Intestinal calcium and bile salts facilitate germination of <i>Clostridium difficile</i> spores

<div><p><i>Clostridium difficile</i> (<i>C</i>. <i>difficile</i>) is an anaerobic gram-positive pathogen that is the leading cause of nosocomial bacterial infection globally. <i>C</i>. <i>difficile</i> infection (CDI) typically occurs after ingestion of infectious spores by a patient that has been treated with broad-spectrum antibiotics. While CDI is a toxin-mediated disease, transmission and pathogenesis are dependent on the ability to produce viable spores. These spores must become metabolically active (germinate) in order to cause disease. <i>C</i>. <i>difficile</i> spore germination occurs when spores encounter bile salts and other co-germinants within the small intestine, however, the germination signaling cascade is unclear. Here we describe a signaling role for Ca²⁺ during <i>C</i>. <i>difficile</i> spore germination and provide direct evidence that intestinal Ca²⁺ coordinates with bile salts to stimulate germination. Endogenous Ca²⁺ (released from within the spore) and a putative AAA+ ATPase, encoded by <i>Cd630_32980</i>, are both essential for taurocholate-glycine induced germination in the absence of exogenous Ca²⁺. However, environmental Ca²⁺ replaces glycine as a co-germinant and circumvents the need for endogenous Ca²⁺ fluxes. <i>Cd630_32980</i> is dispensable for colonization in a murine model of <i>C</i>. <i>difficile</i> infection and <i>ex vivo</i> germination in mouse ileal contents. Calcium-depletion of the ileal contents prevented mutant spore germination and reduced WT spore germination by 90%, indicating that Ca²⁺ present within the gastrointestinal tract plays a critical role in <i>C</i>. <i>difficile</i> germination, colonization, and pathogenesis. These data provide a biological mechanism that may explain why individuals with inefficient intestinal calcium absorption (<i>e</i>.<i>g</i>., vitamin D deficiency, proton pump inhibitor use) are more prone to CDI and suggest that modulating free intestinal calcium is a potential strategy to curb the incidence of CDI.</p></div

Proposed model for the role of calcium in <i>C</i>. <i>difficile</i> germination.

<p>Tc binds to CspC (A) facilitating movement of glycine or calcium through the spore coat and outer membrane (B). Glycine then interacts with an unknown receptor (C) inducing the release of Ca²⁺ from the spore core (D). Ca²⁺ from the environment or the spore core activates CspB (E), which processes pro-SleC (F) subsequently initiating cortex hydrolysis (G). This leads to full core rehydration (H), complete release of DPA (I) and spore outgrowth.</p

Calcium from within the spore is essential for germination in response to Tc-Gly.

<p>Cd630 spores were incubated with 0.2% Tc, 50mM glycine and different concentrations of EGTA (A). Cd630 spores were incubated with the indicated combinations of 0.2% Tc, 50 mM glycine, 1 mM CaCl₂, and 50 μM EGTA (B). EGTA (1mM) pretreated Cd630 spores and non-treated Cd630 spores were incubated with the indicated combinations of chelex-treated germinants, Tc (0.2%) and glycine (50mM) (C). Activation of SleC was assessed by western blot. Cd630 spores were incubated 37°C for 15 minutes with 1% Tc and the indicated combinations of 50mM glycine and 1mM EGTA (D). Germination was tracked by loss of OD at 37°C over the course of one hour. Germination assays were performed in triplicate. Germination assays and western blots are representative of 3 independent spore preps. Error bars are mean plus or minus SD.</p

Tc-CaCl₂ induced germination occurs through the same pathway as Tc-Gly.

