Articles

Characterization of the Dynamic Germination of Individual Clostridium difficile Spores Using Raman Spectroscopy and Differential Interference Contrast Microscopy

Shiwei Wang, Aimee Shen, Peter Setlow, Yong-qing Li
P. de Boer, Editor
DOI: 10.1128/JB.00200-15

ABSTRACT

The Gram-positive spore-forming anaerobe Clostridium difficile is a leading cause of nosocomial diarrhea. Spores of C. difficile initiate infection when triggered to germinate by bile salts in the gastrointestinal tract. We analyzed germination kinetics of individual C. difficile spores using Raman spectroscopy and differential interference contrast (DIC) microscopy. Similar to Bacillus spores, individual C. difficile spores germinating with taurocholate plus glycine began slow leakage of a ∼15% concentration of a chelate of Ca2+ and dipicolinic acid (CaDPA) at a heterogeneous time T1, rapidly released CaDPA at Tlag, completed CaDPA release at Trelease, and finished peptidoglycan cortex hydrolysis at Tlysis. T1 and Tlag values for individual spores were heterogeneous, but ΔTrelease periods (Trelease − Tlag) were relatively constant. In contrast to Bacillus spores, heat treatment did not stimulate spore germination in the two C. difficile strains tested. C. difficile spores did not germinate with taurocholate or glycine alone, and different bile salts differentially promoted spore germination, with taurocholate and taurodeoxycholate being best. Transient exposure of spores to taurocholate plus glycine was sufficient to commit individual spores to germinate. C. difficile spores did not germinate with CaDPA, in contrast to B. subtilis and C. perfringens spores. However, the detergent dodecylamine induced C. difficile spore germination, and rates were increased by spore coat removal although cortex hydrolysis did not follow Trelease, in contrast with B. subtilis. C. difficile spores lacking the cortex-lytic enzyme, SleC, germinated extremely poorly, and cortex hydrolysis was not observed in the few sleC spores that partially germinated. Overall, these findings indicate that C. difficile and B. subtilis spore germination exhibit key differences.

IMPORTANCE Spores of the Gram-positive anaerobe Clostridium difficile are responsible for initiating infection by this important nosocomial pathogen. When exposed to germinants such as bile salts, C. difficile spores return to life through germination in the gastrointestinal tract and cause disease, but their germination has been studied only with population-wide measurements. In this work we used Raman spectroscopy and DIC microscopy to monitor the kinetics of germination of individual C. difficile spores, the commitment of spores to germination, and the effect of germinant type and concentration, sublethal heat shock, and spore decoating on germination. Our data suggest that the order of germination events in C. difficile spores differs from that in Bacillus spores and provide new insights into C. difficile spore germination.

INTRODUCTION

Clostridium difficile is a Gram-positive, spore-forming, strictly anaerobic bacterium that has become a leading cause of antibiotic-associated diarrhea worldwide (1). Antibiotic treatment disrupts the normal colonic flora that typically suppresses C. difficile growth and allows ingested C. difficile spores to germinate, outgrow, and proliferate rapidly in the gastrointestinal tract (2–5). Once established in the gastrointestinal tract, C. difficile secretes two toxins, the enterotoxin TcdA and the cytotoxin TcdB, that cause massive damage to the intestinal epithelium and induce strong inflammatory responses (6). During growth in the gastrointestinal tract, C. difficile strongly induces a transcriptional program that leads to spore formation (7). Spore formation is essential for C. difficile to survive exit from the host and transmit disease because its vegetative cells are exquisitely sensitive to oxygen (8). Furthermore, C. difficile spores are resistant to antibiotics (9), attacks from the host's immune system (10), and disinfectants commonly used in hospital settings due to their metabolic dormancy and intrinsic resistance properties (11–14). Thus, understanding the mechanisms controlling C. difficile spore germination may have practical applications in the management of C. difficile infections as this knowledge may lead to new methods for preventing spore germination or efficiently promoting it to facilitate killing of the less resistant germinated spores.

Although spore germination is essential for C. difficile to initiate disease (15), our knowledge of C. difficile spore biology lags behind that of other well-studied spores, such as those of Bacillus subtilis (16). B. subtilis spore germination can be triggered by a variety of factors, including nutrients, enzymes, hydrostatic pressure, cationic surfactants such as dodecylamine, and a 1:1 chelate of Ca2+ and pyridine-2,6-dicarboxylic acid (dipicolinic acid; DPA) (CaDPA) (17, 18). Nutrient germinants for spores of Bacillus species include l-amino acids, purine nucleosides, and d-sugars as well as some mixtures of such compounds. These compounds trigger germination by binding to and interacting with germination receptors (GRs) present in the spore's inner membrane (IM), leading to a series of events taking place in a defined order (19, 20). First, exposure of spores to nutrient germinants causes a reaction that commits spores to germinate even if the germinant is removed or displaced from its cognate GRs (20–22). This commitment step is followed by release of monovalent cations, as well as the spore core's large pool of CaDPA. Ultimately, the spore's peptidoglycan cortex is degraded by either of two redundant cortex-lytic enzymes (CLEs), CwlJ and SleB, and subsequently the spore core swells and takes up water, leading to initiation of metabolism (17).

