ORCID

Abstract

Microbial species capable of co-existing with healthy individuals, such as the commensal fungus Candida albicans, exploit multifarious strategies to evade our immune defenses. These strategies include the masking of immunoinflammatory pathogen-associated molecular patterns (PAMPs) at their cell surface. We reported previously that C. albicans actively reduces the exposure of the proinflammatory PAMP, β-1,3-glucan, at its cell surface in response to host-related signals such as lactate and hypoxia. Here, we show that clinical isolates of C. albicans display phenotypic variability with respect to their lactate- and hypoxia-induced β-1,3-glucan masking. We have exploited this variability to identify responsive and non-responsive clinical isolates. We then performed RNA sequencing on these isolates to reveal genes whose expression patterns suggested potential association with lactate- or hypoxia-induced β-1,3-glucan masking. The deletion of two such genes attenuated masking: PHO84 and NCE103. We examined NCE103-related signaling further because NCE103 has been shown previously to encode carbonic anhydrase, which promotes adenylyl cyclase-protein kinase A (PKA) signaling at low CO2 levels. We show that while CO2 does not trigger β-1,3-glucan masking in C. albicans, the Sch9-Rca1-Nce103 signaling module strongly influences β-1,3-glucan exposure in response to hypoxia and lactate. In addition to identifying a new regulatory module that controls PAMP exposure in C. albicans, our data imply that this module is important for PKA signaling in response to environmental inputs other than CO2. IMPORTANCE Our innate immune defenses have evolved to protect us against microbial infection in part via receptor-mediated detection of “pathogen-associated molecular patterns” (PAMPs) expressed by invading microbes, which then triggers their immune clearance. Despite this surveillance, many microbial species are able to colonize healthy, immune-competent individuals, without causing infection. To do so, these microbes must evade immunity. The commensal fungus Candida albicans exploits a variety of strategies to evade immunity, one of which involves reducing the exposure of a proinflammatory PAMP (β-1,3-glucan) at its cell surface. Most of the β-1,3-glucan is located in the inner layer of the C. albicans cell wall, hidden by an outer layer of mannan fibrils. Nevertheless, some β-1,3-glucan can become exposed at the fungal cell surface. However, in response to certain specific host signals, such as lactate or hypoxia, C. albicans activates an anticipatory protective response that decreases β-1,3-glucan exposure, thereby reducing the susceptibility of the fungus to impending innate immune attack. Here, we exploited the natural phenotypic variability of C. albicans clinical isolates to identify strains that do not display the response to β-1,3-glucan masking signals observed for the reference isolate, SC5314. Then, using genome-wide transcriptional profiling, we compared these non-responsive isolates with responsive controls to identify genes potentially involved in β-1,3-glucan masking. Mutational analysis of these genes revealed that a sensing module that was previously associated with CO2 sensing also modulates β-1,3-glucan exposure in response to hypoxia and lactate in this major fungal pathogen of humans.

Publication Date

2024-01-23

Publication Title

mBio

Volume

15

Issue

2

ISSN

2161-2129

Acceptance Date

2023-12-11

Deposit Date

2024-08-06

Funding

This work was funded by a program grant to A.J.P.B., N.A.R.G., L.P.E., and M.G.N. from the UK Medical Research Council [www.mrc.ac.uk: MR/M026663/1, MR/M026663/2]. The work was also supported by the Medical Research Council Centre for Medical Mycology [MR/N006364/1, MR/N006364/2], by a grant to C.d.E. from the European Commission [FunHoMic: H2020-MSCA-ITN-2018–812969], and by the Wellcome Trust via Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards [www.wellcome.ac.uk: 075470, 086827, 093378, 097377, 099197, 101873, 102705, 200208, 217163, 224323]. Work in the d’Enfert laboratory was supported by grants from the Agence Nationale de Recherche (ANR-10-LABX-62-IBEID) and the Swiss National Science Foundation (Sinergia CRSII5_173863/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. We are grateful to Jin Pu and Zeynab Heidari in the Centre for Genome-Enabled Biology and Medicine, University of Aberdeen for performing the RNA sequencing, to Attila Bebes and Raif Yuecel in the Cytomics Centre at the University of Exeter, and to Corin Liddle in the Bioimaging Unit in Biosciences at the University of Exeter for support with the fluorescence imaging. Computational analysis of RNA sequencing data was performed with the use of the Maxwell HPC compute cluster at the University of Aberdeen. We also thank Oliver Kurzai for providing the sch9 and rca1 mutants used in this study, and our colleagues in the MRC Centre for Medical Mycology, Aberdeen Fungal Group and the FunHoMic Network for insightful advice. This work was funded by a program grant to A.J.P.B., N.A.R.G., L.P.E., and M.G.N. from the UK Medical Research Council [www.mrc.ac.uk: MR/M026663/1, MR/M026663/2]. The work was also supported by the Medical Research Council Centre for Medical Mycology [MR/N006364/1, MR/N006364/2], by a grant to C.d.E. from the European Commission [FunHoMic: H2020-MSCA-ITN-2018–812969], and by the Wellcome Trust via Investigator, Collaborative, Equipment, Strategic and Biomedical Resource awards [www.wellcome.ac.uk: 075470, 086827, 093378, 097377, 099197, 101873, 102705, 200208, 217163, 224323]. Work in the d’Enfert laboratory was supported by grants from the Agence Nationale de Recherche (ANR-10-LABX-62-IBEID) and the Swiss National Science Foundation (Sinergia CRSII5_173863/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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. A.J.P.B., C.d.E., N.A.R.G., L.P.E., and M.G.N. conceived the project. G.M.A. performed the β-glucan masking screen and RNA work, S.S. analyzed the RNAseq data, Q.M. and A.V.R.R. generated the knockouts, and A.P., I.L., E.H., A.K.K., and C.L. performed the phenotyping of the mutants and data analysis. D.S.C., J.M.B., D.M., C.M., D.E.L., L.J.A., C.L., and C.A.M. provided essential experimental contributions and support such as the design and delivery of microscopy and cytometry and help with data interpretation. A.K.K. and C.M. generated the heatmaps for Figure 1Fig. 1. C.d.E. and M.E.B. provided the clinical isolates central to this study. C.M. and C.d.E. performed genome analyses. G.D.B. provided essential materials for the assays of β-glucan exposure (Fc-Dectin-1) as well as key input to the design of the fungal immunology experiments. A.J.P.B., G.M.A., Q.M., and A.P. wrote the manuscript, and all other authors contributed to the preparation and editing of the manuscript.

Keywords

Candida albicans, NCE103, carbonic anhydrase, immune evasion, pathogen-associated molecular patterns, β-glucan masking, Candida albicans/metabolism, Hypoxia/metabolism, Humans, Lactates/metabolism, Carbon Dioxide/metabolism, Glucans/metabolism, Pathogen-Associated Molecular Pattern Molecules, Cell Wall/metabolism, beta-Glucans

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