeptide in themother cell (112–114).

Activated σE directs the transcription of genes (115)required for the mother cell to (i) engulf the forespore ina phagocytic-like process (spoIID, spoIIP, and spoIIM[116–118]), (ii) localize coat proteins to the forespore(119), (iii) activate σG in the forespore (spoIIIA operon[120]), and (iv) produce and activate σK in the mothercell (sigK, spoIIID, spoIVFA-B, ctpB, and spoIVCA[121–125]). The SpoIIIA proteins form a complex withGerM and the forespore-specific SpoIIQ to form achannel connecting the mother cell and forespore (126–129). This channel, also known as the “feeding tube,”is required to maintain transcriptional potential inthe forespore (126) and, thus, σG activity. SpoIIQ alsocontributes to σG activation by controlling the localiza-tion of SpoIIE, which likely antagonizes the σG-specificanti-sigma factor, CsfB (130). Thus, discrete anti-sigmafactors control the activation of σF and σG, respectively,in B. subtilis.

Engulfment increases σG activity, which couplesits transcriptional program to morphological changes(131). Activated σG directs the transcription of genes inthe forespore (132) required to (i) modify the cortex(133), (ii) prepare the forespore for dormancy (7), and(iii) activate σK via regulated proteolysis by the mothercell-localized protease SpoIVFB (125). Thus, proteolyticsignaling cascades induced by the forespore activate σE

and σK in B. subtilis.σK production depends on σE because σE activates

transcription of spoIIID, which encodes the transcrip-tional regulator that induces sigK expression (134). σE

also controls the expression of spoIVCA, which encodesthe site-specific recombinase necessary to mediate exci-sion of the skin element, a 42-kB prophage-like regionthat disrupts the sigK gene (122). Thus, multiple levelsof σK regulation control the precise timing of its activationin B. subtilis to ensure proper spore assembly, since acti-vated σK induces cortex and coat assembly genes (135).

Taken together, B. subtilis sporulation gene expres-sion is tightly controlled both spatially and temporally,with events in the forespore being coupled to events inthe mother cell. Sporulation sigma factor activation andfunction are further controlled by additional feedbackand feedforward loops to ensure that the timing ofsporulation gene expression is tightly coordinated withmorphological changes (136).

Notably, while most of the gene products that controlsporulation sigma factor activation in B. subtilis areconserved in the clostridia, the functions of some of these

gene products and the timing of their action are notalways conserved. Many of the regulatory loops thatfine-tune the timing of sporulation sigma factor activa-tion and function in B. subtilis are not conserved inclostridial organisms (42, 71) (Fig. 4), suggesting that(i) sporulation sigma factor activity may not be as tightlyregulated in clostridial pathogens relative to B. subtilisand/or (ii) additional regulatory pathways exist. Whilethis review focuses on sporulation sigma factor regula-tion in pathogenic clostridia, this process has also beenexamined in detail in C. acetobutylicum, which revealednotable differences in the regulation of these sigmafactors relative to B. subtilis (reviewed by Al-Hinai et al.[85]).

C. difficileSporulation sigma factor activation has been best stud-ied in C. difficile, where genome-wide transcriptionalanalyses of sigma factor mutants have defined theregulons of sporulation-specific sigma factors (137,138). These factors were shown to function in a com-partment-specific manner similar to that in B. subtilis(Fig. 4) through the development of SNAP tag-basedtranscriptional reporter constructs by the Henriquesgroup (139). Specifically, C. difficile σF and σG activity isrestricted to the forespore, while σE and σK activity isrestricted to the mother cell (139). This regulationresembles B. subtilis (25), with the exception that sigGexpression does not require σF and is likely activatedby Spo0A (137).

The mechanisms underlying bothC. difficile σF and σE

activation likely occur through a mechanism similarto that in B. subtilis based on gene conservation. Con-sistent with this notion, the σF-activating phosphatase,SpoIIE, and the σE-activating protein, SpoIIR, are es-sential for sporulation based on a transposon screen(140). Similar to B. subtilis, C. difficile SpoIIR is re-quired for pro-σE processing (138). However, unlikeB. subtilis, C. difficile σF is not essential for this pro-teolytic activation event because spoIIR is expressedfrom both Spo0A-dependent and σF-dependent pro-moters (138). Thus, C. difficile σE is partially active in asigF mutant (137, 138).

C. difficile σG and σK activation differs markedly fromthat in B. subtilis. Whereas σG activation depends on theSpoIIQ-SpoIIIA channel complex in B. subtilis, C. dif-ficile σG is active in the absence of this channel and itsassociated engulfment defects (141, 142). Notably, σG ispresent but inactive in the absence of σF (137), indicatingthat C. difficile σG activity is posttranslationally acti-vated in the forespore through an unknown mechanism.


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Unlike most spore-forming organisms, C. difficile σK

lacks the N-terminal inhibitory propeptide (143) thattethers B. subtilis pro-σK to the membrane (123). As aresult, C. difficile σK is active upon translation, sinceexpression of sigK from a tet-inducible promoter invegetative C. difficile allows σK-dependent genes to betranscribed in contrast to B. subtilis (144). However,similar to B. subtilis, C. difficile sigK transcription de-pends on the excision of a large skin element thatdisrupts the sigK gene (143) and is activated by σE andSpoIIID (138, 144). In contrast to B. subtilis, the geneencoding the excision recombinase, CD1231, is consti-tutively expressed rather than activated by σE (145).Instead, CD1231 activity is posttranslationally activatedby the CD1234 recombination directionality factor,whose production depends on both σE and SpoIIID.Thus, CD1231 and CD1234 control the timing of SigKproduction and enhance the fidelity of spore assembly(145), similar to B. subtilis (146).

C. difficile sporulation sigma factors generally controlmorphological processes similar to those in B. subtilis,with σF and σE being required for initiating engulfment,and σE and σK being required for coat assembly (139).Unlike B. subtilis, C. difficile σG, but not σK, is requiredfor cortex production, and C. difficile σG is required forengulfment completion at least during sporulation onsolid media (137).

Taken together, C. difficile sporulation sigma factormutants exhibit less intercompartmental signaling, withthe forespore line of gene expression requiring σF and σG,but not σE, and the mother cell line of gene expressionrequiring σE and σK, but not σG (107, 137–139) (Fig. 4).Furthermore, the timing of sporulation sigma factoractivation does not to appear to be as closely coupled tomorphological events as in B. subtilis (25, 107).