The process of an individual spore's germination has been divided into four phases according to a spore's optical intensity in differential interference contrast (DIC) or phase-contrast microscopy, with the different phases ending at times T1, Tlag, Trelease, and Tlysis (23, 24). T1 is the time when slow CaDPA leakage begins after germinant addition and is probably coincident with the time of commitment, Tlag is the time after the start of slow CaDPA leakage when the initiation of very rapid CaDPA release begins, and Trelease is the time for completion of rapid CaDPA release; following Trelease, there is a further small decline in spore refractility due to the hydrolysis of the spore cortex and spore core swelling, and the time when spore refractility becomes constant is termed Tlysis.

While there are some similarities between B. subtilis and C. difficile spore germination, there are a number of notable differences. A major difference is that the C. difficile genome lacks homologs of the GRs that exist in Bacillus species and in many Clostridium species (16, 25). Instead, C. difficile uses the CspC pseudoprotease as a noncanonical germination receptor to directly sense bile salts and activate germination (15). Whereas B. subtilis spores germinate in response to amino acids, nucleosides, and small cations, C. difficile spores germinate in response to specific bile salts, in particular, cholate and its derivatives (taurocholate, glycocholate, cholate, and deoxycholate), with glycine acting as a cogerminant; glycine on its own is insufficient to trigger C. difficile spore germination (26, 27). It should be noted that spores of some C. difficile strains can germinate in rich medium without taurocholate (28), suggesting that the triggers for C. difficile spore germination may be complex. In addition to these differences in germinant sensing, the regulation of cortex hydrolysis differs between C. difficile and B. subtilis (15, 16, 29). C. difficile spores require only a single CLE, SleC (30), which is made as a zymogen that is proteolytically activated by the serine protease CspB, similar to the situation in Clostridium perfringens (31–33). Thus, the environmental signals that trigger germination, the germinant receptors, and the regulation of cortex hydrolysis exhibit notable differences between C. difficile and B. subtilis.

To date the germination of C. difficile spores has been studied only with spore populations although it is known that the generally considerable heterogeneity in germination rates between individuals in spore populations can obscure individual germination events (34). In the current work we have used laser tweezers, Raman spectroscopy, and DIC microscopy to analyze the kinetics of the germination of multiple individual C. difficile spores. The roles of a variety of factors important in spore germination were also investigated, including variations in germination behavior between spores of different strains, responses to different bile salts, the dependence on bile salt and glycine concentrations, effects of sublethal heat shock or decoating prior to germination, and germination by dodecylamine and CaDPA. The results of the study provide new insight into the germination of C. difficile spores.

MATERIALS AND METHODS

Bacterial strains and spore preparation.The C. difficile strains used in this study were ATCC 43593 and ATCC 43601 (35). The PCR ribotype for each C. difficile strain used was ribotype 060 for ATCC 43593 and ribotype 031 for ATCC 43601. Strain JIR8094 and its isogenic sleC mutant were also used to look at the effect of SleC on cortex hydrolysis and spore germination (29). The B. subtilis strain used was strain PS533, an isogenic derivative of a laboratory 168 strain and carrying plasmid pUB110 providing resistance to 10 μg/ml kanamycin (36). Spores of C. difficile were prepared on 70:30 sporulation medium agar (37) and were harvested, purified, and stored as described previously (29). Briefly, C. difficile spores were washed extensively with water and then purified on a Histodenz gradient, followed by subsequent water washes. Spores of B. subtilis were prepared at 37°C on 2× Schaeffer's-glucose medium agar plates and were purified as described previously (38). All spores used in this study were >98% free of sporulating cells, germinated spores, and debris, as observed by phase-contrast microscopy. Where indicated below, C. difficile spores were chemically decoated by incubation of spores at an optical density at 600 nm (OD600) of ∼10 at 37°C in 1% SDS, 2% β-mercaptoethanol, and 0.1 M borate buffer (pH 10.0) (33). After 15 min of incubation, decoated spores were washed twice with 0.1 M borate buffer (pH 10.0) and five times with distilled water, and the washed spores were stored in water.

Spore germination.In order to examine the effects of prior sublethal heat shock on spore germination, C. difficile spores were incubated in water at 65°C for 30 min and then cooled on ice before germination. However, for most experiments, spores were not heat shocked since this treatment did not stimulate spore germination (see Results). Germination was carried out with different kinds and concentrations of bile salts and glycine (obtained from Sigma-Aldrich, Inc., St. Louis, MO) in 10 mM Tris-HCl (pH 7.4)–150 mM NaCl. To check the effects of glycine on C. difficile spore germination, 0.25% taurocholate and variable glycine concentrations were used, as were constant glycine concentrations and variable taurocholate concentrations. The additional bile salts used in this work include deoxycholate, cholate, glycocholate, taurodeoxycholate, chenodeoxycholate, and taurolithocholate. These bile salts were used at 0.5% (∼10 mM) with 19.6 mM glycine. Spore germination was also carried out at 37°C with 60 mM CaDPA made to pH 7.4 with Tris base and with 1.2 mM dodecylamine at 42°C in 25 mM K-HEPES buffer (pH 7.4) as described previously (24).

