Clostridioides difficile Spores: Bile Acid Sensors and Trojan Horses of Transmission

Shen, Aimee

doi:10.1055/s-0040-1701230

“…This allows C. difficile spores to develop to their vegetative form and then grow and secrete highly potent AB-type protein toxins, toxin A (TcdA) and toxin B (TcdB), which are the causative agents and main virulence factors for CDI symptoms [3]. TcdA and TcdB are encoded by Toxins 2023, 15, 384 2 of 15 the tcdA and tcdB genes, respectively, and are expressed in response to environmental cues such as exposure to bile acids [4]. Hypervirulent strains of C. difficile have distinct characteristics compared to other strains.…”

Section: Introductionmentioning

confidence: 99%

Domperidone Protects Cells from Intoxication with Clostridioides difficile Toxins by Inhibiting Hsp70-Assisted Membrane Translocation

Braune-Yan,

Jia,

Wahba

et al. 2023

Toxins

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Clostridioides difficile infections cause severe symptoms ranging from diarrhea to pseudomembranous colitis due to the secretion of AB-toxins, TcdA and TcdB. Both toxins are taken up into cells through receptor-mediated endocytosis, autoproteolytic processing and translocation of their enzyme domains from acidified endosomes into the cytosol. The enzyme domains glucosylate small GTPases such as Rac1, thereby inhibiting processes such as actin cytoskeleton regulation. Here, we demonstrate that specific pharmacological inhibition of Hsp70 activity protected cells from TcdB intoxication. In particular, the established inhibitor VER-155008 and the antiemetic drug domperidone, which was found to be an Hsp70 inhibitor, reduced the number of cells with TcdB-induced intoxication morphology in HeLa, Vero and intestinal CaCo-2 cells. These drugs also decreased the intracellular glucosylation of Rac1 by TcdB. Domperidone did not inhibit TcdB binding to cells or enzymatic activity but did prevent membrane translocation of TcdB’s glucosyltransferase domain into the cytosol. Domperidone also protected cells from intoxication with TcdA as well as CDT toxin produced by hypervirulent strains of Clostridioides difficile. Our results reveal Hsp70 requirement as a new aspect of the cellular uptake mechanism of TcdB and identified Hsp70 as a novel drug target for potential therapeutic strategies required to combat severe Clostridioides difficile infections.

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“…C. difficile spores can survive the antibiotic regimens used to treat C. difficile infections because they are metabolically dormant, and their resistance properties also allow them to survive exposure to commonly used disinfectants. As a result, spores serve as an environmental reservoir for seeding future infections ( 7 - 10 ).…”

Section: Introductionmentioning

confidence: 99%

Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop

Ribis

¹

,

Melo

²

,

Shrestha

³

et al. 2023

mSphere

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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 Ca 2+ , 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 2+ 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 2+ is not essential for inducing C. difficile spore germination because amino acid and Ca 2+ 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: Ca 2+ and amino acids. Prior work suggested that Ca 2+ 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 Ca 2+ is dispensable for inducing C. difficile spore germination, CaDPA can function in a feedforward loop to potentiate the germination of neighboring spores.

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“…Since spores are metabolically dormant, they can survive the antibiotic regimens used to treat C. difficile infections. Their resistance properties also allow them to survive exposure to commonly used disinfectants and persist in the environment to seed future infections (7)(8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

“…C. difficile spores are survive the antibiotic regimens used to treat C. difficile infections because they are metabolically dormant, and their resistance properties also allow them to survive exposure to commonly used disinfectants. As a result, spores serve as an environment reservoir for seeding future infections (7)(8)(9)(10).…”

Section: Introductionmentioning

confidence: 99%

Single-spore germination analyses reveal that calcium released duringClostridioides difficilegermination functions in a feed-forward loop

Ribis

¹

,

Melo

²

,

Shrestha

³

et al. 2022

Preprint

Self Cite

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Clostridioides difficileinfections 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 forC. difficilespore germination, it is currently unclear whether both co-germinant signals are required. One model proposes that divalent cations, particularly Ca2+, are essential for spore 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) do not germinate when germination is induced with amino acid co-germinant and bile acid germinant alone. However, accurately measuring germination in CaDPA-less spores using standard optical density-based germination assays is challenging because these spores are less optically dense. To overcome this issue, 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 show that CaDPA mutant spores still germinate in the presence of amino acid co-germinant and bile acid germinant, although they require higher levels of amino acid co-germinants to induce germination relative to WT spores. This requirement for elevated amino acid co-germinant levels is because CaDPA released during germination can function as a public good to potentiate the germination of other spores within a population. Collectively, these data demonstrate that Ca2+, is not essential forC. difficilespore germination, indicating that parallel co-germinant sensing pathways activate germination

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Clostridioides difficile Spores: Bile Acid Sensors and Trojan Horses of Transmission

Cited by 4 publications

References 101 publications

Domperidone Protects Cells from Intoxication with Clostridioides difficile Toxins by Inhibiting Hsp70-Assisted Membrane Translocation

Domperidone Protects Cells from Intoxication with Clostridioides difficile Toxins by Inhibiting Hsp70-Assisted Membrane Translocation

Single-spore germination analyses reveal that calcium released during Clostridioides difficile germination functions in a feedforward loop

Single-spore germination analyses reveal that calcium released duringClostridioides difficilegermination functions in a feed-forward loop

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