C. perfringens and C. botulinumThe regulation of sporulation sigma factors in C. per-fringens and C. botulinum has not been characterizedextensively, although gene conservation analyses indi-cate that the minimal machinery required for activatingthese sigma factors is conserved (Fig. 4) (136). C. per-fringens σE and σK both undergo proteolytic processingduring sporulation, similar to B. subtilis and C. difficile(65). However, in contrast to these organisms, the sigKgenes in both C. perfringens and C. botulinum do notcontain intervening skin elements. Notably, σK appearsto function at two stages during sporulation in boththese organisms, with the first stage regulating sporula-tion initiation. Indeed, sigK mutants in both organismsdo not appear to initiate asymmetric engulfment (65,

74), unlike in B. subtilis and C. difficile. Thus, the func-tion of σK as an early and late regulator of sporulation inC. perfringens and C. botulinum exhibits similarities toC. acetobutylicum, which uses σK to initiate sporulationthrough its activation of spo0A and during late stagesporulation (147).

In C. perfringens, sigK is expressed from two pro-moters at different stages of sporulation: the upstreamCPR_1739 promoter activates transcription during earlylog phase, in contrast to B. subtilis and C. difficile, whilea σE-dependent promoter controls late-stage sporulationgene expression (65), similar to B. subtilis and C. dif-ficile. C. botulinum sigK transcription is similarly bi-phasic, occurring during late log phase and late-stagesporulation (74). This latter phase of gene expressionoccurs from σF- and σE-dependent promoters (148).Consistent with σK being required early during sporu-lation in both these organisms, C. perfringens σK ac-tivates sigF and sigE expression (65), and C. botulinumσK activates spo0A and sigF expression (74). Further-more, sigK mutants in both organisms fail to completeasymmetric engulfment (65, 74), whereas sigE mutantsare stalled at this stage (65, 148). While transcriptionalanalyses indicate that σK acts upstream of σF, Westernblot analyses have revealed that σK is not detectable inthe absence of σF (86), suggesting that the production (orstability) of these two factors is interdependent.

Another major difference in sporulation gene regu-lation relative to B. subtilis andC. difficile is that σE doesnot activate spoIIID expression in C. perfringens andC. botulinum. Instead, C. botulinum spoIIID tran-scription is σF-dependent (149). Whether σF regulatesspoIIID expression in C. perfringens is unclear, butimmunoblotting indicates that SpoIIID production doesnot require σE or σK (65).

These observations raise the possibility that thesporulation sigma factors exhibit differences in theircompartment-specific activation in C. perfringens andC. botulinum relative to B. subtilis and C. difficile. In-deed, sigK is transcribed early during sporulation inC. perfringens (65), suggesting that σK may also activatesporulation in predivisional C. botulinum cells. Theseobservations raise the question of whether σK must beproteolytically activated at this early stage. Furthermore,since sigK expression is regulated by both σF and σE inC. botulinum (148), is σF activity (and by extensionSpoIIID activity) restricted to the forespore?

Summary of Sporulation RegulationClearly, major differences exist in the functions andregulation of conserved sporulation sigma factors in





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C. difficile, C. perfringens, and C. botulinum relativeto the pathways defined in B. subtilis. C. difficile’s reg-ulatory architecture exhibits greater similarity to B. sub-tilis than to C. perfringens and C. botulinum, whichappear relatively similar to each other. Further study ofC. perfringens and C. botulinum is needed to define theorder of sporulation sigma factor activation, their spe-cific regulons, and the location of their activity. Suchstudies could provide insight into the evolution of di-verse sporulation networks.

SPORE ASSEMBLYThe mechanisms by which spores are physically as-sembled in clostridial organisms also remain poorlydefined, although some progress has been made inC. difficile. This section focuses on factors requiredfor engulfment, coat, and exosporium assembly inC. difficile and the functions of specific coat andexosporium proteins. We also outline the role of thespore-specific small molecule, calcium dipicolonic acid(Ca-DPA), during C. perfringens and C. difficile sporeformation.

EngulfmentThe second major morphological event after asymmetricdivision is engulfment, whereby the smaller forespore isencircled by the mother cell, leaving the forespore free-floating in the mother cell cytosol surrounded by twomembranes (Fig. 1). In B. subtilis, engulfment is medi-ated by peptidoglycan synthesis machinery in the innerforespore membrane working in concert with peptido-glycan degradation machinery localized in the mothercell-derived outer forespore membrane (150, 151). Thedegradation complex consists of the SpoIIP and SpoIIDpeptidoglycan hydrolases (152, 153) in complex withthe transmembrane scaffolding protein, SpoIIM (154).Loss of any of these B. subtilis components preventsengulfment completion and thus heat-resistant sporeformation (116–118). Interestingly, C. difficile SpoIIMis largely dispensable for engulfment and heat-resistantspore formation, in contrast to B. subtilis, while C. dif-ficile SpoIIP and SpoIID are critical for these processes,similar to B. subtilis (155, 156) . While it is unclear whySpoIIM is dispensable forC. difficile engulfment, spoIIDand spoIIP are expressed in different cellular compart-ments in C. difficile relative to B. subtilis (156), whichmay obviate the need for SpoIIM to bring SpoIID andSpoIIP together. C. difficile SpoIIP and SpoIID never-theless have conserved enzymatic activities relative toB. subtilis (152, 155, 157), although SpoIIP undergoes

site-specific cleavage in C. difficile (156), in contrast toB. subtilis (158).

The conserved SpoIIQ-SpoIIIA channel is also re-quired for C. difficile engulfment, unlike in B. subtilis.In B. subtilis, the channel (also known as the “feedingtube”) connects the mother cell and forespore (120, 126,128) and serves as a back-up mechanism for engulfment(159). However, in both organisms the channel is es-sential for maintaining forespore health because theforespore becomes deformed on itself in channel mutants(127, 141).

Coat Assembly in C. difficileThe coat consists of∼80 proteins in B. subtilis (119) thatlocalize to the forespore and form a series of concentricproteinaceous shells around the forespore. However,given that only 25% of these proteins have homologs inC. difficile (160), different pathways likely control coatassembly in these two organisms. Furthermore, C. dif-ficile encodes only two of the nine coat morphogeneticproteins that function to recruit coat proteins to theB. subtilis forespore.