In experiments measuring spores' commitment to germinate, C. difficile spores on a coverslip (see below) were exposed to 0.25% taurocholate and 15.5 mM glycine in 10 mM Tris-HCl (pH 7.4)–150 mM NaCl at 37°C for 1 to 2 min, followed by germinant removal, five rinses with 10 mM Tris-HCl (pH 7.4)–150 mM NaCl by vacuum pump suction, and further incubation at 37°C in 10 mM Tris-HCl (pH 7.4)–150 mM NaCl until 30 min.

Measurement of CaDPA level and Raman spectra of individual spores by laser tweezers Raman spectroscopy.The CaDPA levels of individual spores of various strains were determined by laser tweezers Raman spectroscopy at 25°C (23, 39). Briefly, an individual spore was captured with laser tweezers, and its Raman spectrum was acquired with an integration of 20 s and a laser power of 20 mW at 780 nm. Spectra of 30 individual spores were measured and averaged. The CaDPA level in an individual spore was determined from the peak intensity at 1,017 cm−1 in its Raman spectrum relative to the peak intensity of the same Raman band from a CaDPA solution of known concentration (60 mM) and by multiplying this concentration value by the excitation volume of 1 fl to obtain attomoles of CaDPA/spore (23).

Monitoring germination of multiple individual spores.The kinetics of CaDPA release during germination of individual C. difficile spores optically trapped by laser tweezers was measured simultaneously by Raman spectroscopy and DIC microscopy as described previously (23, 40). The CaDPA level in an individual spore during germination was determined from the intensities of the CaDPA-specific Raman band at 1,017 cm−1, while the intensity of the DIC image was recorded. As found previously (23, 40), the end of the rapid fall in DIC image intensity during spore germination corresponded to the point at which release of CaDPA was complete, and this time point was defined as Trelease. At this time, the DIC image intensity (Irelease) was 25 to 30% of that at T0, when image intensity at T0 was set at 1 and the intensity at the end of measurements was set at zero. Consequently, the CaDPA content of wild-type spores at any time relative to T0 could be estimated from the DIC image intensity, It, as 100% × (It − Irelease)/(1 − Irelease) since the DIC intensity was found to be nearly coincident with the CaDPA level prior to Trelease (see Fig. 2a). In addition to Trelease, a number of other spore germination parameters have been previously described (23, 24, 40), including the following: T1, the time when CaDPA leakage starts; CaDPA leakage, the percent CaDPA released slowly between T1 and Tlag; Tlag, the time after T1 when very rapid CaDPA begins; Trelease, the time when CaDPA release is complete; Tlysis, the time when spore refractility becomes constant; and ΔTrelease and ΔTlysis, which are calculated as Trelease − Tlag and Tlysis − Trelease, respectively.

FIG 1
FIG 1

Average Raman spectra of 30 individual dormant and germinated spores of C. difficile and B. subtilis PS533 (a), difference spectra between dormant and germinated spores (b), and bright-field images of dormant spores (c) are shown. C. difficile spores were germinated at 37°C with 1.2% taurocholate and 15.5 mM glycine in 10 mM Tris-HCl (pH 7.4)–150 mM NaCl for 60 min. B. subtilis spores were heat shocked for 30 min at 70°C, cooled on ice, and then germinated at 37°C with 10 mM l-valine in 25 mM K-HEPES buffer (pH 7.4) for 60 min. The bright-field images of C. difficile and B. subtilis spores were recorded with a video camera. au, arbitrary units.

The germination of multiple individual spores was monitored by DIC microscopy as described previously (34, 40). In brief, spores were spread on the surface of a coverslip that was dried in a vacuum desiccator for ∼10 min, and the coverslips were mounted on and sealed to a microscope sample holder kept at a constant temperature. The DIC images of multiple spores adhered on coverslips were recorded at a rate of 1 frame per 15 s for 30 to 120 min by a digital charge-coupled-device camera (16 bits; 1,600 by 1,200 pixels) following the addition of preheated germinant solution to the spores on the coverslips. The averaged pixel intensity of an area of 40 by 40 pixels that covered each individual spore's DIC image was calculated, the DIC image intensity of each individual spore was plotted as a function of the incubation time with a resolution of 15 s and with initial image intensity at the first time of measurement, T0, normalized to 1, and the intensity at the end of the measurement period was normally set at zero. Invariably, the DIC image intensity had been constant for ≥10 min at the end of measurements. The degree of germination of spore populations was measured by simultaneously monitoring the germination of >200 individual spores by DIC microscopy, and at various times the percentage of these spores that had released their CaDPA was determined as described above.

RESULTS

Raman spectra and average CaDPA levels of individual C. difficile spores.Previous work has found that bands from CaDPA dominate the Raman spectra of individual spores of Bacillus species and C. perfringens (23, 41), and this was also the case for spores of two C. difficile strains (Fig. 1a). The average intensity of the CaDPA-specific 1,017 cm−1 Raman band from ∼30 individual spores indicated that the CaDPA levels in spores of C. difficile ATCC 43593 and ATCC 43601 were 498.1 ± 156.5 and 664.2 ± 162.0 amol/spore, respectively. Both of these values are larger than the value for B. subtilis PS533 spores (432.6 ± 48.6 amol/spore), suggesting that there is more CaDPA in spores of C. difficile species. However, this may be due to the larger size of C. difficile spores than B. subtilis spores, as seen in bright-field images (Fig. 1c). Average Raman spectra of ∼30 individual dormant spores with average germinated spores' Raman spectra subtracted also showed an obvious difference in the ratio between the 1,395 cm−1 and 1,447 cm−1 Raman spectral bands due to CaDPA (Fig. 1b), suggesting that the CaDPA environments in C. difficile and B. subtilis spores are somewhat different.

