Sequence Variation of Candida albicans Sap2 Enhances Fungal Pathogenicity via Complement Evasion and Macrophage M2-Like Phenotype Induction

Abstract

Candida albicans (C. albicans) is an opportunistic pathogen increasingly causing candidiasis worldwide. This study aims to investigate the pattern of systemic immune responses triggered by C. albicans with disease associated variation of Sap2, identifying the novel evasion strategies utilized by clinical isolates. Specifically, a variation in clinical isolates is identified at nucleotide position 817 (G to T). This homozygous variation causes the 273rd amino acid exchange from valine to leucine, close to the proteolytic activation center of Sap2. The mutant (Sap2-273L) generated from SC5314 (Sap2-273V) background carrying the V273L variation within Sap2 displays higher pathogenicity. In comparison to mice infected with Sap2-273V strain, mice infected with Sap2-273L exhibit less complement activation indicated by less serum C3a generation and weaker C3b deposition in the kidney. This inhibitory effect is mainly achieved by Sap2_273L -mediated stronger degradation of C3 and C3b. Furthermore, mice infected with Sap2-273L strain exhibit more macrophage phenotype switching from M0 to M2-like and more TGF-β release which further influences T cell responses, generating an immunosuppressed cellular microenvironment characterized by more Tregs and exhausted T cell formation. In summary, the disease-associated sequence variation of Sap2 enhances pathogenicity by complement evasion and M2-like phenotype switching, promoting a more efficient immunosuppressed microenvironment.

Keywords: Candida albicans; complement evasion; fungal pathogenicity; immunosuppressed cellular environment; sequence variation.

Figure 1

SAP2 sequence variations of C. albicans clinical isolates. a) The patchy distribution of SAP2 variants among 49 clinical isolates. b) Summary of total nucleotide exchanges within the 1197 nucleotide long SAP2 gene and amino acid exchanges in the Sap2 protein identified among 49 clinical isolates. The mutation at nucleotide position 817 (G to T, highlighted in red) existed in all isolates tested, which caused the amino acid exchange at position 273 (valine to leucine). c) Sap2 consists of two active sites, located at 88th aa or 274th aa, respectively, and sequencing results show that the clinically identified variant is located at 273rd aa.

Figure 2

The genetically engineered Sap2‐273L displayed elevated fungal pathogenicity. a) Three different clinical isolates as well as Sap2‐273V and Sap123 ko strains were selected and intravenously injected into wild type C57Bl6 mice (n = 5 per group, 5 × 10⁵ CFU/mouse). Mice were sacrificed at 16 h post infection, kidney, lung, liver, brain, and spleen were therefore taken and homogenized. Samples were pre‐diluted and plated on YPD agar plates, then cultivated at 30 °C for 48 h. The colonies were counted and compared among different groups. b,c) Generation of mutant strain carried V273L variation within Sap2. CRISPR‐cas9 was used to mutate the 817th site within the SAP2 gene from G to T by recombining the template into the genome of the Sap2‐273V strain. d) Sanger sequencing was performed to identify the SAP2 gene of the three strains. e) The growth rate of the Sap2‐273L and Sap2‐273V strains. The equal growth rate of the two strains was observed. f) Sap2 secretion levels by the Sap2‐273L and Sap2‐273V strains were detected by the specific Sap2 antibody. g) The survival rate of mice infected with the Sap2‐273L and Sap2‐273V strains (2 × 10⁵ CFU/mouse) for 15 days, ten mice per group, “n” indicates the number of mice dead in a given day. Data are shown as means ± SD, and from one of three independent experiments. The survival rate of mice was analyzed by a Kaplan–Meier log rank test, and the p values were calculated by the Student's t‐test.