<p>Cd630, Δ<i>gerS</i>, Δ<i>cspB</i>, Δ<i>cspC</i>, and Δ<i>sleC</i> spores were incubated with 0.2% Tc and with (+) or without (-) the indicated co-cogerminant: 50 mM glycine (a) or 60 mM CaCl₂ (b). Germination was measured by loss of OD₆₀₀ at 37°C over the course of one hour. (a-b). Western blot of SleC from WT, Δ<i>gerS</i>, Δ<i>cspB</i>, or Δ<i>cspC</i> spores incubated at 37°C for 15 minutes with 1% Tc and the indicated concentrations of glycine (c) or CaCl₂ (d). Germination assays were performed in triplicate. Germination assays and western blots are representative of 3 independent spore preps. Error bars are mean plus or minus SD.</p

Tc-Gly induced germination is dispensable for <i>in vivo</i> germination of <i>C</i>. <i>difficile</i> spores.

<p>Cefoperazone-treated C57BL/6 mice (n = 8) were infected with 1500 spores of WT or Δ<i>32980</i> by oral gavage. Colonization levels were assessed daily and are presented in total CFU per gram of feces. (a) Multiple t tests were performed and p = 0.23 for each time point tested (a). Ileal contents were collected from Cefoperazone treated C57BL/6 mice (n = 3) and <i>ex vivo</i> germination assays were performed. 1x10³ spores of Cd630 or Δ<i>32980</i> were incubated for one hour at 37°C in ileal contents, calcium depleted ileal contents or calcium depleted ileal contents treated with 15 mM CaCl₂. Samples were then incubated at 65°C for 20 min and then plated on BHIS-Tc plates. Data are presented as % loss of Heat Resistance (b). Free calcium levels of ileal contents, calcium depleted ileal contents or calcium depleted ileal contents treated with 15 mM CaCl₂ were measured using a calcium colorimetric assay. Levels of calcium (mM) were determined using a standard curve (c). Assays were performed in triplicate using ileal contents from three mice. Error bars are mean plus or minus SD. Statistical significance was calculated using Two-way ANOVA. (*) p<0.05 (****) p<0.0001.</p

Exogenous calcium induces <i>C</i>. <i>difficile</i> germination in concert with taurocholate.

<p>Cd630, VPI 10463, or R20291 spores were incubated with the indicated combinations of 0.2% Tc, 60 mM Ca-DPA, 50 mM glycine, 60 mM CaCl₂, or 60 mM DPA (A-G, I, J). Activation of SleC was assessed by western blot analyses. Cd630 spores were incubated for 15 minutes at 37°C with the indicated combinations of 1% Tc, 50 mM glycine, 60 mM CaCl₂, 60 mM DPA, or 60 mM Ca-DPA. Spores were subsequently lysed and assayed for levels of pro-SleC and SleC (K). <i>Bacillus anthracis</i> strain Sterne 34F₂ spores were incubated with the indicated combinations of either 60 mM CaCl₂, 60 mM DPA, or 60 mM CaDPA (H). Germination was measured either by tracking the loss of OD₆₀₀ over time (A-H), measuring loss of heat resistance at 37°C after 1 hour (I), or measuring release of DPA at 37°C after 1 hour (J). Germination assays were performed in triplicate. Germination assays and western blots are representative of three independent spore preps. Error bars are mean plus or minus SD. Statistical analysis was performed using one-way ANOVA. (****) p<0.0001.</p

<i>Cd630_32980</i> is required for Tc-Gly induced germination.

<p>WT and Δ<i>32980</i> spores were incubated with 0.2% Tc and the indicated concentrations of glycine (a-b) or CaCl₂ (c-d) with germination was measured by loss of OD₆₀₀ at 37°C over the course of an hour. Western blot assessing SleC activation from WT and Δ<i>32980</i> spores incubated at 37°C for 15 minutes with 1% Tc and the indicated concentrations of glycine (e) or CaCl₂ (f). %pro-SleC and % SleC relative densities were calculated for each lane using ImageJ by determining the ratio of each band to the total density for the two bands combined. Blots are representative of 3 experiments. Germination assays were performed in triplicate and are representative of 3 independent spore preps. Error bars are mean plus or minus SD.</p