Kinetics of germination of individual C. difficile spores with taurocholate and glycine.Given the dominant CaDPA-specific band in the Raman spectrum of C. difficile spores and previous methods developed to follow the germination of individual spores of Bacillus species (23, 40), we used laser tweezers Raman spectroscopy and DIC microscopy to analyze the dynamic germination of individual C. difficile spores. The results with a single optically trapped C. difficile spore germinating with taurocholate and glycine showed that the fall in the spore's DIC image intensity corresponded to the release of CaDPA (Fig. 2a). In addition, this C. difficile spore gave a kinetic germination curve with the same four phases seen with spores of Bacillus species, including T1, Tlag, Trelease, and Tlysis (Fig. 2). The same results were also obtained when the germination of multiple individual optically trapped wild-type C. difficile spores was followed by monitoring CaDPA release and loss of DIC image intensity (Fig. 3 and Table 1). Notably, 13 to 16% of total spore CaDPA was released between T1 and Tlag, similar to results with nutrient germination of spores of Bacillus species (24). Given the good correspondence between CaDPA release and loss of DIC image intensity in spore germination, in further experiments we used DIC microscopy to monitor the germination of multiple individual C. difficile spores.

FIG 2
FIG 2

Germination of a single optically trapped C. difficile spore monitored by Raman spectroscopy and DIC microscopy. An optically trapped C. difficile ATCC 43593 spore was germinated with 1.2% taurocholate and 15.5 mM glycine, and DIC images and Raman spectra were recorded, all as described in Materials and Methods. The trapped spore appears as two bright spots in the DIC image due to the differential interference of the illumination polarized light (40). (a) The germination curves were determined from either the loss in spore DIC image intensity or the intensities of the CaDPA-specific Raman band at 1,017 cm−1. The DIC image and Raman band intensities in arbitrary units (a.u.) were normalized to 1 based on the respective values at the first time of measurement, and DIC image intensities at the end of the experiment were set at 0. T1, Tlag, Trelease, and Tlysis for this spore are indicated. (b) Sequential Raman spectra during germination of the single trapped spore were obtained as described in Materials and Methods.

FIG 3
FIG 3

Germination of multiple individual optically trapped C. difficile spores as monitored simultaneously by DIC microscopy and Raman spectroscopy. C. difficile ATCC 43593 spores were germinated with 1.2% taurocholate and 15.5 mM glycine, and spore germination was monitored, all as described in Materials and Methods. Germination curves were determined from the loss in spore DIC image intensity (a) or the intensities of the CaDPA-specific Raman bands at 1,017 cm−1 (b). The DIC image and Raman band intensities in arbitrary units (a.u.) were normalized to 1 based on the respective values at the first time of measurement, and DIC image intensities at the end of the experiment were set at 0. Trelease (arrow) is indicated for each individual spore.

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TABLE 1

Kinetic parameters of C. difficile spores with or without heat shock and germinating with taurocholate and glycinea

The germination of spores of the C. difficile wild-type strain JIR8094 and its isogenic sleC derivative were also examined to assess the effects of SleC on the overall germination process of C. difficile spores (see Fig. S4 in the supplemental material). Strikingly, while ∼90% of the wild-type spores germinated in ∼20 min with taurocholate plus glycine, at most 1% of sleC spores germinated in this time and even with higher germinant concentrations; even after 600 min, only ∼5% of the sleC spores had germinated (see Fig. S4a and b). It was also striking that the individual sleC spores that germinated did not show any cortex hydrolysis period (ΔTlysis) in the curves of DIC image intensity and displayed much higher absolute DIC image intensity at the end of the experiment than the wild-type spores (see Fig. S4c and d). This suggests that sleC spores do not undergo any cortex hydrolysis process, similar to C. perfringens sleC mutant spores (41). It was also notable that the ΔTrelease period for CaDPA in the wild-type spores that germinated was ∼2.5 min, while for the sleC spores that germinated this value was much larger at ∼40 min. The accelerated CaDPA ΔTrelease times by cortex hydrolysis have also been seen for germinating B. subtilis and Bacillus megaterium spores (42, 43).

