Ultrastructure Variability of the Exosporium Layer of Clostridiumdifficile Spores from Sporulating Cultures and Biofilms

Marjorie Pizarro-Guajardo,a,b Paulina Calderón-Romero,a,b Daniel Paredes-Sabjaa,b

Microbiota-Host Interactions and Clostridia Research Group, Departamento de Ciencias Biológicas, Universidad Andres Bello, Santiago, Chilea; Center for Bioinformaticsand Integrative Biology, Facultad de Ciencias Biológicas, Universidad Andres Bello, Santiago, Chileb

ABSTRACT

The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developedand developing countries. The metabolically dormant spore form is considered the morphotype responsible for transmission,infection, and persistence, and the outermost exosporium layer is likely to play a major role in spore-host interactions duringrecurrent infections, contributing to the persistence of the spore in the host. A recent study (M. Pizarro-Guajardo, P. Calderón-Romero, P. Castro-Córdova, P. Mora-Uribe, and D. Paredes-Sabja, Appl Environ Microbiol 82:2202–2209, 2016, http://dx.doi.org/10.1128/AEM.03410-15) demonstrated by transmission electron microscopy the presence of two ultrastructural morpho-types of the exosporium layer in spores formed from the same sporulating culture. However, whether these distinct morphotypesappeared due to purification techniques and whether they appeared during biofilm development remain unclear. In this commu-nication, we demonstrate through transmission electron microscopy that these two exosporium morphotypes are formed undersporulation conditions and are also present in spores formed during biofilm development. In summary, this work provides de-finitive evidence that in a population of sporulating cells, spores with a thick outermost exosporium layer and spores with a thinoutermost exosporium layer are formed.

IMPORTANCE

Clostridium difficile spores are recognized as the morphotype of persistence and transmission of C. difficile infections. Spores ofC. difficile are intrinsically resistant to all known antibiotic therapies. Development of spore-based removal strategies requires adetailed knowledge of the spore surface for proper antigen selection. In this context, in this work we provide definitive evidencethat two types of spores, those with a thick outermost exosporium layer and those with a thin outermost exosporium layer, areformed in the same C. difficile sporulating culture or during biofilm development.

Infections caused by Clostridium difficile are the leading cause ofnosocomial antibiotic-associated diarrhea in developed and de-

veloping countries (1, 2). Mortality rates of C. difficile infections(CDIs) may reach up to 5%; however, the recurrence of the infec-tion, which may reach up to 25, 40, and 65% of cases after a first,second, and third episode of CDI, respectively, has become thecurrent main clinical challenge (3). The main factors in recurrenceof CDI include (i) an irreversible loss of function of the gut mi-crobiota due to antibiotic therapy, leading to a loss of the coloni-zation resistance barrier against enteric pathogens (4, 5), and (ii)the formation of metabolically dormant spores during the courseof infection (6, 7). These newly formed C. difficile spores have beenshown to be essential for the transmission of the disease to a newsusceptible host and for the persistence of C. difficile in the host,leading to recurrent infection (6).

Biofilms are sessile surface-associated microbial communitiesthat represent the predominant state of bacteria in nature (8). C.difficile biofilms have been shown to be more resistant than plank-tonic cells to antibiotics (9). During in vivo infection in the mousemodel, C. difficile resides in multicellular communities (biofilms)(10) in which spores have been shown to form (11). Although notdemonstrated experimentally, it has been suggested that develop-ment of spores might be relevant for biofilm formation, since ithas been reported that a Spo0A mutant (Spo0A is a transcriptionfactor that controls spore formation) is defective in biofilm for-mation (12). Recently, the presence of two exosporium proteins(i.e., CdeC and the N-terminal domain of BclA1) was detected by

indirect immunofluorescence analysis of spores from C. difficilebiofilms (13). However, whether the ultrastructure of biofilm-formed spores is similar to that of sporulating cultures remainsunclear.