The two coat morphogenetic proteins shared betweenB. subtilis and C. difficile are SpoVM and SpoIVA.SpoVM functions as a landmark protein in B. subtilisby recognizing the positive curvature of the foresporemembrane and embedding itself within this membrane(161, 162). SpoVM recruits SpoIVA to the foresporeand is required for SpoIVA to use its ATPase activity(163) to polymerize around the forespore (161). SpoIVApreferentially localizes SpoVM to the outer foresporemembrane (164) and recruits the coat morphogeneticprotein, SpoVID, to the forespore by binding SpoVID’sC-terminal region A (164). These three proteins form thebasement layer of the B. subtilis coat (165). SpoVID inturn recruits the inner coat morphogenetic protein, SafA(166), and outer coat morphogenetic protein, CotE(167). B. subtilis spoIVA and spoVM mutants mis-localize the coat and fail to make cortex, so they cannotproduce heat- or chloroform-resistant spores (168–170).Furthermore, spoIVA and spoVM mutants are activelylysed by a bacilli-specific (171) quality control mecha-nism mediated by CmpA (172).

C. difficile spoIVAmutants resemble a B. subtilis IVAmutant in mislocalizing the coat and failing to produceheat-resistant spores (173, 174). However, in contrast toB. subtilis, C. difficile spoIVA mutants produce cortex(170, 174), consistent with the absence of CmpA in theclostridia (175). Nevertheless, it is unclear why C. dif-ficile spoIVA mutants are heat sensitive, given that theyproduce visible cortex.


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Unlike SpoIVA, C. difficile SpoVM is largely dis-pensable for spore formation. C. difficile spoVM mu-tants exhibit an ∼3-fold defect in heat- and chloroform-resistant spore formation, make cortex, and properlylocalize SpoIVA around the forespore in most cells, incontrast to the 6-log defect in heat and chloroform re-sistance observed in a B. subtilis spoVM mutant (169,173). While it is unclear whether SpoVM encases theC. difficile forespore as it does in B. subtilis (161),C. difficile SpoVM directly binds SpoIVA in recombi-nant coaffinity purification analyses (173), so C. difficilemay use a redundant factor to substitute for loss ofSpoVM.

While SpoVM is largely dispensable for C. difficilespore formation, the clostridia-specific CD3567 wasidentified as a coat morphogenetic protein because it isessential for proper coat localization and heat-resistantspore formation but not cortex formation (174).CD3567 was identified based on spore proteomic anal-yses (176) and targeted mutagenesis (174) and directlybinds to SpoIVA through CD3567’s C-terminal LysMdomain, so it was renamed SipL (SpoIVA interactingprotein L) (174). Fluorescent protein fusion analysesindicate that SipL is necessary for SpoIVA to encase theforespore, while SpoIVA is necessary for SipL to localizeto the forespore (M. Touchette and A. Shen, unpub-lished data). Thus, even though clostridia-specific SipL(174) lacks sequence homology with bacilli-specificSpoVID (41), aside from their C-terminal LysMdomains, the two proteins are functional homologsbecause they directly bind SpoIVA, encase the forespore,and recruit coat proteins to the forespore (164, 174).

C. difficile SpoIVA and SipL likely comprise the coatbasement layer (Fig. 5), but the specific coat proteinsthey recruit to the forespore are unknown. Analyses ofSpoIVA and SipL localization in engulfment mutantsindicate that SpoIVA and SipL can localize to the fore-spore membrane in the absence of engulfment (156),while the outer coat protein CotE (CD1433) localizes tothe cytosolic polymerized coat visible by phase-contrastmicroscopy (141). These results indicate that SpoIVAand SipL can adhere to the forespore independent ofengulfment but that outer layer coat proteins requireengulfment completion to stay associated with theforespore (156).

Additional coat proteins have been identified in sporeproteomic analyses, some of which have been shown tobe surface-localized (176–179). While coat proteins areenriched in these analyses, cytosolic contaminants in-evitably become encased as the outer layers are assem-bled in the mother cell cytosol (176–179). The functions

of many of these coat proteins have not been deter-mined. CotA has been implicated in spore assembly andheat and ethanol resistance (180). Nevertheless, enzy-matic activities have also been determined for severalcoat proteins (180, 181). For example, alanine racemaseinterconverts L- and D-alanine (as well as L- and D-serine)and alters the sensitivity of C. difficile spores to the D-alanine cogerminant (181).

C. difficile CotE, which is unrelated to the B. subtilisouter coat morphogenetic protein, CotE (119), degradesmucin and promotes spore binding to intestinal epithe-lial cells (182). Loss of CotE or its C-terminal mucinasedomain reduces virulence in a hamster model of infec-tion, indicating that the spore surface actively regulatesC. difficile colonization and disease (182). Given thatC. difficile cotE mutant spores do not have obviousultrastructural or resistance property defects in vitro(180), these analyses highlight the importance of ana-lyzing spore function in the context of infection.

Exosporium Assembly in C. difficileThe C. difficile exosporium layer directly contacts thecoat, unlike the exosporium of Bacillus cereus groupspores, which have an interspace gap between the exo-sporium and the coat (160, 183, 184). Since the C. dif-ficile exosporium is closely associated with the sporecoat (Fig. 5), it has been challenging to identify spe-cific components. However, by sonicating spores, theParedes-Sabja lab enriched for exosporium proteins (178)such as the (i) cysteine-rich proteins, CdeC (CD1067)(185), CdeM (CD1581) (64), and CdeA (CD2375)and (ii) the collagen-like proteins, BclA1, BclA2, andBclA3 (186, 187). Traces of coat proteins such as CotA,CotB, CotD, and CotE were also observed, which mayindicate that they are part of the coat/exosporium in-terface (178).

While it is unclear how the C. difficile exosporiumis assembled, genetic analyses have identified exosporiummorphogenetic proteins. The cysteine-rich proteins, CdeCand CdeM, have been implicated in exosporium mor-phogenesis (D. Paredes-Sabja, unpublished data). Sporesdeficient in CdeC have a defective coat that is permeableto lysozyme, have a higher core water content, and aremore susceptible to ethanol and heat than wild-typespores (185). Interestingly, cysteine-rich proteins areessential for the morphogenesis of the outer crust ofB. subtilis and the exosporium of the B. cereus group(188–190). These Bacillus spp. cysteine-rich proteinsself-assemble into two-dimensional crystalline layers(189, 191) that correlate with the two-dimensional crys-talline basal layer underneath the hairy nap (extensions)





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on B. anthracis spores. Both CdeC and CdeM formdimers, trimers, and higher molecular weight complexes(178, 185, 192), suggesting that a similar self-assemblymechanism might govern the assembly of the outer layersof C. difficile spores.