Effect of heat shock on C. difficile spore germination.A short treatment at a high but generally sublethal temperature termed a heat shock can generally potentiate and synchronize germination of spores of Bacillus and at least some Clostridium species (44). Although the mechanism of this effect is unknown, it is generally seen only for GR-dependent spore germination (45). Examination of the effects of such a heat shock on C. difficile spores showed that heat shock actually decreased the extent of spore germination with taurocholate plus glycine (Fig. 4; Table 1). In addition, while the kinetic parameters of ATCC 43593 spore germination were similar for spores with and without heat shock, the heat-shocked ATCC 43601 spores had much larger values of T1, Tlag, and Trelease (Fig. 4 and 5; Table 1). However, ΔTrelease times (corresponding to rapid CaDPA release) of ATCC 43601 spores were relatively unaffected by heat shock, as were the amounts of CaDPA leakage between T1 and Tlag (Table 1). The lack of stimulation of C. difficile spore germination by a heat shock is consistent with several previous reports examining germination of spore populations (15, 46), and together these results suggest that heat activation is not important in C. difficile spore germination, at least for the spores of strains that have been studied to date.

FIG 4
FIG 4

Effect of heat shock on germination of spores of two C. difficile strains. ATCC 43593 and ATCC 43601 spores (>200 each) with or without heat shock were germinated at 37°C with 0.25% taurocholate and either 15.5 mM glycine (a) or 19.6 mM glycine (b), and spore germination percentages were determined by DIC microscopy, all as described in Materials and Methods.

FIG 5
FIG 5

Germination curves of multiple individual C. difficile spores with or without a prior heat shock. ATCC 43593 and ATCC 43601 spores were germinated with 0.25% taurocholate and either 15.5 mM (a and b) or 19.6 mM (c and d) glycine for 30 min, and DIC image intensities of multiple individual spores were determined, all as described in Materials and Methods. The image intensities (a.u.) were normalized to 1 based on the respective values at the first time of measurement, and image intensities at the end of the experiment were set at 0. T1, Tlag, Trelease, and Tlysis are indicated for a single spore.

Effects of taurocholate and glycine concentrations and various bile salts on C. difficile spore germination.Previous work found that taurocholate and glycine act as cogerminants for C. difficile spore germination (26). Initially, we examined the germination of multiple individual spores of the two C. difficile strains with different concentrations of taurocholate and glycine (Fig. 6). In the presence of 0.25% taurocholate, the extent of germination of the C. difficile spores increased with increasing glycine concentrations up to 15.5 and 19.6 mM glycine for ATCC strains 43593 and 43601, respectively (Fig. 6a and c). When the glycine concentration was held constant at 15.5 and 19.6 mM glycine for ATCC 43593 and 43601, respectively, the extent of spore germination increased until the taurocholate concentration reached 0.25% (Fig. 6b and d). In the presence of 15.5 mM glycine, T1, Tlag, and Trelease times and the CaDPA leakage between T1 and Tlag of ATCC 43593 spore germination were all reduced when taurocholate concentrations were increased from 0.05% to 0.25% although ΔTrelease and ΔTlysis (corresponding to presumed cortex hydrolysis) times were relatively unchanged (Table 2; also data not shown). In the presence of 0.25% taurocholate, T1, Tlag, and Trelease times of ATCC 43593 spore germination decreased markedly when glycine concentrations increased from 9.1 to 15.5 mM although ΔTrelease and ΔTlysis times and the amounts of CaDPA leakage between T1 and Tlag were relatively unchanged (Table 3). Notably, low concentrations of glycine (1.3 mM) were insufficient to stimulate germination in the presence of 0.1% taurocholate for both C. difficile strains even after incubation for 120 min (data not shown).

FIG 6
FIG 6

Effects of glycine and taurocholate concentrations on C. difficile spore germination. More than 200 individual ATCC 43593 or ATCC 43601 C. difficile spores were germinated at with 0.25% taurocholate and various glycine concentrations (a and c) or with 15.5 mM (b) or 19.6 mM (d) glycine and various taurocholate concentrations. Spore germination was monitored by DIC microscopy, all as described in Materials and Methods.

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TABLE 2

Kinetic parameters of C. difficile spores germinating with constant glycine and variable taurocholate concentrationsa

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TABLE 3

Kinetic parameters of C. difficile spore germination with constant taurocholate and various glycine concentrationsa

In addition to effects of concentrations of bile salts and glycine on spore germination rates, different bile salts also have various effects on C. difficile spore germination (26, 28). Analysis of the effects of different bile salts on C. difficile spore germination showed that, among the bile salts tested, only taurodeoxycholate resulted in germination at an extent similar to that of taurocholate (Fig. 7). This finding contrasted with the prior observation that taurodeoxycholate is ∼70% less effective at activating C. difficile strain 630 spores (27) and may reflect strain-specific differences, as has been observed by previous groups (28). Deoxycholate, cholate, or glycocholate partially activated germination although the magnitude of this germination differed somewhat between the spores of the two C. difficile strains and was always significantly less than that with taurocholate (Fig. 7; Tables 4 and 5). The major differences in the kinetic parameters of the germination of the C. difficile spores with the different bile salts were in the T1, Tlag, and Trelease times and the amounts of CaDPA leakage between T1 and Tlag, while ΔTrelease and ΔTlysis times were essentially identical for the five bile salts that induced at least some spore germination (Tables 4 and 5 and data not shown). As found in previous work, chenodeoxycholate (47) and taurolithocholate (27) did not trigger germination of C. difficile spores (Fig. 7). Consistent with the prior observation indicating that chenodeoxycholate is a competitive inhibitor of taurocholate for C. difficile UK1 spores, 0.25% chenodeoxycholate significantly inhibited germination stimulated by treatment with 0.25% taurocholate plus glycine of spores of both the C. difficile ATCC 43593 and 43601 strains. In contrast, 0.25% taurolithocholate failed to inhibit germination of spores of both strains under the same conditions (compare Fig. S1 in the supplemental material with Fig. 4), consistent with prior observations with C. difficile 630 spores (27).

