Imaging Clostridioides difficile Spore Germination and Germination Proteins

Abstract

Clostridioides difficile spores are the infective form for this endospore-forming organism. The vegetative cells are intolerant to oxygen and poor competitors with a healthy gut microbiota. Therefore, in order for C. difficile to establish infection, the spores have to germinate in an environment that supports vegetative growth. To initiate germination, C. difficile uses Csp-type germinant receptors that consist of the CspC and CspA pseudoproteases as the bile acid and cogerminant receptors, respectively. CspB is a subtilisin-like protease that cleaves the inhibitory propeptide from the pro-SleC cortex lytic enzyme, thereby activating it and initiating cortex degradation. Though several locations have been proposed for where these proteins reside within the spore (i.e., spore coat, outer spore membrane, cortex, and inner spore membrane), these have been based, mostly, on hypotheses or prior data in Clostridium perfringens. In this study, we visualized the germination and outgrowth process using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and used immunogold labeling to visualize key germination regulators. These analyses localize these key regulators to the spore cortex region for the first time. IMPORTANCE Germination by C. difficile spores is the first step in the establishment of potentially life-threatening C. difficile infection (CDI). A deeper understanding of the mechanism by which spores germinate may provide insight for how to either prevent spore germination into a disease-causing vegetative form or trigger germination prematurely when the spore is either in the outside environment or in a host environment that does not support the establishment of colonization/disease.

Keywords: Clostridium difficile; cortex; endospores; germination; imaging.

FIG 1

C. difficile spore structure and cell morphology visualized by TEM and SEM. Spores (A to F) and vegetative cells (G to I) derived from the C. difficile R20291 strain were embedded in epoxy resin, sectioned, and imaged by TEM (A, B, C, G, and H) or were chemically dried, sputter coated, and imaged by SEM (D, E, F, and I). IM, inner membrane; GCW, germ cell wall; OM, outer membrane; EXO, exosporium.

FIG 2

C. difficile cortex significantly thins within 5 min of germinant addition. C. difficile spores were germinated in rich medium supplemented with 10 mM TA and 30 mM glycine. A sample was taken prior to the addition of germinants (T = 0 min) and 5 min after germinant additions (T = 5 min). (A) Representative images of T = 0 min spores and T = 5 min spores. Boxes represent areas of cortex thickness measurement, with magnified view with measurement scale bars shown underneath. (B) Average cortex thickness was measured in cross-sectioned and longitudinally sectioned spores, at T = 0 min (T₀) and T = 5 min (T₅). n = 10 spores of each section orientation counted under both conditions (total n = 20). Cortex thickness was measured using the Fiji Scale Bar tool. The asterisk indicates a P value of <0.0001 as determined by one-way ANOVA using Tukey’s multiple-comparison test. “ns” indicates no significance.

FIG 3

Monitoring C. difficile spore germination over time (T = 0 to T = 60 min). Dormant (T = 0 min) C. difficile R20291 spores or spores germinated for 5, 30, and 60 min (T = 5, T = 30, and T = 60 min) were embedded in epoxy resin, sectioned, and imaged by TEM.

FIG 4

Monitoring C. difficile spore germination over time (T = 90 min and T = 180 min). C. difficile R20291 spores germinated for T = 90 min and T = 180 min were embedded in epoxy resin, sectioned, and imaged by TEM. Arrows indicate cells exiting the coat/exosporium layer.

FIG 5

C. difficile outgrowth occurs inside the coat/exosporium layers. Representative images of spores and vegetative cells derived from the C. difficile R20291 strain, germinated for 180 min, fixed, chemically dried, sputter coated, and imaged by SEM.

FIG 6

CspB, CspA, CspC, and SleC are located within the spore cortex. Spores derived from C. difficile R20291, C. difficile ΔcspBAC, C. difficile ΔsleC, C. difficile ΔcspBAC pcspBA_FLAG, and C. difficile ΔsleC psleC_FLAG were fixed; embedded in acrylic resins; sectioned; immunolabeled with antibodies specific for the CspB, CspA, CspC, and SleC proteins or the FLAG epitope; and then imaged by TEM. (A) Representative images of anti-FLAG-immunolabeled spores derived from the CspBA_FLAG (left)- and SleC_FLAG (right)-expressing strains. (B) Spores derived from the above-mentioned strains were imaged, and gold particles were counted. For each sample immunolabeled in this way, we imaged 100 random spores on the grid and counted the gold labels present. Counts for the spores derived from the C. difficile R20291, C. difficile ΔcspBAC, and C. difficile ΔsleC strains labeled with anti-CspB, anti-CspA, anti-CspC, and anti-SleC primary and gold-conjugated secondary antibodies. For clarity, only the counts for the gold particles located in the cortex are shown since the counts for the coat layer and the core do not reach statistical significance. Counts were compared to the counts observed in the deletion mutant. (C) Counts for the spores derived from the C. difficile R20291, C. difficile ΔcspBAC pcspBA_FLAG, and C. difficile ΔsleC psleC_FLAG strains, labeled with anti-FLAG primary and gold-conjugated secondary antibodies, and in various spore regions. The data represent the average from 3 or 4 biological replicates, and the error bars represent the standard errors of the means. **, P < 0.0001, and *, P < 0.05, as determined by one-way ANOVA using Šidák’s multiple-comparison test. An additional, representative, image for each strain can be found in Fig. S1 in the supplemental material. WT, wild type.