Biology subjects

Ribis, J. W.

Publications and source records attributed to Ribis, J. W..

2 recordsLinked to original sources

bioRxiv2022

Single-spore germination analyses reveal that Clostridioides difficile uses parallel pathways to sense distinct classes of co-germinants during spore germination.

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. ImportanceClostridioides 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.

microbiology↗

bioRxiv2022

Development of a dual fluorescent reporter system in Clostridioides difficile reveals a division of labor between virulence and transmission gene expression

The bacterial pathogen Clostridioides difficile causes gastroenteritis through its production of toxins and transmits disease through its production of resistant spores. Toxin and spore production are energy-expensive processes that are regulated by multiple transcription factors in response to many nutritional inputs. While toxin and sporulation genes are both heterogeneously expressed in only a subset of C. difficile cells, the relationship between these two sub-populations remains unclear. To address whether C. difficile coordinates the generation of these sub-populations, we developed a dual transcriptional reporter system that allows toxin and sporulation gene expression to be simultaneously visualized at the single-cell level using chromosomally-encoded mScarlet and mNeonGreen fluorescent transcriptional reporters. We then adapted an automated image analysis pipeline to quantify toxin and sporulation gene expression in thousands of individual cells in different media conditions and genetic backgrounds. These analyses revealed that toxin and sporulation gene expression rarely overlap during growth on agar plates, but broth culture increases this overlap in a manner dependent on the multifunctional RstA transcriptional regulator. Our results suggest that certain growth conditions promote a "division of labor" between transmission and virulence gene expression, highlighting how these subpopulations are influenced by environmental inputs. Given that recent work has revealed population-wide heterogeneity for numerous cellular processes in C. difficile, we anticipate that our dual reporter system will be broadly useful for determining the overlap in these subpopulations. IMPORTANCEClostridioides difficile is an important nosocomial pathogen that causes severe diarrhea by producing toxins and is transmitted by producing spores. While both processes are crucial for C. difficile disease, only a subset of cells express toxins and/or undergo sporulation. Whether C. difficile coordinates the relationship between these energy-expensive processes remains unknown. We developed a dual fluorescent reporter system coupled with an automated image analysis pipeline to rapidly characterize expression two genes of interest across thousands of bacterial cells. Using this reporter system, we discovered that toxin and sporulation gene expression appear to undergo a "division of labor" in certain growth conditions, particularly during growth on agar plates. Since C. difficile specializes into subpopulations for numerous vital cellular processes, this novel dual reporter system will enable future studies aimed at understanding how C. difficile coordinates various subpopulations throughout its infectious disease cycle.

microbiology↗