The imminent relevance of C. difficile spores in the infectiouscycle raises numerous questions regarding its assembly, composi-tion, and fate in the host. In this context, recent studies have re-vealed that the ultrastructure of C. difficile spores is similar to thatof other Gram-positive bacteria (13, 14). The outermost layer is anelectron-dense layer that, in most epidemic strains, is covered byhair-like projections and has been shown to be ultrastructurallystable (13). Underlying the exosporium layer is the spore coat,which has laminations (i.e., lamellae) similar to those present inother bacterial spores, although it differs in protein compositionfrom the spore coat of other bacterial species (13, 14). The

Received 14 May 2016 Accepted 7 July 2016

Accepted manuscript posted online 29 July 2016

Citation Pizarro-Guajardo M, Calderón-Romero P, Paredes-Sabja D. 2016.Ultrastructure variability of the exosporium layer of Clostridium difficile spores fromsporulating cultures and biofilms. Appl Environ Microbiol 82:5892–5898.doi:10.1128/AEM.01463-16.

Editor: D. W. Schaffner, Rutgers, The State University of New Jersey

Address correspondence to Daniel Paredes-Sabja,daniel.paredes.sabja@gmail.com.

Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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C. difficile spore coat, as similarly reported for Bacillus subtilis (15),exhibits enzymatic digestion resistance to proteases (i.e., protei-nase K and trypsin) (16), which might be related to the apparentresistance of C. difficile spores to macrophages (17).

The outermost exosporium layer of C. difficile spores isthought to play important roles in host-spore interaction and tohold spore ligands involved in spore-interaction with host cells(18). However, only recently has the outermost exosporium layerbegun to be characterized. The composition of the exosporiumlayer of C. difficile spores has been recently identified (19), reveal-ing a complex composition of 184 proteins, among which severalproteins known to be involved in pathogenesis and evasion ofimmunity in other pathogens were identified (e.g., elongation fac-tor Tu and alpha-enolase) (19). Notably, our recent results haveshown that during spore formation, C. difficile is capable of pro-ducing spores with two distinctive morphotypes of the exospo-rium layer, either thin or thick (13). However, whether these twodifferent morphotypes of the exosporium layer emerge directlyfrom the mother cell and whether these two morphotypes areformed during biofilm development remain unclear. In this work,through transmission electron microscopy, we have evaluated theultrastructural variability of C. difficile spores formed under spo-rulation conditions and during biofilm development and demon-strate that two morphotypes of the exosporium layer are formedsimultaneously in a sporulating culture or during biofilm devel-opment.

MATERIALS AND METHODSBacterial strains. The C. difficile strain used in this study was the epidemicstrain R20291 (ribotype 027), which caused an outbreak and has beendescribed elsewhere (20, 21). C. difficile was routinely grown under anaer-obic conditions in an anaerobic chamber (Coy, USA) in 3.7% brain heartinfusion supplemented with 0.5% yeast extract (BHIS) broth or on BHISagar plates.

Sporulating culture and spore preparations. Sporulating cultureswere prepared by plating a 1:500 dilution of an overnight culture onto 3%Trypticase soy-0.5% yeast extract (TY) agar plates and incubated for 5days at 37°C under anaerobic conditions. One-half of the total plates werecarefully harvested in sterile distilled water and fixed with 3% glutaralde-hyde in 0.1 M cacodylate buffer for further processing for transmissionelectron microscopy as described below. The other half of the plates wereharvested with ice-cold sterile distilled water, washed (18,625 � g for 10min) five times with sterile distilled water, and purified with 50% Nyco-denz as previously described (22). Pure spores were immediately fixed andprocessed for analysis by transmission electron microscopy as describedbelow.

