Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans

CV Giuraniuc; C Parkin; MC Almeida; M Fricker; P Shadmani; S Nye; S Wehmeier; S Chawla; T Bedekovic; L Lehtovirta-Morley; D Richards; NA Gow; AC Brand

doi:10.1101/2023.04.20.537637

Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans

CV Giuraniuc, C Parkin, MC Almeida, M Fricker, P Shadmani, S Nye, S Wehmeier, S Chawla, T Bedekovic, L Lehtovirta-Morley, D Richards, NA Gow, AC Brand^* (Corresponding Author)

^*Corresponding author for this work

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Abstract

Calcium (Ca2+) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca2+-dynamics in fungi are poorly understood due to lack of effective real-time Ca2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans, and used live-cell imaging to observe both dynamic Ca2+ spiking and slower changes in non-spiking Ca2+-GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca2+-GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca2+-GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H2O2 abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H2O2 was dependent on Cap1, extracellular Ca2+, and calcineurin but not on its downstream target, Crz1. Ca2+-dynamics were not affected by H2O2 in the hog1Δ or yvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca2+-GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca2+-influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans.

Original language	English
Article number	e01157-23
Number of pages	25
Journal	mBio
Volume	14
Issue number	5
Early online date	26 Sept 2023
DOIs	https://doi.org/10.1101/2023.04.20.537637 https://doi.org/10.1128/mbio.01157-23
Publication status	Published - 31 Oct 2023

Bibliographical note

Acknowledgements
AB conceived the project and wrote the manuscript. CVG conceived the experimental design. SW designed the GCaMP reporter. AM, KL, LV-M, SC and TB constructed strains and optimised imaging. MF developed the image analysis software. CVG and CP carried out the microfluidics experiments and imaging analysis. NG assisted with preparation of the manuscript. PS, SN and DMR developed and undertook the theoretical data analysis and contributed to the interpretation of the results.

Funding
AB, CG and TB were funded by the Wellcome Trust [Grant number 206412/A/17/Z]. AB and DR were supported by a Wellcome Trust Institutional Strategic Support Award (WT204909/Z/16/Z). CP was funded by a University of Exeter studentship (113516). This work was also supported by a Royal Society URF (UF080611), an MRC NIRG (G0900211/90671) and the MRC-Centre for Medical Mycology at the University of Exeter (MR/N006364/2). DR was funded by the Medical Research Council (MR/P022405/1). SN was supported by the Medical Research Council via the GW4 BioMed2 DTP (MR/W006308/1). MCA was supported by a European Commission ITN ‘FungiBrain’ studentship (607963). LL and SC were funded by a Wellcome Trust Institutional Strategic Support Award to the University of Aberdeen. NG acknowledges support of Wellcome Trust Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards (101873, 200208, 215599, 224323). NG and AB thank the MRC (MR/M026663/2) for support. This study/research is funded by the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

Data Availability Statement

Supplemental material - mbio.01157-23-s0001.docx
Fig. S1-S7; Tables S1 and S2.

Access to Document

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10.1128/mbio.01157-23Licence: CC BY

Giuraniuc_etal_M_Dynamic_Calcium_Mediated_VoR
© 2023 Giuraniuc et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license. https://creativecommons.org/licenses/by/4.0/
Final published version, 21.4 MBLicence: CC BY

Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans - Supplementary Movies
Giuraniuc, V. (Creator), Parkin, C. (Creator), Cruz Almeida, M. (Creator), Fricker, M. (Creator), Shadmani, P. (Creator), Nye, S. (Creator), Wehmeier, S. (Creator), Chawla, S. (Creator), Bedekovic, T. (Creator), Lehtovirta-Morley, L. (Creator), Richards, D. (Creator), Gow, N. A. (Creator) & Brand, A. C. (Creator), Zenodo, 1 Jun 2023
DOI: 10.5281/zenodo.7994336, https://zenodo.org/record/7994336 and one more link, https://zenodo.org/record/8179089 (show fewer)
Dataset
Analysis Software: Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans
Giuraniuc, V. (Creator), Parkin, C. (Creator), Cruz Almeida, M. (Creator), Fricker, M. (Creator), Shadmani, P. (Creator), Nye, S. (Creator), Wehmeier, S. (Creator), Chawla, S. (Creator), Bedekovic, T. (Creator), Lehtovirta-Morley, L. (Creator), Richards, D. (Creator), Gow, N. A. (Creator) & Brand, A. C. (Creator), Zenodo, 1 Jun 2023
DOI: 10.5281/zenodo.7995489, https://zenodo.org/record/7995489
Dataset

Cite this

Giuraniuc, CV., Parkin, C., Almeida, MC., Fricker, M., Shadmani, P., Nye, S., Wehmeier, S., Chawla, S., Bedekovic, T., Lehtovirta-Morley, L., Richards, D., Gow, NA., & Brand, AC. (2023). Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans. mBio, 14(5), Article e01157-23. https://doi.org/10.1101/2023.04.20.537637, https://doi.org/10.1128/mbio.01157-23

Giuraniuc, CV, Parkin, C, Almeida, MC, Fricker, M, Shadmani, P, Nye, S, Wehmeier, S, Chawla, S, Bedekovic, T, Lehtovirta-Morley, L, Richards, D, Gow, NA & Brand, AC 2023, 'Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans', mBio, vol. 14, no. 5, e01157-23. https://doi.org/10.1101/2023.04.20.537637, https://doi.org/10.1128/mbio.01157-23

