Systematic Evaluation of Parameters Important for Production of Native Toxin A and Toxin B from Clostridioides difficile

Aminzadeh, Aria; Jørgensen, René

doi:10.3390/toxins13040240

Cited by 6 publications

(8 citation statements)

References 53 publications

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“…Native TcdA was purified from C. difficile strain R20291 (NCTC13366) as described in Aminzadeh and Jørgensen (2021). Briefly, the firststage seed culture was prepared by inoculating sterilized tryptoneyeast extract (TY) (Formedium, Hunstanton, NK, UK) medium with C. difficile glycerol stock and incubating unagitated for 24 h at 37°C under anaerobic conditions.…”

Section: Protein Purificationmentioning

confidence: 99%

High‐resolution structure of native toxin A from Clostridioides difficile

et al. 2021

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Clostridioides difficile infections have emerged as the leading cause of healthcare-associated infectious diarrhea. Disease symptoms are mainly caused by the virulence factors, TcdA and TcdB, which are large homologous multidomain proteins. Here, we report a 2.8 A resolution cryo-EM structure of native TcdA, unveiling its conformation at neutral pH. The structure uncovers the dynamic movement of the CROPs domain which is induced in response to environmental acidification. Furthermore, the structure reveals detailed information about the interaction area between the CROPs domain and the tip of the delivery and receptor-binding domain, which likely serves to shield the C-terminal part of the hydrophobic pore-forming region from solvent exposure. Similarly, extensive interactions between the globular subdomain and the Nterminal part of the pore-forming region suggest that the globular subdomain shields the upper part of the pore-forming region from exposure to the surrounding solvent. Hence, the TcdA structure provides insights into the mechanism of preventing premature unfolding of the pore-forming region at neutral pH, as well as the pH-induced inter-domain dynamics.

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Section: Protein Purificationmentioning

confidence: 99%

High‐resolution structure of native toxin A from Clostridioides difficile

et al. 2021

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“…As a comparison to the TY liquid media tested above, we analyzed the behavior of the dual reporter strains on TY plates (Fig 7 ) . Even though TY broth is used to induce toxin gene expression (19), when wild-type cells were growth on TY agar, they were 5-fold less likely to induce toxin (9% vs. 41% of the population was “Toxin-ON” on TY agar vs. TY broth), and the magnitude of toxin gene expression was also ∼2-fold lower on TY agar vs. TY broth (Figs 6 & 7). In contrast, wild-type cells were twice as likely to induce sporulation on TY plates (62% vs. 33% of cells were “Sporulation-ON” on TY agar vs. TY broth).…”

Section: Resultsmentioning

confidence: 99%

“…When the P tcdA::mNG -P sipL::mSc dual reporter strains were grown overnight in TY broth, which is traditionally used to induce maximal toxin gene expression (19), 27 ± 5% of the wild-type population was solely “toxin-ON” and 21 ± 6% of the population was solely “Sporulation-ON” ( Fig 6). Simultaneous expression of both tcdA and sipL was observed in 11 ± 3% of the population (Fig 6, blue arrows) such that 41 ± 6% of the overall bacterial population was “Toxin-ON” (similar to what we observed in the single reporter strains in TY broth, Fig 4).…”

Section: Resultsmentioning

confidence: 99%

“…When the PtcdA::mNG-PsipL::mSc dual reporter strains were grown overnight in TY broth, which is traditionally used to induce maximal toxin gene expression (19), 27 ± 5% of the wild-type population was solely "toxin-ON" and 21 ± 6% of the population was solely About one-third of the population was "Sporulation-ON," indicating that a sizable proportion of cells induce sporulation genes in TY broth, a property that was not possible to accurately quantify in our earlier analyses with the single PtcdA::mNG toxin reporter (Fig 4). In TY broth, ∆tcdR induced the sipL reporter at low levels, with only 2 ± 1% of cells being identified as "Sporulation-ON" (Fig 6C).…”

Section: Transmission and Virulence Gene Expression Exhibit A Divisio...mentioning

confidence: 99%

“…Numerous environmental signals control toxin and sporulation gene expression in C. difficile . For example, both sets of genes are repressed by glucose and preferentially induced during stationary phase in C. difficile , suggesting that nutrient starvation induces these processes (19). The levels of c-di-GMP, autoinducing peptides, and other signaling molecules also modulate these processes (20, 21).…”

Section: Introductionmentioning

confidence: 99%

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

Donnelly

Shrestha

Ribis

et al. 2022

Preprint

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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.

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