Biofilm formation. Biofilm formation was evaluated by previouslydescribed protocols (12, 23). Briefly, overnight cultures grown in BHIS(3.7% brain heart infusion broth supplemented with 0.5% yeast extract)were diluted 1:100 in 24-well polystyrene plates (Greiner Bio-One, Stutt-gart, Germany) containing fresh BHIS broth and incubated at 37°C underanaerobic conditions for 5 days. Some wells were used to confirm thepresence of biofilm biomass, stained with crystal violet, and quantified atan optical density of 570 nm with a spectrophotometer (ELx800; BioTek)as previously described (13). Other wells were used to harvest biofilmmaterial. Briefly, wells were gently rinsed three times with sterile phos-phate-buffered saline (PBS) to remove nonbiofilm material, and biofilmmass was removed by mechanical scraping, immediately fixed with 3%glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 16 h at 4°C, andprocessed for transmission electron microscopy as described below.

Transmission electron microscopy. Purified spores, sporulating cul-ture, and fixed biofilm samples (3% glutaraldehyde in 0.1 M cacodylatebuffer, pH 7.2) were stained for 30 min with 1% tannic acid. Samples were

further processed and embedded in Spurr resin as previously described(18). Thin sections of 90 nm were obtained with a microtome, placed onglow discharge carbon-coated grids, and double-lead stained with 2%uranyl acetate and lead citrate. Grids were analyzed with a Philips Tecnai12 Bio Twin electron microscope.

To quantify the percentage of spores with a thick or thin exospo-rium layer, a total of 100 spores from randomly selected transmissionelectron micrographs (TEMs) (approximately 10 fields at a magnifica-tion of �10,500) were analyzed. To distinguish spores with a thick andthose with a thin exosporium, we established the following criteria: (i)spores with a thick exosporium were defined as having a basal electron-dense exosporium layer at least three-fourths larger than the spore coatwhile (ii) spores with a thin exosporium layer were defined as those with abasal exosporium layer one-half the size of the spore coat. Two replicateswere observed with essentially similar results.

To analyze the length of the spore layers, 60,000� transmission elec-tron micrographs of 19 representative spores with thin and thick exospo-ria were randomly selected. For each spore, the length of the layers wasquantified at 6 different locations.

Statistical analysis. Data were determined to be nonparametric basedon the Q-Q plot and the Shapiro-Wilk test; therefore, to test for statisticalsignificance between two groups, the nonparametric unpaired Mann-Whitney test was used.

RESULTSTwo morphotypes of the exosporium layer are present in spo-rulating cultures of epidemic C. difficile strain R20291. Re-cently, two distinctive thicknesses of the exosporium layer (i.e., athin and a thick exosporium) were reported to appear in purifiedspore suspensions of various epidemic C. difficile strains analyzedby transmission electron microscopy (13). In order to rule out thepossibility that the spore purification conditions (see Materialsand Methods), which include a Nycodenz purification step of C.difficile spores, could be implicated in the removal of exosporiummaterial and lead to the appearance of these two morphotypes,transmission electron micrographs (TEMs) of carefully preparedunwashed sporulating cultures and purified spore suspensionswere examined. As expected, TEMs confirmed the presence ofboth exosporium morphotypes, spores with a thick and those witha thin exosporium-like layer, in the unwashed sporulating culture(Fig. 1A). Depending on how the spores were sliced (i.e., transver-sal or longitudinal), the distribution of the exosporium layer ex-hibited notable differences. In transversal spore sections, a regularand homogeneously distributed electron-dense and thick exospo-rium layer can be observed surrounding the spore (Fig. 1B). Inlongitudinal spore sections, a thicker electron-dense layer at oneof the spore poles becomes evident (Fig. 1C). We also observedthat nearly 37% of the spores with a thin exosporium layer weresurrounded with cellular debris (Fig. 1D), while 67% were free ofcellular debris (Fig. 1E and data not shown). In contrast, none ofthe analyzed spores with a thick exosporium layer had cellulardebris. Spores from both exosporium morphotypes had the dis-tinctive hair-like extensions (Fig. 1B to E). Collectively, these re-sults clearly indicate that both morphotypes of exosporium layers,thick and thin, are formed during sporulation and are not due toan artifact during spore purification.

