Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop
infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for spore germination, it is currently unclear whether both co-germinan...
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Published in | mSphere Vol. 8; no. 4; p. e0000523 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
American Society for Microbiology
24.08.2023
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Subjects | |
Online Access | Get full text |
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Summary: | infections begin when its metabolically dormant spores germinate in response to sensing bile acid germinants alongside amino acid and divalent cation co-germinants in the small intestine. While bile acid germinants are essential for
spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca
, are essential for inducing germination, while another proposes that either co-germinant class can induce germination. The former model is based on the finding that spores defective in releasing large stores of internal Ca
in the form of calcium dipicolinic acid (CaDPA) cannot germinate when germination is induced with bile acid germinant and amino acid co-germinant alone. However, since the reduced optical density of CaDPA-less spores makes it difficult to accurately measure their germination, we developed a novel automated, time-lapse microscopy-based germination assay to analyze CaDPA mutant germination at the single-spore level. Using this assay, we found that CaDPA mutant spores germinate in the presence of amino acid co-germinant and bile acid germinant. Higher levels of amino acid co-germinants are nevertheless required to induce CaDPA mutant spores to germinate relative to WT spores because CaDPA released by WT spores during germination can function in a feedforward loop to potentiate the germination of other spores within the population. Collectively, these data indicate that Ca
is not essential for inducing
spore germination because amino acid and Ca
co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE
spore germination is essential for this major nosocomial pathogen to initiate infection.
spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca
and amino acids. Prior work suggested that Ca
is essential for
spore germination based on bulk population analyses of germinating CaDPA mutant spores. Since these assays rely on optical density to measure spore germination and the optical density of CaDPA mutant spores is reduced relative to WT spores, this bulk assay is limited in its capacity to analyze germination. To overcome this limitation, we developed an automated image analysis pipeline to monitor
spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca
is dispensable for inducing
spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 The authors declare no conflict of interest Present address: Department of Biological Sciences, Saint Anselm College, Manchester, New Hampshire, USA |
ISSN: | 2379-5042 2379-5042 |
DOI: | 10.1128/msphere.00005-23 |