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. 2017 Jan 26;85(2):e00832-16.
doi: 10.1128/IAI.00832-16. Print 2017 Feb.

Phagosomal Neutralization by the Fungal Pathogen Candida albicans Induces Macrophage Pyroptosis

Slavena Vylkova  1 , Michael C Lorenz  1
Affiliations

Phagosomal Neutralization by the Fungal Pathogen Candida albicans Induces Macrophage Pyroptosis

Slavena Vylkova et al. Infect Immun. .

Abstract

The interaction of Candida albicans with the innate immune system is the key determinant of the pathogen/commensal balance and has selected for adaptations that facilitate the utilization of nutrients commonly found within the host, including proteins and amino acids; many of the catabolic pathways needed to assimilate these compounds are required for persistence in the host. We have shown that C. albicans co-opts amino acid catabolism to generate and excrete ammonia, which raises the extracellular pH, both in vitro and in vivo and induces hyphal morphogenesis. Mutants defective in the uptake or utilization of amino acids, such as those lacking STP2, a transcription factor that regulates the expression of amino acid permeases, are impaired in multiple aspects of fungus-macrophage interactions resulting from an inability to neutralize the phagosome. Here we identified a novel role in amino acid utilization for Ahr1p, a transcription factor previously implicated in regulation of adherence and hyphal morphogenesis. Mutants lacking AHR1 were defective in growth, alkalinization, and ammonia release on amino acid-rich media, similar to stp2Δ and ahr1Δ stp2Δ cells, and occupied more acidic phagosomes. Notably, ahr1Δ and stp2Δ strains did not induce pyroptosis, as measured by caspase-1-dependent interleukin-1β release, though this phenotype could be suppressed by pharmacological neutralization of the phagosome. Altogether, we show that C. albicans-driven neutralization of the phagosome promotes hyphal morphogenesis, sufficient for induction of caspase-1-mediated macrophage lysis.

Keywords: Ahr1; Candida albicans; alkalinization; macrophages; pyroptosis.

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Figures

FIG 1
FIG 1
C. albicans AHR1 is important for growth on amino acids and for in vitro pH neutralization. C. albicans strains were grown in YNB plus 1% CAA, pH 4.0. (A) The growth of the cells was assessed by measuring the OD600 at the indicated time points. (B) The pH of the growing cultures was measured. The experiment was performed in triplicate. (C) Ammonia release from strains grown on YAC medium, pH 4.0, was assessed as described in Materials and Methods.
FIG 2
FIG 2
Cells lacking AHR1 are defective in hyphal morphogenesis during phagocytosis. FITC-stained C. albicans wild-type, ahr1Δ, ahr1Δ stp2Δ, ahr1Δ+AHR1, and stp2Δ strains were cocultured with J774A.1 macrophages for 60 min. Nonphagocytosed fungal cells were stained with calcofluor white, and then the cocultures were fixed and imaged. The percentage of phagocytosed hyphae (A, B) and hyphal length (C, D) were scored as described in Materials and Methods. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
FIG 3
FIG 3
Ahr1p is required for alkalinization of the macrophage phagosome. FITC-stained C. albicans wild-type, ahr1Δ, ahr1Δ stp2Δ, ahr1Δ+AHR1, and stp2Δ strains were cocultured with LysoTracker red-preloaded J774A.1 macrophages for 60 min and imaged. (A) To estimate the phagosomal pH, we measured the LysoTracker red intensity as described in Materials and Methods. Results are reported as mean values ± SDs from triplicate experiments. ***, P < 0.001. (B) Representative images of cocultures incubated in the absence (left) or presence (right) of the vATPase inhibitor BafA.
FIG 4
FIG 4
C. albicans strains defective in phagosomal neutralization fail to induce macrophage pyroptosis. C. albicans wild-type strain SC5314 and ahr1Δ, stp2Δ, and ahr1Δ stp2Δ mutant and complemented strains were pretreated or not with 50 nM BafA for 30 min. To calculate macrophage lysis, the C. albicans strains were cocultured with the immune cells for 5 h in the absence (no treatment) or presence of a caspase-1 inhibitor (z-YVAD-fmk). The extracellular release of lactate dehydrogenase (A, C) and IL-1β (B, D) was measured as described in Materials and Methods. The experiments were performed in triplicate. *, P < 0.05; ***, P < 0.001.
FIG 5
FIG 5
Alkalinization of the phagosome by C. albicans is required for macrophage killing and induction of pyroptotic cell death. C. albicans SC5314 and the ssy1Δ, dur1,2Δ, and ato5Δ mutants and their complemented strains were cocultured with J774A.1 macrophages for 5 h in a 96-well plate in the presence or absence of the caspase-1 inhibitor z-YVAD-fmk. Some of the wells were pretreated with 50 nM BafA for 30 min prior to coculture. The vATPase inhibitor was removed after 1 h of coculture, and all of the cultures were incubated for an additional 4 h. Macrophage lysis (A) and the release of IL-1β (B) were assessed as described in Materials and Methods. The experiments were performed in triplicate.
FIG 6
FIG 6
Phagosomal neutralization triggers C. albicans-driven macrophage pyroptosis. Upon interaction with macrophages, C. albicans cells trigger activation of the NLRP3 inflammasome and expression of pro-IL-1β. Within the phagosome, the fungal cell takes up amino acids (AA), metabolizes them, and expels NH4+ into the phagosomal milieu, a process that is dependent on the transcription factors Stp2p and Ahr1p. This results in neutralization of the phagosome and hyphal morphogenesis, with the latter contributing to membrane distention and the loss of immune cell integrity. LDH and the proinflammatory cytokine IL-1β are released from the cell and serve as a danger signal to uninfected macrophages to react to infection. Most of the phagocytosed cells defective in pH modulation, such as cells of the ahr1Δ stp2Δ mutant, reside in acidic phagosomes in yeast form, fail to activate the inflammasome, and are cleared more readily by the macrophages. These cells restore the ability to form hyphae and to damage the immune cell upon pharmacological neutralization of the phagosome with the vATPase inhibitor bafilomycin A.
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