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title = "Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans",

abstract = "Calcium (Ca2+) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca2+-dynamics in fungi are poorly understood due to lack of effective real-time Ca2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans, and used live-cell imaging to observe both dynamic Ca2+ spiking and slower changes in non-spiking Ca2+-GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca2+-GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca2+-GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H2O2 abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H2O2 was dependent on Cap1, extracellular Ca2+, and calcineurin but not on its downstream target, Crz1. Ca2+-dynamics were not affected by H2O2 in the hog1Δ or yvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca2+-GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca2+-influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans.",

author = "CV Giuraniuc and C Parkin and MC Almeida and M Fricker and P Shadmani and S Nye and S Wehmeier and S Chawla and T Bedekovic and L Lehtovirta-Morley and D Richards and NA Gow and AC Brand",

note = "Acknowledgements AB conceived the project and wrote the manuscript. CVG conceived the experimental design. SW designed the GCaMP reporter. AM, KL, LV-M, SC and TB constructed strains and optimised imaging. MF developed the image analysis software. CVG and CP carried out the microfluidics experiments and imaging analysis. NG assisted with preparation of the manuscript. PS, SN and DMR developed and undertook the theoretical data analysis and contributed to the interpretation of the results. Funding AB, CG and TB were funded by the Wellcome Trust [Grant number 206412/A/17/Z]. AB and DR were supported by a Wellcome Trust Institutional Strategic Support Award (WT204909/Z/16/Z). CP was funded by a University of Exeter studentship (113516). This work was also supported by a Royal Society URF (UF080611), an MRC NIRG (G0900211/90671) and the MRC-Centre for Medical Mycology at the University of Exeter (MR/N006364/2). DR was funded by the Medical Research Council (MR/P022405/1). SN was supported by the Medical Research Council via the GW4 BioMed2 DTP (MR/W006308/1). MCA was supported by a European Commission ITN {\textquoteleft}FungiBrain{\textquoteright} studentship (607963). LL and SC were funded by a Wellcome Trust Institutional Strategic Support Award to the University of Aberdeen. NG acknowledges support of Wellcome Trust Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards (101873, 200208, 215599, 224323). NG and AB thank the MRC (MR/M026663/2) for support. This study/research is funded by the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. ",

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AU - Parkin, C

AU - Almeida, MC

AU - Fricker, M

AU - Shadmani, P

AU - Nye, S

AU - Wehmeier, S

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AU - Bedekovic, T

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AU - Brand, AC

N1 - Acknowledgements AB conceived the project and wrote the manuscript. CVG conceived the experimental design. SW designed the GCaMP reporter. AM, KL, LV-M, SC and TB constructed strains and optimised imaging. MF developed the image analysis software. CVG and CP carried out the microfluidics experiments and imaging analysis. NG assisted with preparation of the manuscript. PS, SN and DMR developed and undertook the theoretical data analysis and contributed to the interpretation of the results. Funding AB, CG and TB were funded by the Wellcome Trust [Grant number 206412/A/17/Z]. AB and DR were supported by a Wellcome Trust Institutional Strategic Support Award (WT204909/Z/16/Z). CP was funded by a University of Exeter studentship (113516). This work was also supported by a Royal Society URF (UF080611), an MRC NIRG (G0900211/90671) and the MRC-Centre for Medical Mycology at the University of Exeter (MR/N006364/2). DR was funded by the Medical Research Council (MR/P022405/1). SN was supported by the Medical Research Council via the GW4 BioMed2 DTP (MR/W006308/1). MCA was supported by a European Commission ITN ‘FungiBrain’ studentship (607963). LL and SC were funded by a Wellcome Trust Institutional Strategic Support Award to the University of Aberdeen. NG acknowledges support of Wellcome Trust Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards (101873, 200208, 215599, 224323). NG and AB thank the MRC (MR/M026663/2) for support. This study/research is funded by the National Institute for Health and Care Research (NIHR) Exeter Biomedical Research Centre (BRC). The views expressed are those of the author(s) and not necessarily those of the NIHR or the Department of Health and Social Care. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission.

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N2 - Calcium (Ca2+) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca2+-dynamics in fungi are poorly understood due to lack of effective real-time Ca2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans, and used live-cell imaging to observe both dynamic Ca2+ spiking and slower changes in non-spiking Ca2+-GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca2+-GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca2+-GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H2O2 abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H2O2 was dependent on Cap1, extracellular Ca2+, and calcineurin but not on its downstream target, Crz1. Ca2+-dynamics were not affected by H2O2 in the hog1Δ or yvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca2+-GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca2+-influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans.

AB - Calcium (Ca2+) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca2+-dynamics in fungi are poorly understood due to lack of effective real-time Ca2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans, and used live-cell imaging to observe both dynamic Ca2+ spiking and slower changes in non-spiking Ca2+-GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca2+-GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca2+-GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H2O2 abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H2O2 was dependent on Cap1, extracellular Ca2+, and calcineurin but not on its downstream target, Crz1. Ca2+-dynamics were not affected by H2O2 in the hog1Δ or yvc1Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca2+-GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca2+-influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans.

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DO - 10.1101/2023.04.20.537637

M3 - Article

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JO - mBio

JF - mBio

IS - 5

M1 - e01157-23

ER -

Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans

Abstract

Bibliographical note

Data Availability Statement

Access to Document

Fingerprint

Datasets

Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans - Supplementary Movies

Analysis Software: Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans

Cite this