The C. difficile BclA proteins have also been impli-cated in exosporium assembly: spores lacking eitherBclA1, BclA2, or BclA3 produce defective exosporiums(186) and exhibit heat-resistance defects (186, 193). TheBclA proteins likely compose the hair-like extensions ofC. difficile spores (187), since these proteins producehair like-projections on B. anthracis spores (160). Fur-

thermore, like B. anthracis BclA (194), C. difficile BclA3is glycosylated (193). C. difficile BclA orthologues alsoappear to have a topology similar to that of B. anthracisBclA, because both proteins use their N-terminal do-mains to localize to the spore surface (187).

Exosporium Function in C. difficileAs the outermost layer of C. difficile spores, the exo-sporium may contact host component(s) that contributeto the persistence of C. difficile spores in the host (195,196). In transmission electron microscopy analyses ofclinically relevant C. difficile strains, the exosporium

FIGURE 5 Spore coat and exosporium structure inC. difficile. (A, B) Transmission electronmicroscopy sections of C. difficile spores highlighting (from outside to inside) the bumpy,outermost exosporium (Ex) layer with its hair-like projections (HPs), outer coat (OC), innercoat (IC), cortex layer (Cx), germ cell wall (GCW), inner forespore membrane (IM), andspore core (cytosol). (C) Scanning electron microscopy of C. difficile spores reveals thebumpy surface created by the exosporium. Images used without modification from Rabiet al. (280) under Creative Commons BY 4.0. (D) Schematic of spore coat layers high-lighting morphogenetic factors identified as being important for the assembly of specificlayers. Assembly of the outermost exosporium depends on the BclA collagen-likeproteins, which likely create hair-like projections on the spore surface (186, 187), CdeC(185), and CdeM (D. Paredes-Saja, unpublished data). The proteins that make up the outerand inner coat layers are unknown, but CotA and themucinase, CotE, have been shown tobe surface accessible (180, 182). SpoIVA (IVA) and SipL are interacting coat morphogeneticproteins that are essential for recruiting coat proteins to the forespore and forming heat-resistant spores (173, 174). The specific proteins recruited by SpoIVA, SipL, CdeC, andCdeM remain unknown.


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layer appears as electron-dense “bumps” on the sporesurface with hair-like extension (185, 196). Notably,two exosporium morphotypes are observed in clonalpopulations: thin and thick electron-dense layers, al-though both have hair-like extensions (197, 198). Thisobservation raises the possibility that the different mor-photypes may have different roles during C. difficileinfection, such as spore persistence and immune evasion.Consistent with this hypothesis, loss of the exosporiumprotein, CdeM (CD1581), decreases C. difficile fitnessin gnotobiotic mice (64), and loss of individual BclAproteins, particularly BclA1, results in decreased colo-nization in mice (186).

CLOSTRIDIAL SPORE GERMINATIONOverview of Spore Germination andOutgrowthDuring spore germination, metabolically dormant sporeslose their resistance properties and transform into met-abolically active cells. The low water content of the sporecytosol, known as the core, (∼25 to 40%) is criticalto this resistance because it prevents metabolism (7).Ca-DPA transport is essential for dehydrating the core,while the modified peptidoglycan cortex layer is essentialfor maintaining this partially dehydrated state. Thus,spore germination requires the removal of this cortex layerto allow core hydration and metabolism to resume (28).

Spore germination begins when spores sense smallmolecules termed germinants, which trigger a signalingcascade that leads to cortex degradation, release of Ca-DPA, core hydration, and degradation of SASPs boundto the chromosome. Althoughmany germination-relatedproteins are conserved in the clostridial pathogens, no-table differences in their function and mechanisms ofaction have been identified in C. difficile, C. perfringens,and C. botulinum. As discussed below, the order inwhich cortex hydrolysis and core hydration occurs dif-fers between these species and is even strain-specificin the case of C. botulinum. Furthermore, C. difficilespore germination has several unique features to its sig-naling pathway that are specific to C. difficile and/orPeptostreptococcaceae family members.

Germinant Sensing and SignalingEnvironmental signalsIn most spore-forming bacteria, germinants are nutrientsignals such as amino acids, monosacharides, nucleo-sides, salts, and organic acids (28). In contrast, C. diffi-cile responds to cholate-derived bile acids, which areproduced exclusively in the mammalian gut (199, 200).

Taurocholate is the most potent of the cholate-derivedgerminants, while chenodeoxycholate is an efficientcompetitive inhibitor of taurocholate-mediated germi-nation (201). Bile salt-induced spore germination maynot be unique to C. difficile, since taurocholate can en-rich for clostridial species from fecal samples (202), andspores from Paeniclostridium sordellii, a Peptostrep-tococcaceae family member, germinate in response tosome bile acids (203). Notably, amino acid and calciumion cogerminants enhance taurocholate-induced C. dif-ficile spore germination, with glycine and calcium ionsbeing the most potent of these small molecules (199,204, 205).

In C. perfringens and C. botulinum, germinant spec-ificity is species and strain-specific. Universal germinantsfor C. perfringens food-poisoning and nonfoodborneisolate spores include L-cysteine, L-serine, L-threonine,and a mixture of L-asparagine and KCl, while uniquegerminants for food-poisoning isolate spores are L-asparagine, L-glutamine, KCl, and the cogerminantsNa+ and Pi (206–208). Bicarbonate is a unique co-germinant for nonfoodborne spores (209). L-alanine,L-cysteine, L-methionine, L-serine, L-phenylalanine, andglycine can induce spore germination of group I pro-teolytic C. botulinum, although L-lactate and bicarbon-ate ions can act as cogerminants (210, 211). Spores ofgroup II nonproteolytic C. botulinum germinate in re-sponse to L-alanine, L-cysteine, L-serine, L-threonine, andglycine, with L-lactate serving as a cogerminant (211,212).

Germinant selectivity is likely influenced by adapta-tion to specific environmental niches. For example, theresponsiveness of C. perfringens food-poisoning isolate,but not nonfoodborne isolate, spores to KCl or NaPiimplies that food-poisoning isolates have adapted tofood niches (i.e., processed meat products) where KCland NaPi are highly abundant (10). Also, the findingthat bicarbonate is a unique cogerminant for nonfood-borne spores, which germinate better than food-poisoningisolate spores in the presence of cultured intestinal epi-thelial cells (196, 210), suggests that nonfoodborneisolate spores are better adapted to germinate in thehost’s intestinal epithelium environment, where bicar-bonate is more prevalent (213). Similarly, the respon-siveness of C. difficile spores to taurocholate may allowC. difficile to sense favorable conditions in the gut, sincetaurocholate levels are increased in the dysbiotic gutduring antibiotic treatment (21).

