FAST and splitFAST! Already well described in solventogenic and acetogenic Clostridia strains… Recently disclosed in pathogenic C. difficile… Now in Clostridium perfringens! Thanks to Prof. Stephen Melville and team of Virginia Tech and researchers of the Lawrence Berkeley National Laboratory. Preprint in bioRxiv 2024.
Clostridium perfringens is a Gram-positive anaerobic bacterial pathogen of humans and animals. Hence, to secrete folded proteins to the environment, they have to overcome the physical barrier of the thick peptidoglycan layer. One mechanism known in Gram-negative bacteria to export proteins from the periplasm through the outer membrane to the extracellular environment is the so-called Type II secretion system (TTSS). It acts as a “piston” pumping folded proteins through the outer membrane to the environment. Yet, Clostridium perfringens, like most clostridia, has thin appendages called type IV pili (T4P) reminiscent of the TTSS though Gram-positive.
The authors here present evidence that C. perfringens effectively uses one set of T4P genes to secrete proteins. It may even be responsible for secreting the main biofilm protein BsaA.
To investigate the cellular location of BsaA, the authors have selected the chemigenetic fluorogenic protein reporter FAST. FAST is indeed well documented in anaerobic conditions. Moreover, one single construct allows to discriminate proteins located in the periplasm or outside cells. Here, the authors constructed FAST gene fusions with the bsaA and/or pilT genes. Indeed the FAST protein binds a soluble fluorogen which, upon binding, exhibits a large increase in fluorescence allowing localization of the fusion proteins. After expressing above constructs, they exposed the cells to two different fluorogens, tfCoral, which is membrane permeable, and tfAmber-NP which is impermeable to membranes. As a result, they could access to the location of expressed proteins, cytoplasmic or extracellular.
By the way, this reminds me of the work already published on Listeria excretion monitoring through the periplasm.
Besides tfCoral and tfAmber-NP used above, The Twinkle Factory offers a wide range of commercial fluorogens, permeant or not, and quenchers, for FAST and derivatives, splitFAST, greenFAST & redFAST, frFAST. Expect more soon in the near-infrared zone. On top of those, the range of reagents now includes fluorogenic molecular glues for CATCHFIRE, our reversible and self-reporting protein dimerizer.
More reading on FAST in Clostridia
- bioRxiv 2024 – Type IV pili-associated secretion of a biofilm matrix protein from Clostridium perfringens that forms intermolecular isopeptide bonds
- PLOS Pathogens 2024 – The multiplicity of thioredoxin systems meets the specific lifestyles of Clostridia
- ACS Synth. Biol. 2022 – Establishment of Green- and Red-Fluorescent Reporter Proteins Based on the Fluorescence-Activating and Absorption-Shifting Tag for Use in Acetogenic and Solventogenic Anaerobes
- mBio 2020 – Interspecies Microbial Fusion and Large-Scale Exchange of Cytoplasmic Proteins and RNA in a Syntrophic Clostridium Coculture
- Appl. Environ. Microbiol. 2019 – A Strongly Fluorescing Anaerobic Reporter and Protein-Tagging System for Clostridium Organisms Based on the Fluorescence-Activating and Absorption-Shifting Tag Protein (FAST)
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