Spore Cortex Hydrolysis Precedes Dipicolinic Acid Release during Clostridium difficile Spore Germination

Abstract

Bacterial spore germination is a process whereby a dormant spore returns to active, vegetative growth, and this process has largely been studied in the model organism Bacillus subtilis. In B. subtilis, the initiation of germinant receptor-mediated spore germination is divided into two genetically separable stages. Stage I is characterized by the release of dipicolinic acid (DPA) from the spore core. Stage II is characterized by cortex degradation, and stage II is activated by the DPA released during stage I. Thus, DPA release precedes cortex hydrolysis during B. subtilis spore germination. Here, we investigated the timing of DPA release and cortex hydrolysis during Clostridium difficile spore germination and found that cortex hydrolysis precedes DPA release. Inactivation of either the bile acid germinant receptor, cspC, or the cortex hydrolase, sleC, prevented both cortex hydrolysis and DPA release. Because both cortex hydrolysis and DPA release during C. difficile spore germination are dependent on the presence of the germinant receptor and the cortex hydrolase, the release of DPA from the core may rely on the osmotic swelling of the core upon cortex hydrolysis. These results have implications for the hypothesized glycine receptor and suggest that the initiation of germinant receptor-mediated C. difficile spore germination proceeds through a novel germination pathway. IMPORTANCEClostridium difficile infects antibiotic-treated hosts and spreads between hosts as a dormant spore. In a host, spores germinate to the vegetative form that produces the toxins necessary for disease. C. difficile spore germination is stimulated by certain bile acids and glycine. We recently identified the bile acid germinant receptor as the germination-specific, protease-like CspC. CspC is likely cortex localized, where it can transmit the bile acid signal to the cortex hydrolase, SleC. Due to the differences in location of CspC compared to the Bacillus subtilis germinant receptors, we hypothesized that there are fundamental differences in the germination processes between the model organism and C. difficile. We found that C. difficile spore germination proceeds through a novel pathway. Clostridium difficile (a Gram-positive, spore-forming, strict anaerobe) has become a significant threat to antibiotic-treated or immunocompromised hosts. Antibiotics are known to disrupt the colonic microbiota, and this perturbation permits C. difficile colonization (1, 2). Due to the strict anaerobic nature of C. difficile cells, spores are generally thought to be the infectious agent (only the spore can survive for extended periods of time in the aerobic environment outside a host) (3, 4). Because the spore form is noninfectious, spores must germinate to actively growing bacteria which initiate infection (5, 6). Thus, germination by C. difficile spores represents one of earliest steps in the pathogenesis of this organism.Endospore germination has been extensively studied in Bacillus sp. and, more recently, in clostridia (7,8). In the spore core,...

Detecting Cortex Fragments During Bacterial Spore Germination

“…Under alkaline conditions (Step 2.10), this enol form can react with the adjacent acetyl to generate an oxazole derivative which reacts with Ehrlich's reagent (Step 4.3) to generate a purple color 15,20 . This method described above yields consistent, reliable results for the detection of cortex fragments from germinating spores, both B. subtilis and C. difficile 9,16,17 . The assay requires special attention to ensure the reaction times, temperatures and procedures are performed with precision.…”

“…Note: Should DPA need to be monitored, remove a separate 100 μl sample for the detection of DPA in the solution using an assay based on terbium fluorescence 9,18,19 . 7.…”

“…Heat activate C. difficile spores at 65 °C for 30 min and store on ice. Note: C. difficile spores can be produced and purified as described previously 7,9,10 . 2.…”

“…A previous report suggests that much of this change in OD is due to the release of DPA from the spore 12 . During our recent study, we sought to compare the timing of C. difficile spore germination and monitored the release of DPA and cortex fragments 9 . For this study it was critical to monitor the release of cortex fragments as they began to be released by germinating spores.…”

“…In many spore-forming bacteria, these germinants are amino acids, sugars, nucleotides, ions or combinations thereof 2 . C. difficile spore germination is initiated by combinations of certain bile acids, [e.g., taurocholic acid (TA)] and amino acids (e.g., glycine) [7][8][9][10] . Though there are differences between the C. difficile germination pathway and the pathways studied in other spore-forming bacteria, such as B. subtilis, common to all is the absolute requirement to degrade the spore cortex to allow a vegetative cell to grow from the germinated spore 2,8 .…”