Abstract
Fluorescence microscopy is a valuable tool to study a broad variety of bacterial cell components and dynamics thereof. For Clostridioides difficile, the fluorescent proteins CFPopt, mCherryOpt and phiLOV2.1, and the self-labelling tags SNAPCd and HaloTag, hereafter collectively referred as fluorescent systems, have been described to explore different cellular pathways. In this study, we sought to characterize previously used fluorescent systems in C. difficile cells. We performed single cell analyses using fluorescence microscopy of exponentially growing C. difficile cells harbouring different fluorescent systems, either expressing these separately in the cytosol or fused to the C-terminus of HupA, under defined conditions. We show that the intrinsic fluorescence of C. difficile cells increases during growth, independent of sigB or spo0A. However, when C. difficile cells are exposed to environmental oxygen autofluorescence is enhanced. Cytosolic overexpression of the different fluorescent systems alone, using the same expression signals, showed heterogeneous expression of the fluorescent systems. High levels of mCherryOpt were toxic for C. difficile cells limiting the applicability of this fluorophore as a transcriptional reporter. When fused to HupA, a C. difficile histone-like protein, the fluorescent systems behaved similarly and did not affect the HupA overproduction phenotype. The present study compares several commonly used fluorescent systems for application as transcriptional or translational reporters in microscopy and summarizes the limitations and key challenges for live-cell imaging of C. difficile. Due to independence of molecular oxygen and fluorescent signal, SNAPCd appears the most suitable candidate for live-cell imaging in C. difficile to date.
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The datasets and biological materials generated during and/or analysed during the current study are available from the authors on reasonable request.
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Analyses were performed using publicly available tools as indicated in “Materials and Methods” section.
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Acknowledgements
We thank Dr. Gillian Douce, Glasgow University, for kindly providing the phiLOV2.1 plasmid; Dr. Craig Ellermeier for kindly providing the mCherryOpt containing vector pDSW1728; Dr. Adriano Henriques, ITQB-UNL, for kingly providing the pFT46 for the SNAPCd expression; and Dr. Jeroen Corver for helpful discussions.
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This work was supported by a Vidi Fellowship to W.K.S. (864.10.003) from the Netherlands Organization for Scientific Research (NWO) and a Gisela Thier Fellowship to W.K.S. from the Leiden University Medical Center.
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A.F., R.D., B.W. and W.K.S contributed to construction of the vectors and strains used in this study. A.P. conducted the sample preparation, microscopy and data analysis in this study. A.P. and A.F. conducted the expression analysis through in-gel detection. A.P. and W.K.S designed the study and wrote the manuscript with the contributions of all authors.
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Financial interests: WKS has received speaker fees from Promega. All other authors: none to declare. Non-financial interests: none.
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Oliveira Paiva, A.M., Friggen, A.H., Douwes, R. et al. Practical observations on the use of fluorescent reporter systems in Clostridioides difficile. Antonie van Leeuwenhoek 115, 297–323 (2022). https://doi.org/10.1007/s10482-021-01691-8
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DOI: https://doi.org/10.1007/s10482-021-01691-8