Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop
ABSTRACT Clostridioides difficile 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 C. difficile spore germination, i...
| Main Authors: | , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-08-01
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| Series: | mSphere |
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| Online Access: | https://journals.asm.org/doi/10.1128/msphere.00005-23 |
| _version_ | 1797730251341561856 |
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| author | John W. Ribis Luana Melo Shailab Shrestha David Giacalone Enrique E. Rodriguez Aimee Shen Amy Rohlfing |
| author_facet | John W. Ribis Luana Melo Shailab Shrestha David Giacalone Enrique E. Rodriguez Aimee Shen Amy Rohlfing |
| author_sort | John W. Ribis |
| collection | DOAJ |
| description | ABSTRACT Clostridioides difficile 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 C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, 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 Ca2+ 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 Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile 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 C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores. |
| first_indexed | 2024-03-12T11:42:31Z |
| format | Article |
| id | doaj.art-58a4263d6df446edbd5ffdd39386a791 |
| institution | Directory Open Access Journal |
| issn | 2379-5042 |
| language | English |
| last_indexed | 2024-03-12T11:42:31Z |
| publishDate | 2023-08-01 |
| publisher | American Society for Microbiology |
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| series | mSphere |
| spelling | doaj.art-58a4263d6df446edbd5ffdd39386a7912023-08-31T14:26:41ZengAmerican Society for MicrobiologymSphere2379-50422023-08-018410.1128/msphere.00005-23Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loopJohn W. Ribis0Luana Melo1Shailab Shrestha2David Giacalone3Enrique E. Rodriguez4Aimee Shen5Amy Rohlfing6Department of Molecular Biology and Microbiology, Tufts University School of Medicine , Boston, Massachusetts, USADepartment of Molecular Biology and Microbiology, Tufts University School of Medicine , Boston, Massachusetts, USADepartment of Molecular Biology and Microbiology, Tufts University School of Medicine , Boston, Massachusetts, USADepartment of Molecular Biology and Microbiology, Tufts University School of Medicine , Boston, Massachusetts, USATufts University , Boston, Massachusetts, USADepartment of Molecular Biology and Microbiology, Tufts University School of Medicine , Boston, Massachusetts, USADepartment of Molecular Biology and Microbiology, Tufts University School of Medicine , Boston, Massachusetts, USAABSTRACT Clostridioides difficile 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 C. difficile spore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, 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 Ca2+ 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 Ca2+ is not essential for inducing C. difficile spore germination because amino acid and Ca2+ co-germinant signals are sensed by parallel signaling pathways. IMPORTANCE Clostridioides difficile spore germination is essential for this major nosocomial pathogen to initiate infection. C. difficile spores germinate in response to sensing bile acid germinant signals alongside co-germinant signals. There are two classes of co-germinant signals: Ca2+ and amino acids. Prior work suggested that Ca2+ is essential for C. difficile 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 C. difficile spore germination using time-lapse microscopy. With this analysis pipeline, we demonstrate that, although Ca2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.https://journals.asm.org/doi/10.1128/msphere.00005-23Clostridioides difficilespore germinationtime-lapse microscopyautomated image analysisco-germinantDPA |
| spellingShingle | John W. Ribis Luana Melo Shailab Shrestha David Giacalone Enrique E. Rodriguez Aimee Shen Amy Rohlfing Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop mSphere Clostridioides difficile spore germination time-lapse microscopy automated image analysis co-germinant DPA |
| title | Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop |
| title_full | Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop |
| title_fullStr | Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop |
| title_full_unstemmed | Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop |
| title_short | Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop |
| title_sort | single spore germination analyses reveal that calcium released during clostridioides difficile germination functions in a feedforward loop |
| topic | Clostridioides difficile spore germination time-lapse microscopy automated image analysis co-germinant DPA |
| url | https://journals.asm.org/doi/10.1128/msphere.00005-23 |
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