Candida albicans Yeast, Pseudohyphal, and Hyphal Morphogenesis Differentially Affects Immune Recognition

Abstract

Candida albicans is a human opportunist pathogen that can grow as yeast, pseudohyphae, or true hyphae in vitro and in vivo, depending on environmental conditions. Reversible cellular morphogenesis is an important virulence factor that facilitates invasion of host tissues, escape from phagocytes, and dissemination in the blood stream. The innate immune system is the first line of defense against C. albicans infections and is influenced by recognition of wall components that vary in composition in different morphological forms. However, the relationship between cellular morphogenesis and immune recognition of this fungus is not fully understood. We therefore studied various vegetative cell types of C. albicans, singly and in combination, to assess the consequences of cellular morphogenesis on selected immune cytokine outputs from human monocytes. Hyphae stimulated proportionally lower levels of certain cytokines from monocytes per unit of cell surface area than yeast cells, but did not suppress cytokine response when copresented with yeast cells. Pseudohyphal cells induced intermediate cytokine responses. Yeast monomorphic mutants had elevated cytokine responses under conditions that otherwise supported filamentous growth and mutants of yeast and hyphal cells that were defective in cell wall mannosylation or lacking certain hypha-specific cell wall proteins could variably unmask or deplete the surface of immunostimulatory ligands. These observations underline the critical importance of C. albicans morphology and morphology-associated changes in the cell wall composition that affect both immune recognition and pathogenesis.

Keywords: Candida albicans; cell wall; cytokine; immune recognition; morphogenesis.

Figure 1

TNFα stimulation with Candida albicans yeast and hyphal cells by human peripheral blood mononuclear cells (PBMCs). (A) Cytokine production by human PBMCs stimulated with yeasts cells (Y) of C. albicans NGY152 grown at 25°C (closed circles) or hyphae (H) grown at 37°C (closed squares) in RPMI 1640 + 2.5% fetal calf serum (FCS). Error bars = SEM (n = 3). (B) Dose-dependent stimulation of TNFα production by human PBMCs when stimulated with different forms of C. albicans NGY152 grown in RPMI 1640 + 2.5% FCS. Heat-killed yeasts (HKY), closed circles; live yeasts (LY), open circles; HK hyphae (HKH), closed squares; live hyphae (LH), open squares. Error bars = SEM (n = 6). In these experiments, a total of 5 × 10⁵ PBMCs were used in each treatment and so the MOI varied from 0.0002:1 to 2:1. (C) Cytokine production by human PBMCs stimulated with the different morphological forms of C. albicans NGY152. In each treatment, an inoculum of 1 × 10⁶ cells/ml was used. Results are means ± SEM (n = 6; *p < 0.05).

Figure 2

Cell surface area (SA) of hyphal forming cells negatively correlated with TNFα stimulation. (A) Cell SA and cell size of Candida albicans SC5314 yeast (Y) or hyphae (H) grown at either 25 or 37°C for 1.5, 2.5, 3.5, and 4 h (*p < 0.05). Error bars are SEM (n > 50 individual cells). (B) Relationship between TNFα production and extent of cell elongation expressed as the ratio of the hyphal cell area and the mother yeast cell area for C. albicans SC5314 and NGY152, and Candida dubliniensis CD36.

Figure 3

TNFα production by human peripheral blood mononuclear cells stimulated with morphological mutants of Candida albicans mutants or other yeast species. (A) C. albicans NGY152 (WT), JKC19 (cph1Δ), HLC52 (efg1Δ), HLC54 (cph1Δ/efg1Δ), WYZ12.2 (hgc1Δ), and Bca2-10 (tup1Δ). Results are means ± SEM (n = 6; *p < 0.05). (B) TNFα stimulation by non-hypha-forming species Saccharomyces cerevisiae (S288C) and Candida glabrata (ATCC 2001) compared to hypha-forming species C. albicans (NGY152) and Candida dubliniensis (CD36) (*p < 0.05). Error bars = SEM (n = 9).

Figure 4

Stimulation of cytokine production by human peripheral blood mononuclear cells (PBMCs) interacting with Candida albicans yeast cells and hyphae. Human PBMCs in RPMI 1640 were preincubated 1 h with either C. albicans NGY152 yeast cells or hyphae and then stimulated with a second cell type for 24 h. HKY, heat-killed yeast; LH, live hyphae; HKH, heat-killed hyphae (*p < 0.05; n = 12).

Figure 5

Cytokine production stimulated by cell wall mutants of yeast and hyphal cells. (A) Cytokine production stimulated by human PBMCs with LY—live yeast; HKY—heat-killed yeast; LH—live hyphae; HKH—heat-killed hyphae; Candida albicans mnn4Δ and chs3Δ and control strains, NGY152 and CAF2-1. (B) Cytokine stimulation with mannosylation defective mutants, pmr1Δ, mnt1Δ/mnt2Δ, and mns1Δ. (C) als3Δ, hwp1Δ, hyr1Δ, and ece1Δ. Results presented as mean ± SEM (n = 6), and the asterisks indicate comparison of the wild-type values and the mutant strains (*p < 0.05).

Figure 6

Cytokine production by hPBMCs stimulated by different Candida albicans morphological forms. (A) Yeasts (i–ii), pseudohyphae (iii–iv), or hyphae (v–vi) of C. albicans NGY152 were grown in RPMI 1640 (pH 7) for 6 h at 25, 30, and 37°C, respectively, fixed, and stained with 25 µg/ml CFW to visualize the cell wall. Scale bars = 10 µm. (B) Morphological index (MI) of yeast, pseudohyphal, and hyphal cells was measured. Error bars are SEMs (averages from 50 cell measurements and 4 biological replicates) (*p < 0.05; **p < 0.01). (C) TNFα cytokine production elicited by human peripheral blood mononuclear cells (PBMCs) with HK NGY152 of yeast (Y), pseudohypha (PH), and hypha (H) cells. Each replicate (R1–R4) were averaged as total values. Data are means ± SEM (n > 6; *p < 0.05). (D) IL-6, IL-1β, IL-1α, and IL-10 cytokine production elicited by human PBMCs with HK of yeast (Y), pseudohypha (PH), and hypha (H) cells. Data are means ± SEM (n > 6; *p < 0.05). (E) Correlation between TNFα or IL-1β cytokine and MI. Results are Person R values (n = 4 biological replica).