While classical germinants are directly sensed throughgerminant receptors, nonnutrient germinants can arti-ficially trigger germination of many bacterial species





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independent of germinant receptors. These include thecationic surfactant dodecylamine (28, 214), lysozyme(196), and Ca-DPA (215). While Ca-DPA activatesClostridium sporogenes (a proxy for C. botulinumgroup I [5]) spore germination, it does not activateC. difficile spore germination (205, 216, 217) and is arelatively weak activator of C. perfringens spore germi-nation (218). Interestingly, although high hydrostaticpressure can induce Bacillus spp. spore germinationindependent of germination receptors, high pressurealone is not sufficient to induce spore germination ofC. sporogenes (219), C. perfringens (220), and C. diffi-cile (220). For C. perfringens and C. difficile, the inef-fectiveness of high pressure to induce germination islikely due to differences in their germination mechanismrelative to Bacillus spp. as discussed below.

Transmembrane germinant receptorsin C. perfringens and C. botulinumAlmost all bacterial spores sense environmental signalsthrough germinant receptors localized in the spore’sinner membrane; the exception to this rule is C. difficileas described below. Germinant receptors usually con-sist of three protein subunits (A, B, and C) encodedin a tricistronic operon (gerABC). GerA and GerB aretransmembrane proteins, and GerC is a lipoprotein.Loss of any one of these components generally elimi-nates germinant receptor function (28, 196).

Many spore-forming bacteria encode multiple tricis-tronic operons, which are thought to determine thegerminant specificity of given strains. While B. subtilisencodes three major germinant receptors, there are di-verse arrangements for ger genes and operons in theBacillales and Clostridiales (see 28, 196). For example,C. perfringens has a monocistronic gerAA that is distantfrom the gerK locus, which encodes a bicistronic gerKA-KC operon upstream of the oppositely oriented mono-cistronic gerKB gene (196, 206). While many Clostrid-ium species encode monocistronic germinant receptorgenes, studies of C. perfringens provided the firstevidence that functional germinant receptors can consistof a single subunit. Indeed, the lipoprotein GerKCis the sole and essential germinant receptor for allknown nutrient- and nonnutrient-induced germinationof SM101 and F4969 spores (221, 222), in contrast toB. subtilis, in which all three subunits are essential forfunctional germinant receptor formation. Nevertheless,although GerAA and GerKB can play auxiliary roles infood-poisoning strain SM101 spore germination (206,222, 223), GerAA is required for nonfoodborne strainF4969 spore germination.

Canonical GerA family germinant receptors localizeto the spore’s inner membrane in several species, in-cluding B. subtilis (224) and C. botulinum (225). TheGerKC subunit is present in the inner membrane ofC. perfringens spores at ∼250 molecules/spore (221);the abundance and relative stoichiometry of the GerKA,GerKB, and GerAA subunits is unclear.

gerA family operons can also contain an additionalgerAD gene, which encodes a novel protein of 50 to80 residues with two highly conserved transmembranesequences in some Bacillales and Clostridiales species(196). Although GerAD is required for functional ger-minant receptor formation in Bacillus spp. (226, 227),their role in Clostridiales spore germination remainsunclear.

Genomic analyses of C. botulinum strains have iden-tified four gerABC subtypes (gerX type1 to 4) (211).gerX indicates that the germinant recognized by thereceptor encoded in the locus is unknown. Notably,although group II C. botulinum (gIICb) encodes onlyone predicted canonical germinant receptor despite be-ing able to respond to many amino acids, deletion ofgerBAC from gIICb strain NCTC 11219 does not affectgermination in response to nutrient or nonnutrient ger-minants (228). Thus, unidentified germinant receptorsappear to mediate spore germination in gIICb in theabsence of gerABC.

Germinant receptor-mediated spore germination isheat-activatable through a mechanism that is thought toinvolve conformational changes in the inner membranegerminant receptors and that potentiate their activation(28). Accordingly, bothC. perfringens andC. botulinumspore germination can be enhanced by a transient heatshock (229, 230). In contrast, C. difficile spore germi-nation is not heat-activatable, consistent with the ab-sence of germinant receptor genes in its genome (217,231).

A pseudoprotease, CspC, is the bile saltgerminant receptor in C. difficileSince C. difficile lacks germinant receptors, it was un-clear how it senses bile salt germinants, which arestructurally distinct from the nutritional germinantssensed by germinant receptor-encoding spore-formers.Joseph Sorg’s group identified the elusive C. difficilegerminant receptor using a genetic selection for germi-nation-defective mutants (232). This screen identifiedseven point mutations in cspC, which encodes a pseu-doprotease from the subtilisin-like serine protease fam-ily, and two nonsense mutations in cspBA, whichencodes a fusion protein consisting of CspB and CspA


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subtilisin-like serine proteases (Fig. 6). While the CspBprotease is catalytically active, bothC. difficileCspA andCspC are pseudoproteases because they harbor twopoint mutations in their catalytic triads (233). Subse-quent work revealed that CspBA is required for CspCincorporation into spores such that the cspBA nonsensemutations identified in the screen effectively lead to lossof CspC (234, 235).

CspC was further implicated as the direct receptorfor bile salts using a second genetic screen for alteredspore germinant specificity. By selecting for sporesthat germinate in response to the germination inhibi-tor, chenodeoxycholate (201), the Sorg group deter-

mined that a single point mutation in CspC (G457R)could change the ability of C. difficile spores to sensecholate versus deoxycholate derivatives (232). Al-though direct binding of bile acids to CspC has yet tobe demonstrated biochemically, germinant binding togerminant receptors has not been established in anyorganism.