FIG 7
FIG 7

Germination of C. difficile spores with different bile salts. Spores of C. difficile strains ATCC 43593 and ATCC 43601 were germinated with different bile salts at 0.5% plus 19.6 mM glycine, and germination of >200 spores was monitored by DIC image intensity changes, all as described in Materials and Methods.

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TABLE 4

Kinetic parameters of C. difficile ATCC 43593 spore germination with different bile saltsa

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TABLE 5

Kinetic parameters of C. difficile ATCC 43601 spore germination with different bile salts

C. difficile spore germination with CaDPA and dodecylamine.In addition to GR-dependent germinants, spores of Bacillus and some Clostridium species can also germinate with the GR-independent germinants CaDPA and dodecylamine (18–20, 48, 49). To test whether C. difficile spores were similarly responsive to these nonnutritional GR-independent germinants, the germination of C. difficile spores in the presence of CaDPA or dodecylamine was also tested. This analysis showed that spores of both C. difficile strains exhibited no (<0.3% with 300 spores examined) germination with CaDPA in 60 min, under conditions where B. subtilis spores routinely germinated >90% (data not shown). In contrast, intact spores of both C. difficile strains germinated with dodecylamine. ATCC 43593 spores had much lower average T1 times than ATCC 43601 spores (Fig. 8; Table 6). Notably, dodecylamine germination failed to decrease DIC image intensity after Trelease in either intact or decoated spores (Fig. 8c to f; see also Fig. S2 in the supplemental material), suggesting that cortex lysis either does not take place or takes place in parallel with CaDPA release during dodecylamine germination. However, the fact that the absolute DIC image intensity fell much less during dodecylamine germination than during germination with taurocholate plus glycine (see Fig. S3a and b in the supplemental material) strongly suggests that there is minimal cortex lysis during dodecylamine germination. There was also more CaDPA leakage between T1 and Tlag during dodecylamine germination than during germination by taurocholate plus glycine (Tables 1 and 6). Notably, the absolute DIC image intensity of C. difficile spores germinating with taurocholate plus glycine and chenodeoxycholate fell by an amount similar to that of spores germinating with taurocholate plus glycine (see Fig. S3a and c), indicating that chenodeoxycholate inhibits not only cortex hydrolysis but also CaDPA release. This observation is consistent with the observation that a cspC mutant fails to release DPA (15) as well as activate SleC cleavage (data not shown).

FIG 8
FIG 8

Germination of C. difficile spores with dodecylamine. Spores of C. difficile strains (ATCC 43593 and ATCC 43601), either intact or decoated, were germinated with dodecylamine as described in Materials and Methods. (a and b) Germination of >200 intact and decoated spores as a function of time. (c to f) DIC intensities of 20 individual intact spores as a function of time were measured as described in Materials and Methods. In panels c to f, T1, Tlag, and Trelease for one spore are indicated.

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TABLE 6

Kinetic parameters of intact or decoated C. difficile spores germinating with 1.2 mM dodecylaminea

The reasons for differences in rates of dodecylamine germination of spores of different strains are not known. Since chemical decoating can greatly increase rates of Bacillus spore germination with dodecylamine (48), we tested the effects of chemical decoating on C. difficile spore germination. While decoating had no major effect on the overall rate of the dodecylamine germination of the ATCC 43593 spore population (Fig. 8a), chemical decoating greatly increased ATCC 43601 spore germination with dodecylamine (Fig. 8b). In addition, decoating significantly decreased the average times between T1 and Tlag for the dodecylamine germination of spores of both C. difficile strains (Table 6). As seen with intact spores germinating with dodecylamine, there was also no obvious cortex lysis period during dodecylamine germination of decoated C. difficile spores (Fig. 8e and f), consistent with the observation that decoating removes C. difficile spores' only cortex-lytic enzyme, SleC (data not shown). Indeed, as expected, the decoated C. difficile spores exhibited <0.3% germination with 0.5% taurocholate or taurodeoxycholate plus 19.6 mM glycine when 300 spores were examined for 60 min (data not shown).

Measurement of commitment of C. difficile spores to germinate.A notable feature of B. subtilis spores is their irreversible commitment to germination, followed by some slow CaDPA leakage after a short exposure to germinant, with commitment taking place at approximately T1 (22, 24, 50). Since C. difficile spores also showed a T1 time followed by some slow CaDPA leakage, we examined if C. difficile spores also showed commitment after exposure to taurocholate and glycine for various periods, followed by germinant removal, extensive rinsing, and further incubation in buffer (Fig. 9). Like Bacillus spores, C. difficile spores given a short germinant exposure continued to release CaDPA after germinant removal. Indeed, spores given a 2-min exposure to germinants that gave only ∼10% germination at the time of germinant removal ultimately reached ∼76% germination (Fig. 9). The commitment phenomenon in germination with taurocholate plus glycine was not eliminated if 0.25% chenodeoxycholate was added after committed spores were rinsed and prior to subsequent incubation (data not shown). Taken together, these results suggest that C. difficile spores, similar to B. subtilis spores, also commit to germination shortly after exposure to germinant.