C. difficile spores formed during biofilm development ex-hibit both thick and thin exosporium morphotypes. To addresswhether these two exosporium morphotypes were also formedduring biofilm development, biofilm biomass was carefully har-vested and analyzed for the presence of C. difficile spores by trans-mission electron microscopy. The presence of C. difficile spores in

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biofilm biomass was evident, and spores with a thick or a thinexosporium layer were also detectable (Fig. 2A). We also identifiedspores with a large amount of electron-dense exosporium mate-rial, which accounted for 40% of the spores with a thick exospo-rium morphotype formed during biofilm development (Fig. 2Band C). Similarly, as in unwashed sporulating cultures, in biofilmbiomass we observed two types of spores with a thin exosporiumlayer: (i) those with hair-like extensions and little if any electron-dense exosporium material (Fig. 2D) and (ii) those either sur-rounded by an aberrantly assembled exosporium material or as-sociated with cellular debris from lysed mother cells (Fig. 2E). Insummary, these results clearly indicate that spores formed duringbiofilm formation (i) have an exosporium layer with hair-like fil-aments and (ii) may have either a thick or a thin exosporium layeras an outer surface.

Spore purification removes a slight amount of hair-like ex-tensions and electron-dense material. To more rigorously deter-mine the similarities between spores from unwashed sporulatingcultures and purified spores, the lengths of the outermost layerswere quantified (Fig. 3A). Notably, the proportion of spores witha thick or a thin exosporium layer in unwashed sporulating cul-tures (i.e., 29% with a thick layer and 71% with a thin layer) wassimilar to that of purified spore suspensions (i.e., 23% with a thicklayer and 77% with a thin layer) (Fig. 3A). Within the spores witha thick exosporium, those from unwashed sporulating culture had

an exosporium layer 60 nm thicker than that of purified spores(Fig. 2B). Differences were observed in both components of theexosporium layer. The hair-like extensions and the electron-denselayer of spores from unwashed sporulating cultures were 27 and41 nm longer, respectively, than those in spores from purifiedspores (Fig. 3B). On the other hand, spores with a thin exospo-rium layer obtained from unwashed sporulating cultures had aslightly thicker exosporium layer (i.e., 12 nm) than those frompurified spore suspensions (Fig. 3B), which seems to be due to theslightly shorter hair-like extensions (i.e., 8 nm) observed in puri-fied spores (Fig. 3B). Analysis of the spore coat layer revealed nosignificant differences between spores with a thick or a thin exo-sporium layer from unwashed sporulating culture and purifiedspore suspensions (Fig. 3B). In summary, these observations in-dicate that spore purification procedures might cause a slight de-crease in the length of the hair-like filaments and a reduction inthe thickness of the exosporium in both morphotypes.

Thinner spore coat layer is evident in biofilm-formed C. dif-ficile spores. To gain more insight on how the outer spore layers ofbiofilm-formed spores differ from those formed under traditionalsporulation conditions, the lengths of the hair-like extensions,exosporium, and spore coat were analyzed. Electron micrographsof unwashed sporulating cultures prepared at the same time asbiofilms were prepared and were the same as those used in theexperiment shown in Fig. 3. A significant difference in the thick-

FIG 1 Presence of two distinctive morphotypes of the exosporium layer in sporulating cultures of C. difficile R20291 spores. (A) Representative electronmicrograph of unwashed sporulating cultures of R20291 demonstrates the presence of spores with a thick exosporium layer (black arrows) and spores with a thinexosporium layer (white arrows). (B to E) Magnified transmission electron micrographs of spores with a thick (B and C) or a thin (D and E) exosporium layer.These are representative micrographs from two independent spore crops with essentially similar morphotypes. Bars, 500 nm.