ACTIVATION OF CORTEX HYDROLYSISIN CLOSTRIDIAL PATHOGENSJust as clostridial pathogens use two mechanisms forsensing germinants, these organisms use two mechanisms

FIGURE 6 Putative locations of germination regulators in C. difficile and C. perfringens.Germinant signaling proteins, CspC (pseudoprotease and germinant receptor) and itsdownstream effectors, the CspB protease, and cortex hydrolase, SleC, are all produced inthemother cell under the control of either σE or σK (137–139). CspB is produced as a fusionto the pseudoprotease, CspA, which is critical for CspC incorporation into mature spores(233–235); all three Csp proteins are incorporated into mature spores. GerG is requiredfor optimal incorporation of CspC, CspB, and CspA into mature spores (253). The GerSlipoprotein (248) is produced in the mother cell and does not directly participate in sporegermination (O. Diaz and A. Shen, unpublished data), Q9even though it is required for sporegermination to proceed. The ATP/GTP binding protein CD3298 presumably localizes tothe cytosolic face of the outer forespore membrane and regulates calcium release andpossibly internalization (205). Germinant sensing induces the proteolytic activation ofSleC by CspB in both organisms, but CspA and/or CspC can cleave SleC in C. perfringens(marked in brackets) (218, 242, 245), since they are active proteases unlike their cognatepartners in C. difficile (233). C. perfringens also produces inner membrane-boundgerminant receptors, similar to most spore-forming organisms, in the forespore, incontrast to the soluble CspC protein used by C. difficile to sense germinant. The loca-tions of all proteins in mature spores is putative, with the exception of SleC, whichhas been shown to localize to the C. perfringens cortex region by immuno-electronmicroscopy (251).





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to degrade their cortex: (i) proteolytic activation of thecortex hydrolase, SleC, and (ii) DPA-mediated activa-tion of the cortex hydrolases CwlJ and/or SleB (Fig. 7)(28). C. perfringens and C. difficile use the SleC path-way, while C. botulinum uses either mechanism in astrain-specific manner, with groups I and III using CwlJ/SleB (211, 230, 236) and groups II and IV presumablyusing the SleC pathway. While cortex hydrolases arethought to specifically recognize the muramic-δ-lactammodification that characterizes cortex peptidoglycan(237), the CspB-SleC cortex hydrolysis pathway is onlyfound in the clostridia, whereas the CwlJ/SleB systemis present in the bacilli and some members of the clos-tridia (196).

Proteolytic Activation of the SleC CortexHydrolase in C. perfringens and C. difficileSleC was first identified as a cortex hydrolase in bio-chemical fractionations of germinating C. perfringensexudates (238, 239) and was subsequently shown tohave lytic transglycosylase and amidase activities (240).SleC’s long N-terminal predomain acts as an intramo-lecular chaperone to ensure proper folding of its hy-drolase domain (241). The predomain is removed byproteolysis in a YabG-dependent manner (at least inC. difficile [235]) to generate the pro-SleC zymogen. Thisimmature form remains in mature spores until ger-minant signaling induces the proteolytic removal SleC’sinhibitory propeptide to generate active SleC (242).

FIGURE 7 Schematic of spore germination signaling pathways. Germinants that aresensed by B. subtilis, C. botulinum, C. perfringens, and C. difficile are shown in dark blue;C. botulinum group I and III germinants are shown in the brackets (and include L-Ala).Amino acid and calcium ion cogerminants are not pictured for C. difficile (199, 204, 205).Germinant receptors are shown in green. The signaling pathway between B. subtilis and C.botulinum groups 1 and III (far left) differs from the other clostridial organisms mainly withrespect to cortex hydrolase (shown in orange) activation mechanisms, with SleC beingactivated by proteolytic cleavage by Csp proteases, and the CwlJ and SleB cortexhydrolases being activated directly or indirectly by DPA release. Accordingly, the order ofcortex hydrolysis and DPA release via SpoVAC differs between these two types ofmechanisms. C. botulinum groups II and IV encode germinant receptors with variablenumbers of A and B components. Adapted from reference 183.


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The proteases responsible for activating SleC wereidentified as CspA, CspB, and CspC in biochemicalfractionations of C. perfringens strain S40 germinatingexudates (242, 243). These three proteases are membersof the subtilisin-like serine protease family (243) and areencoded by a tricistronic operon immediately upstreamof the sleC gene inC. perfringens strain S40 (244). Whilenonfoodborne C. perfringens strains encode this tricis-tronic operon, C. perfringens food-poisoning strainSM101 encodes cspB alone upstream of sleC (4, 245,246). Genetic analyses in SM101 revealed that C. per-fringens CspB is sufficient to proteolytically activateSleC, since cspB mutant spores exhibit 104-fold ger-mination and cortex hydrolysis defects relative to thewild type (245) and fail to process pro-SleC into activeSleC in response to germinants (245).

SleC is the major cortex hydrolase in C. perfringens,since sleC mutant spores have a 103 germination defectrelative to the wild type (218). The SleM cortexhydrolase (247) functions as an accessory cortex hy-drolase, since a sleM mutant exhibits wild-type germi-nation, but a sleC sleM double mutant has a 100-foldmore severe germination defect than the sleC singlemutant (218).

The CspB protease and SleC cortex hydrolase havesimilar functions in C. difficile, since loss of CspB leadsto an ∼103 to 105 germination defect (233, 234), andcomplementation with the wild-type cspB, but not acatalytic mutant, restores germination and pro-SleCcleavage (233). Loss of sleC also results in a similar∼103-fold germination defect relative to the wild type(234) and an inability to hydrolyze cortex (216, 248).However, in contrast with the tricistronic cspA-cspB-cspC operon in some C. perfringens strains, C. difficilestrains (and other Peptostreptococcaceae family mem-bers) universally encode cspB fused to cspA, with cspCencoded downstream of the cspBA fusion gene (cspBA-cspC) (235).

SleC and Csp proteins are produced in the mother cellof both C. perfringens and C. difficile (137–139, 249).Western blot analyses of spore fractions inC. perfringensand C. difficile indicate that CspB and SleC localize toa coat-extractable fraction (248, 250). However, C.perfringens SleC can be visualized in the cortex region ofmature spores using immune-electron microscopy (251),indicating that fractions previously assumed to comprisecoat proteins alone extract proteins from the cortex re-gion. Interestingly, neither C. perfringens nor C. difficileSleC harbor signal sequences, raising the question ofhow they are targeted to the intermembrane space (as-suming Csp proteins are targeted to this region).

The crystal structure of C. perfringens CspB re-vealed that Csp proteins consist of three main domains:a long N-terminal prodomain, a subtilisin fold, andan internal jelly roll domain (233, 243). Like othersubtilisin-like proteases, C. perfringens and C. difficileCspB have a catalytic triad consisting of Asp, His, andSer, and the prodomain undergoes autoprocessing (233,243). However, unlike almost all previously studiedsubtilisin-like serine proteases, both C. perfringensand C. difficile CspB remain bound to their prodomainfollowing autoprocessing in vitro (233, 243). Theprodomain sterically inhibits recombinant CspB activity(233), but it is unclear whether it stays bound to CspB inmature spores. Regardless, the prodomain presumablymust be removed during spore germination to allowCspB to bind its presumed substrate, SleC. The centraljelly roll domain within the subtilase domain is anotherunique feature of CspB; this domain markedly increasesthe conformational rigidity of CspB in vitro and isnecessary for stable CspB production in C. difficile(233).