FIG 9
FIG 9

Commitment in C. difficile spore germination. ATCC 43593 spores were exposed to 0.25% taurocholate and 15.5 mM glycine in 10 mM Tris-HCl (pH 7.4)–150 mM NaCl at 37°C for short periods (arrows denote ends of germinant exposures), followed by germinant removal, rinsing with vacuum suction, and further incubation, as described in Materials and Methods. The exposure periods were as follows: 1 to 0.83 min (red), 2 to 1 min (blue), 3 to 1.5 min (pink), and 4 to 2 min (green). ATCC 43593 spores were also germinated continuously (black) with 0.25% taurocholate and 15.5 mM glycine. In all cases, the germination of >200 spores was monitored by DIC microscopy as described in Materials and Methods.

DISCUSSION

Analysis of the germination of individual C. difficile spores indicated that both the kinetics and the various stages of this process were essentially the same as with individual spores of a number of Bacillus species, Geobacillus stearothermophilus, and Clostridium perfringens (Fig. 1 and 2) (24, 41, 51). This result was somewhat surprising because the signal transduction pathway in germination of at least Bacillus spores is quite different from that in C. difficile spore germination (17, 18, 52). In Bacillus sp. spore germination, binding of nutrient germinants to their germinant receptors triggers CaDPA release, and this in turn triggers cortex lysis. However, in C. difficile spores, binding of bile salt germinants to CspC triggers cortex lysis and CaDPA release (15), with the proviso that the precise mechanism of action of the cogerminant glycine is not known.

As a result, while the overall kinetics of C. difficile spore germination resembled that of B. subtilis, the order of CaDPA release appeared to differ between these two organisms. During C. difficile spore germination, CaDPA release appears to precede cortex lysis, or at least the great majority of cortex lysis (Fig. 8; see also Fig. S3 in the supplemental material). How can CaDPA release precede cortex hydrolysis during C. difficile spore germination? One simple explanation is that only a small amount of cortex hydrolysis is needed to trigger CaDPA release, and this cortex hydrolysis is not reflected in changes in the DIC image intensity of the spores, perhaps because there are no such changes until later. Indeed, while GR-dependent CaDPA release can take place in Bacillus spore germination even in the absence of both redundant CLEs, values of ΔTrelease are increased 5- to 10-fold simply by the loss of one specific CLE, CwlJ (42, 43), and this was also seen in this work with sleC mutant C. difficile spores. Thus, it seems likely that there is some cortex hydrolysis during taurocholate-induced C. difficile spore germination, beginning perhaps at or even before T1, that causes changes in spore IM permeability or leakiness of the CaDPA channel, and then CaDPA leakage begins. This CaDPA leakage, combined with further cortex hydrolysis, could trigger faster CaDPA release. Notably, the same kinetics of GR-dependent germination are also seen in the germination of C. perfringens spores, in which GR-dependent germinants can trigger some CaDPA release even in the absence of SleC although loss of both SleC and the auxiliary CLE SleM greatly reduced CaDPA release in GR-dependent germination (31).

It was also notable that while germination of C. difficile spores was slowed tremendously in the absence of SleC, a small percentage of the sleC mutant spores released CaDPA but did not exhibit cortex hydrolysis. The latter observation is consistent with the low viability of sleC mutant C. difficile spores and the restoration of significant viability by inclusion of lysozyme in plating medium (30). An important question then concerns the mechanism of the germination of the small percentage of sleC mutant spores that germinated in 10 h, as measured by their release of CaDPA, as this is perhaps analogous in some ways to the spontaneous germination of small percentages of B. subtilis spores that lack all GRs (53).

Another surprising result was that there was no evidence of cortex hydrolysis during dodecylamine germination of C. difficile spores, in contrast to clear cortex hydrolysis during dodecylamine germination of Bacillus spores (42, 48). Notably, there also does not appear to be any cortex hydrolysis during C. perfringens spore germination with dodecylamine (41). In Bacillus spore germination with dodecylamine, this agent is thought to bind to and activate the spore's IM channel for CaDPA composed of SpoVA proteins (17, 18). Many of these SpoVA proteins are highly conserved in spore-forming members of Bacillales and Clostridiales including C. difficile (16). Dodecylamine may bind specifically to the conserved SpoVAC protein, which was recently shown to have mechanosensitive properties (54). As noted above, in Bacillus spores CaDPA release activates CLEs, at least CLE probably directly, such that cortex hydrolysis is triggered in the process of dodecylamine germination. However, during germination of Clostridiales spores, neither CaDPA release nor its absence from spores appears to trigger CLE action (16) (Fig. 8; see also Fig. S3 in the supplemental material). Whereas DPA-less B. subtilis spores that lack the SpoVA proteins essential for CaDPA uptake in sporulation spontaneously germinate very rapidly during sporulation (55), DPA-less spoVA C. perfringens spores are completely stable (56). Presumably this will also be the case for spoVA C. difficile spores. Consequently, C. difficile spores triggered directly to release CaDPA may have no way to efficiently activate the hydrolysis of their cortex peptidoglycan and thus cannot activate events downstream of CaDPA release. In further support of this simple model for Clostridium spore germination, the rapid GR-dependent CaDPA release from C. perfringens spores as measured by ΔTrelease values is not altered notably by loss of SleC (41), in contrast to the large increases in ΔTrelease values due to loss of the CLE CwlJ (42, 43).