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ness of the exosporium layer was evident upon comparison ofthick-exosporium spores obtained from biofilms and those fromunwashed sporulating cultures, with estimated thicknesses of 150and 216 nm, respectively (Fig. 3C). Thick-exosporium sporesfrom biofilms had hair-like extensions and an electron-dense layer34 and 36 nm thinner, respectively, than those from unwashedsporulating cultures (Fig. 3C). In contrast, we found no significantdifference in the lengths of the exosporium layers of thin-exospo-rium spores obtained from biofilms and those from unwashedsporulating cultures (Fig. 3C). Differences were observed betweenthe inner, but not external, coat layer of thick-exosporium sporesfrom biofilms and those from unwashed sporulating cultures,where biofilm spores had a 6-nm-thinner inner coat (i.e., 12 ver-sus 18 nm, respectively) (Fig. 3A and C). Thin-exosporium sporesfrom biofilms had thinner spore coat layers than did spores fromunwashed sporulating cultures, with the external and inner coatlayers of biofilm spores being 18 nm (i.e., 24 versus 42 nm) and 4nm (i.e., 9 versus 13 nm) thinner, respectively, than those ofspores from unwashed sporulating cultures (Fig. 3C). In sum-mary, these results demonstrate that, during biofilm develop-ment, C. difficile also forms spores with a thick or a thin exospo-rium layer and that, particularly, spores with a thin exosporiumlayer formed during biofilm development have a thinner sporecoat than those formed under standard sporulating conditions.

DISCUSSION

The relevance of C. difficile spores to the pathogenesis of the dis-ease is well known (6, 7, 14), increasing the need to develop spore-based strategies to eradicate C. difficile from the host and clinicallyrelevant surfaces. In this context, we have recently provided evi-dence of two different morphotypes of the exosporium layer thatappear from a monoclonal culture (13). In this work, we haveaddressed whether these two different exosporium morphotypesare indeed formed during sporulation and whether they also arisein spores formed during biofilm formation.

A first contribution of this work is that the two ultrastructuralmorphotypes (i.e., thick and thin) of the exosporium layer weredetectable in unwashed sporulating cultures and therefore werenot an artifact of the extensive washing of spore suspensions.These observations are strongly supported by transmission elec-tron micrographs of unwashed sporulating cultures of the C. dif-ficile R20291 strain showing both morphotypes and reinforce ourprevious hypothesis that an intrinsic regulatory mechanism(s)might drive the formation of each exosporium morphotype on acell-to-cell basis (13). Although a slight amount of hair-like exten-sion and exosporium material was removed from spores with athick exosporium morphotype during the purification process,our electronic micrographs demonstrate that this was not suffi-cient to contribute to the appearance of the thin exosporium mor-

FIG 2 Presence of two distinctive morphotypes of the exosporium layer in biofilms of C. difficile R20291 spores. (A) Representative electron micrographs ofbiofilms of R20291 demonstrate the presence of spores with a thick exosporium layer (black arrows) and spores with a thin exosporium layer (white arrows). (Bto E) Magnified transmission electron micrographs of spores with a thick (B and C) or a thin (D and E) exosporium layer. These are representative micrographsfrom two independent biofilm formation experiments and spore crops with essentially similar morphotypes. Bars, 500 nm.

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photype. Notably, the frequency of appearance of both exospo-rium morphotypes in our previous work revealed that thefrequency was strain dependent and that in R20291 spores nearlyone-fourth and three-fourths of the spore population had a thickand a thin exosporium morphotype, respectively (13). Here, we

observed a similar proportion of both morphotypes in unwashedsporulating cultures and purified spore suspensions, where one-third and two-thirds of the spores had the thick and thin exospo-rium morphotypes, respectively. How the assembly of these twomorphotypes of the exosporium layer is regulated during sporu-