CspB activity has been proposed to require calciumbased on the observation that calcium is a criticalcogerminant during taurocholate-induced C. difficilespore germination (205). Depletion of calcium in vitroand in intestinal extracts reduces wild-type C. difficilespore germination and abrogates spore germination in astrain lacking CD3298, an ATP/GTP binding proteinrequired for releasing calcium during germination(205). While these data indicate that calcium is a keyadjuvant during C. difficile spore germination, the pre-cise stage regulated by calcium is unclear, since it couldaffect CspB activity, CspC activation, germinant per-meability, or as-yet-unidentified events. Whether calci-um is required for C. perfringens spore germinationremains to be tested.

Collectively, germinant signaling triggers CspB-dependent processing of inactive pro-SleC into activeSleC, which subsequently degrades the cortex in bothC. perfringens and C. difficile. It is unclear how germi-nant signaling leads to CspB activation, especially sincethese two organisms use different germinant receptors.The topology of these receptors also differs betweenthese organisms, with Ger receptors being embeddedin the C. perfringens forespore-derived membrane, andsoluble C. difficile CspC being made in the mother cell(Fig. 6) (28, 200). C. difficile CspC has been hypothe-sized to be transported across the mother cell-derivedmembrane into the intermembrane space so that it canactivate CspB through protein-protein interactions (200,234, 252, 253). CspB in C. perfringens and C. difficile





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is presumably also transported into the intermembranespace so that it can activate its substrate SleC, whichhas been localized to this region in C. perfringensby immuno-electron microscopy (251). Clearly, manyquestions regarding the molecular details of this processremain to be addressed.

DPA-Mediated Activation of Cortex Hydrolasesin Group I C. botulinumGene conservation predicts that C. botulinum groups IIand IV activate cortex hydrolysis via CspB-mediatedproteolytic activation of pro-SleC (211), but group I(and likely group III based on homology) use the par-tially redundant cortex hydrolases, CwlJ and SleB, toremove their cortex layer (211, 230, 236). Both hydro-lases are produced in their mature form, but their acti-vation appears to be tied to Ca-DPA release as observedin B. subtilis (28, 254). C. sporogenes (a proxy for groupI C. botulinum) cwlJ mutant spores do not germinatein response to Ca-DPA, unlike sleB mutant spores (230,255) and similar to analogous mutants in B. subtilis(256). In B. subtilis, SleB activity is directly inhibited byYpeB (254), but the mechanism by which SleB is acti-vated during germination is unknown. C. botulinumgroup I sleB and ypeBmutants exhibit 2- to 3-log defectsin spore germination based on colony forming units,whereas cwlJ mutant spores produce wild-type levels ofcolonies on rich media (236). In contrast, cwlJ and sleBmutant spores in C. sporogenes have similar germina-tion defects in single-spore germination analyses (230).Regardless, CwlJ and SleB likely have partially over-lapping functions during group I C. botulinum sporegermination, so a double mutant lacking both enzymeswould presumably exhibit a severe germination defectsimilar to that in B. subtilis (257). Interestingly, bothgroup I and group III C. botulinum spp. can encodemultiple SleB homologs (211), suggesting that theremay be additional redundancy in regulating cortexhydrolysis.

Ca-DPA and Spore Formation versusGerminationCa-DPA is a spore-specific molecule that makes up 5 to15% of spore dry weight (256). It is synthesized in themother cell from lysine biosynthesis intermediates (258)and transported across the outer and inner foresporemembranes by SpoVV (259) and SpoVAC (260, 261),respectively, in exchange for water. While the partialdehydration of the core by Ca-DPA transport into theforespore confers heat resistance to mature spores (256,261, 262), Ca-DPA is differentially required for spore

formation. In B. subtilis, mutants defective in syn-thesizing or transporting Ca-DPA cannot stably formspores due to premature germination and cortex hy-drolase activation (256, 259, 260). Similar to B. subtilis,C. sporogenes spoVA mutants also produce unstablespores, presumably due to premature SleB activation(211). In contrast, C. difficile mutants defective in syn-thesizing or transporting Ca-DPA can stably formspores, although they are less dense than wild-typespores (261). Interestingly, Ca-DPA synthesis is neces-sary for C. perfringens spore formation (263), but Ca-DPA transport is not (262). Since Ca-DPA does notstrongly induce C. perfringens germination (262), pre-sumably because it uses the CspB-SleC pathway toactivate cortex hydrolysis, it is unclear why DPA is re-quired for spore formation in C. perfringens but notin C. difficile. This difference may reflect the differ-ent mechanisms used by these organisms to synthesizeCa-DPA. While C. difficile uses dihydro-dipicolinatesynthase (DpaAB or SpoVFAB) to produce Ca-DPA,C. perfringens uses an electron transfer flavoprotein,EftA, to make this molecule. Since C. botulinum lacksSpoVFAB homologs and likely uses EftA, DPA-lessC. botulinum spores likely will not be stably produced.

Cortex Hydrolysis Precedes Ca-DPA Releasein C. difficileWhile Ca-DPA release activates cortex hydrolysis ingroup I C. botulinum and other CwlJ/SleB-controlledsystems, cortex hydrolysis is necessary for Ca-DPA re-lease in C. difficile (261). Cortex hydrolysis activates themechanosensitive SpoVAC channel (264) to release Ca-DPA from the core, since C. difficile spores germinatedin high osmolyte solutions degrade their cortex normallybut release Ca-DPA more slowly (265). Furthermore,C. difficile sleC mutant spores do not release Ca-DPA(216, 217). A similar mechanosensing phenomenon mayoccur in C. perfringens, since a sleC-sleM double mutantand a cspB mutant exhibit major defects in Ca-DPArelease (218, 245). Thus, the CspB-SleC cortex degra-dation pathway likely induces Ca-DPA release, while theorder of these events is reversed in spores that use CwlJand SleB to degrade their cortex (28).

Events after Cortex Hydrolysis andCa-DPA ReleaseWhile recent studies have provided critical insight intothe early stages of germination in clostridial pathogens,namely germinant sensing, cortex hydrolysis, andCa-DPA release, little is known about subsequentevents. Ca-DPA release in C. difficile leads to core


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hydration based on the observation that the opticaldensity of germinating dpaAB– and spoVAC– sporesdecreases by ∼50% relative to the wild type, whereasthe optical density of sleC– spores remains unchanged(261). Core hydration allows metabolism to resume,and transcript levels increase within 15 min of germi-nation (231).