The sensitive Raman spectroscopy and DIC microscopy techniques used in this study allowed us not only to measure DPA release and cortex hydrolysis in real time but also to analyze for the first time the responsiveness of individual C. difficile spores to different germinants. Until now, germinant responsiveness of C. difficile spores was generally restricted to population-wide measurements using OD600 or bulk DPA release assays. Using assays of individual spores, we observed that spores of different C. difficile strains exhibited marked differences in their responsiveness to heat treatment (Fig. 4; Table 1) and bile salts plus glycine (Fig. 6 and 7; Tables 4 and 5), similar to results with spores of other C. difficile strains (28). The most notable difference in the germination of the spores of the two strains examined in this work was in their germination with dodecylamine as ATCC 43601 spores were much slower to respond to this germinant than ATCC 43593 spores (Fig. 8; Table 6). Since this difference was not seen with decoated spores, this suggests that precise structures of spores' outer layers, the coats and/or outer membranes, are major factors in spores' rates of germination with dodecylamine. Differences in the permeability of the outer layers of C. difficile spores between strains may lead to differential responsiveness to bile salt germinants (52, 57), especially since the differences in dodecylamine sensitivity of the ATCC 43593 and 43601 strains largely disappeared following decoating. However, the exact mechanism restricting dodecylamine access to the spore's inner membrane in intact spores is not yet clear. In spores of Bacillus species, the GerP proteins, a group of small proteins likely present in the spore coat, are thought to be crucial to ensure rapid access of some germinants to spores' IMs (58, 59). However, proteins analogous to GerP proteins have not been identified in Clostridium spores.

A second notable finding in this work was that C. difficile spores exhibited no detectable germination with exogenous CaDPA (data not shown). This is in contrast to spores of Bacillus species in which exogenous CaDPA or CaDPA released from the spore core activates the CLE CwlJ, perhaps directly. In contrast, CaDPA appears to activate a GR in C. perfringens spores (49). Given this finding, the absence of IM GRs in C. difficile spores (16) would then be consistent with these spores' lack of germination with CaDPA. This would further suggest that CaDPA plays no direct role in the signaling pathways in C. difficile spore germination, as suggested above based on the results with dodecylamine germination.

The final notable observation in this work is that C. difficile spores showed the phenomenon of commitment in spore germination, whereby brief treatment with bile salts plus glycine resulted in germination events much later than and well after germinant removal. Prior work also found that a short exposure to taurocholate or deoxycholate enhanced colony formation by C. difficile spores, and it was suggested that this indicated that C. difficile spores also exhibit commitment to germinate (26). Bacillus spores also exhibit commitment in GR-dependent germination (18, 22, 24, 32, 50) although the mechanism of this commitment is not known. However, in Bacillus spores, commitment appears to parallel a large change in the permeability of either the spore IM or the CaDPA channel, such that CaDPA leakage begins at about the time of commitment. Since C. difficile spores also exhibited slow CaDPA leakage early in germination, it is tempting to speculate that this is also associated with commitment in C. difficile spore germination. The mechanism of establishing commitment is, however, unclear, although one obvious possible mechanism is that commitment reflects the activation of significant CspB protease activity by bile salts plus glycine. In this model, activated CspB protease persists after germinant removal and catalyzes pro-SleC activation, which leads to cortex hydrolysis followed by slow leakage of CaDPA release that transitions to fast CaDPA release. Since the precise signal transduction pathway in C. difficile spore germination is not yet clear, testing this model will yield important insights into the mechanisms regulating C. difficile spore germination.

ACKNOWLEDGMENTS

This work was supported by a Department of Defense Multidisciplinary University Research Initiative through the U.S. Army Research Laboratory and the U.S. Army Research Office under contract number W911F-09-1-0286 (P.S. and Y.-Q.L.) and by a grant from the Army Research Office under contract number W911NF-12-1-0325. A.S. is a Pew Scholar in the Biomedical Sciences, supported by The Pew Charitable Trusts, and is supported by award number R01GM108684 from the National Institute of General Medical Sciences.

The content is solely the responsibility of the authors and does not necessarily reflect the views of the Pew Charitable Trusts, the National Institute of General Medical Sciences, or the National Institutes of Health.

FOOTNOTES

    • Received 14 March 2015.
    • Accepted 27 April 2015.
    • Accepted manuscript posted online 4 May 2015.
  • Address correspondence to Yong-qing Li, liy{at}ecu.edu.

  • Citation Wang S, Shen A, Setlow P, Li Y-Q. 2015. Characterization of the dynamic germination of individual Clostridium difficile spores using Raman spectroscopy and differential interference contrast microscopy. J Bacteriol 197:2361–2373. doi:10.1128/JB.00200-15.

  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/JB.00200-15.

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