FIG 3 Analysis of the length of the outermost layers of thick and thin exosporium spores from unwashed sporulating cultures and biofilms. (A)Representative transmission electron micrographs of spores from unwashed sporulating culture, purified spore suspensions, and biofilm of C. difficilestrain R20291. Bars, 200 nm for whole and selected spore sections, respectively. Ex, exosporium layer (i.e., hairs and electron-dense layer [ED]); EC,external coat; IC, internal coat. The percentage of spores with thick (black bar) or thin (gray bar) exosporium layers is depicted below the micrographs(n � 100 spores). Data are representative of two spore preparations with similar results. (B) The thicknesses of the exosporium and the outer and innercoat layers of C. difficile spores from unwashed sporulating cultures with a thick (solid white bar) or a thin (solid gray bar) exosporium and of spores frompurified spore suspensions with a thick (diagonally striped white bar) or a thin (diagonally striped gray bar) exosporium were estimated from transmissionelectron micrographs of at least 10 individual spores. Error bars denote standard errors of the means. Asterisks denote statistically significant differences(n.s., not significant; **, P � 0.01; ****, P � 0.0001). (C) The thicknesses of the exosporium and the outer and inner coat layers of C. difficile spores fromunwashed sporulating cultures with a thick (solid white bar) or a thin (solid gray bar) exosporium and of spores from biofilm biomass with a thick(diagonally striped white bar) or a thin (diagonally striped gray bar) exosporium were estimated by transmission electron micrographs of at least 10individual spores. Error bars denote standard errors of the means. Data on the length of the filaments of the outer layers of spores from unwashedsporulating cultures are the same as those in panel B and are used for comparative purposes. Asterisks denote statistically significant differences (n.s., notsignificant; *, P � 0.05; **, P � 0.01; ***, P � 0.001; ****, P � 0.0001).

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lation is a question that remains to be answered in order to beginunderstanding the dynamics of appearance of these morphotypesand their roles during an infection.

Transmission electron micrographs of biofilms of the epi-demic R20291 strain demonstrate that (i) C. difficile sporesformed during biofilms also have an exosporium layer and hair-like extensions and (ii) spores with both exosporium morpho-types also appear during the development of C. difficile biofilms.These observations are meaningful, since previous evidence (11)suggested that spores formed during biofilm development lackedthe outermost exosporium-like layer commonly observed in var-ious epidemic strains (13, 14). However, whether the compositionof the outermost surface layers of biofilm-formed spores is similarto that of traditionally prepared spores or to that of in vivo-pre-pared spores remains unclear. Remarkably, the proportion ofthick to thin exosporium spores differed from that observed insporulating cultures and purified spores, where a 1:1 ratio ofspores with a thick and a thin exosporium layer was observed inbiofilms. These differences might indicate that slight variations inthe regulatory mechanism(s) in exosporium assembly might oc-cur during biofilm development, leading to a variation in the ratioof thick to thin exosporium morphotypes. This hypothesis mightbe consistent with the thinner spore coat layers observed in bio-film-formed spores than in spores formed under traditional spo-rulating conditions, particularly between spores with a thin exo-sporium layer.

In summary, this work complements our previous study(13) by providing convincing evidence that, independently ofthe conditions under which spores are formed, C. difficile pro-duces spores with two different morphotypes of the exospo-rium layer. This should be considered in the development ofspore removal strategies based on exosporium proteins to en-sure efficient development of novel strategies to combat C.difficile infections.

ACKNOWLEDGMENTS

This work was supported by grants from Fondo Nacional de Ciencia yTecnología de Chile (FONDECYT grant 1151025), from the ResearchOffice of Universidad Andres Bello (DI-641-15/R 2015) (to D.P.-S.), andfrom the Fondo Nacional de Ciencia y Tecnología de Chile, DoctoralFellowship CONICYT 21151202 to M.P.-G.

FUNDING INFORMATIONThis work, including the efforts of Paulina Calderón-Romero, was fundedby MINEDUC | Comisión Nacional de Investigación Científica y Tec-nológica (CONICYT) (21140380). This work, including the efforts ofMarjorie Pizarro-Guajardo, was funded by MINEDUC | Comisión Nacio-nal de Investigación Científica y Tecnológica (CONICYT) (21151202).This work, including the efforts of Daniel Paredes-Sabja, was funded byMINEDUC | Comisión Nacional de Investigación Científica y Tec-nológica (CONICYT) (1151025).

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