In B. subtilis, Ca-DPA release activates the Gprgermination protease (266), which degrades SASPscoating the nucleoid. The amino acids liberated bySASP degradation provide substrates for both cata-bolism and metabolism (267). It is currently unclearwhether Ca-DPA activates Gpr homologs in clostridialpathogens.

Loss of Resistance PropertiesDuring GerminationThe hydration of the core during spore germinationallows metabolism to resume while concomitantly lead-ing to loss of resistance. This is because the resistanceproperties of spores can largely be attributed to (i) thelow water content of metabolically dormant spores and(ii) the coating of the genetic material by SASPs, whichcollectively prevent metabolic activity in the spore core.Indeed, B. subtilis mutant spores with higher watercontent or lacking SASP proteins are more sensitive toheat (256, 268) and in the latter case are more sensitiveto UV irradiation (269, 270). Although B. subtilis pro-duces three major types of SASP proteins (α, β, and γ),which are encoded by multiple genes (269), SspA andSspB (α and β, respectively) are the major SASPs coat-ing the chromosome and the primary contributors toB. subtilis spores’ resistance to UV and solar radiation(270).

Similar to B. subtilis, C. difficile and C. perfringensmutant spores with higher spore water content are moresusceptible to heat (261, 262, 271) and UV irradiation,possibly because increased hydration reduces SASPbinding to DNA (262, 271). Consistent with this idea,C. perfringens food-poisoning isolates with extremeresistance properties (e.g., type A chromosomal cpe andtype C Darmbrand strains) produce an Ssp4 variant(also known as SASP4) that binds DNA with higheraffinity than Ssp4 variants from more heat-sensitiveC. perfringens isolates (272, 273). Mutational analy-ses indicate that this Ssp4 variant is a major determi-nant controlling the extreme heat resistance of certainfood-poisoning strains. Interestingly, Ssp4 exhibits only∼20% sequence identity relative to the three otherSASP proteins (Ssp1 to Ssp3) produced byC. perfringensstrains, suggesting that this Ssp4 variant may be a

specific adaptation of food-poisoning isolates (e.g.,SM101). Indeed, it should be noted that Ssp1 to Ssp3have been shown to confer UV resistance to C. per-fringens food-poisoning strain SM101 spores using an-tisense RNA silencing methods (274).

C. botulinum encodes four SASPs, CBO1789,CBO1790, CBO3048, and CBO3145. This latter pro-tein exhibits the lowest sequence conservation and isdispensable for resistance to nitrous acid (275) and thusmay function similarly to B. subtilis SASP γ in acting as afood source for the outgrowing spore (276). Mutationalanalyses indicate that CB01789 and CPB01790 are pri-marily responsible for conferring resistance to nitrousacid, an oxidizing agent (275).

The role of SASPs in conferring resistance to C. dif-ficile spores has not been examined, although RNA-Seqanalyses suggest that SspA and SspB are the predo-minant SASPs produced (137). C. difficile spores areless resistant to heat than B. subtilis and C. perfringensspores, since C. difficile spores are readily killed attemperatures above 80°C (261).

CONCLUSIONS AND UNIQUE FEATURESOF C. DIFFICILE SPORE GERMINATIONDiverse pathways are clearly used by group I C. botu-linum, C. perfringens, and C. difficile to mediate sporegermination. The pathway used by group IC. botulinumspore germination most resembles Bacillus spp. germi-nation, with germinant receptors sensing amino acidsand stimulating Ca-DPA release followed by cortexhydrolysis by CwlJ and SleB (28). While C. perfringensspore germination also uses germinant receptors to sensenutrient germinants such as amino acids, germinantsensing activates the CspB-SleC cortex hydrolysis path-way, after which most Ca-DPA is released. C. difficilespore germination is the most divergent, with its uniquegerminant receptor, the CspC pseudoprotease, sensingbile acids. Like C. perfringens, germinant sensing acti-vates the CspB-SleC cortex hydrolysis pathway, and thechange in pressure caused by the resulting cortex hy-drolysis induces SpoVAC to release Ca-DPA.

Although CspA, CspB, and CspC are conserved inmany clostridial organisms, they are produced as singleprotease domains in the Clostridiaceae and Lachnos-piraceae families and as a CspB-CspA fusion protein inthe Peptostreptococcaceae family (235). Furthermore,CspB is the only active serine protease, with CspC andthe CspA domain being pseudoproteases. Since CspA iscritical for incorporating the germinant receptor andpseudoproteases, CspC, into mature spores (234, 235),





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the catalytic site mutations within CspA and CspC,which are largely conserved across Peptostreptococ-caceae family members (235), appear to confer uniquefunctions to these proteins.

Recent studies have identified two unique proteinsthat modulate C. difficile spore germination. GerG(CD0311) is a C. difficile-specific gel-forming proteinthat is necessary for incorporating CspA, CspB, andCspC into mature spores, while GerS (CD3464) is aPeptostreptococcace family-specific lipoprotein that isnecessary for cortex hydrolysis (248) because it is nec-essary for generating the muramic-δ-lactam required forSleC to recognize its cortex substrate (Diaz and Shen,unpublished data).

While the Csp pseudoproteases, GerG, and GerSare distinguishing features of the C. difficile spore ger-mination pathway, it is likely that additional novelregulators specific to certain clostridial families will beidentified as this fascinating developmental process isstudied using the genetic tools recently developed forstudying clostridial pathogens (140, 277–279).

ACKNOWLEDGMENTSResearch reported in this article was funded by award numbersR21AI126067 and R01AI22232 from the National Institutesof Allergy and Infectious Disease (NIAID), award numberR01GM108684 from the National Institutes of General MedicalSciences, and a Pew Scholar in the Biomedical Sciences grant fromthe Pew Charitable Trusts to A.S.; R01 AI116933 from NIAID toA.N.E.; N.L. Tartar Foundation and the Agricultural ResearchFoundation of Oregon State University to M.R.S; and FondecytRegular 1151025 from the Fondo Nacional de Ciencia yTecnología de Chile to D.P.S.The content is solely the responsibility of the authors and does

not necessarily reflect the views of the Pew Charitable Trusts,NIAID, or the National Institutes of Health. The funders had norole in study design, data collection and interpretation, or thedecision to submit the work for publication.

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