Figure 3

Genetically engineered Sap2‐273L strain potentially modulated complement activation and early inflammatory cytokine pattern. a,b) C3a level in the blood of fungal infected mice. Mice were intravenously infected with Sap2‐273L (n = 6), Sap2‐273V (n = 6) strains, or PBS (n = 3) (1 × 10⁵ CFU/mouse). At day 2, day 7, and day 12 post infection, C3a in the blood was detected by western blotting and compared by integrated density. c–e) Correlation between blood C3a level and fungal burden in mice. Mice were intravenously infected with Sap2‐273L (n = 15), Sap2‐273V strains (n = 20), or PBS (n = 5) (1 × 10⁵ CFU/mouse) and sacrificed on day 2 post infection. Blood levels of C3a were detected by ELISA (c), the fungal burden in the kidney was counted and compared between groups (d), and then the correlation between blood C3a level and fungal burden was analyzed (e). f) C3b deposition (yellow) was detected by immunofluorescent staining of the kidney slides. g) The levels of different cytokines released in the serum of fungal infected mice. Mice were intravenously injected with the same amount of Sap2‐273L, Sap2‐273V strains, or PBS (n = 8 per group) (1 × 10⁵ CFU/mouse). The cytokines in serum at day 2 post infection were detected by cytometric bead array. Data are shown as means ± SD, and from one of three independent experiments. p values were analyzed by Student's t‐test, *p < 0.05, **p < 0.01.

Figure 4

Site mutated Sap2_273L, by potentially degrading C3 and C3b, displayed stronger complement inhibitory effects. a,b) Sap2 mediated concentration‐dependent degradation of C3/C3b. Different doses (0.25, 0.5, 1, 2 µg) of purified Sap2_273V and Sap2_273L proteins were incubated with purified human C3 or C3b (0.5 µg), afterward, the mixtures were separated by SDS‐PAGE under reducing conditions, and the degradation products of C3 (a) and C3b (b) were detected by western blotting using polyclonal goat anti human C3 antibody. c) Effect of V273L variation of Sap2 on MAC formation, PBS indicated a negative background control without NHS in the sample. d) Effect of V273L variation of Sap2 on C3b/iC3b deposition, PBS indicated a negative background control without NHS in the sample. e) Effect of V273L variation of Sap2 on C3a release, PBS indicated a negative background control without NHS in the sample. f) Effect of V273L variation of Sap2 on C3b/iC3b mediated phagocytosis of C. albicans by THP‐1. The phagocytosis of Sap2‐273V strain by THP‐1 cells was quantified by flow cytometry as double positive cells (FITC⁺, DiD⁺), PBS indicated a negative background control without C. albicans. Data are shown as means ± SD, and from one of three independent experiments. p values were analyzed by Student's t‐test, **p < 0.01.

Figure 5

Genetically engineered Sap2‐273L strain triggered a stronger immunosuppressed microenvironment. Mice were intravenously injected with an equal amount of Sap2‐273L (n = 4), Sap2‐273V strains (n = 4), or PBS (n = 3) (1 × 10⁵ CFU/mouse). At day 12 post infection, a) CD4⁺/CD8⁺ T cells (green/red) infiltration and C3b deposition (yellow) were detected by immunofluorescent staining of the kidney. b) The early activation of CD4⁺/CD8⁺T cells in B (blood) and S (spleen) was analyzed by flow cytometry. CD4⁺CD69⁺ population indicated the early activation of CD4⁺ T cells, and CD8⁺CD69⁺ population was early activation of CD8⁺ T cells. c,d) The differentiation of CD4+ T help cells in the B (blood) (c) and S (spleen) (d) analyzed by flow cytometry. Th1 cells, Th17, and Treg cells were classified by the positive expression of T‐bet, RORγT, and FoxP3. Tfh cells were identified by the high expression of CXCR5 and PD‐1. e) The exhaustion of CD4⁺/CD8⁺ T cells in B (blood) and S (spleen) was detected by expression of immune checkpoint molecule PD‐1. f) The levels of different cytokines released in serum at day 12 post infection. Mice were intravenously infected with the same amount of Sap2‐273L, Sap2‐273V strains, or PBS (n = 8 per group) (1 × 10⁵ CFU/mouse). The cytokines in serum were detected by cytometric bead array. Data are shown as means ± SD, and from one of three independent experiments. The p values were calculated by the Student's t‐test.

Figure 6

C. albicans triggered immunosuppressed T cell responses by inducing macrophage M2‐like phenotype switch. a) The types of macrophages in blood and spleen were examined by flow cytometry using CD11c, CD11b, and F4/80⁺ as detection markers, n = 4 per group. b,c) The expression of CD206 in RAW cells stimulated by Sap2‐273L, Sap2‐273V strains, or LPS was detected by flow cytometry. d) The cytokine ARG‐1 level was detected by qPCR. e) Co‐localization of BMDM cells (CD206⁺, pink) and C. albicans (CFSE⁺, green) was detected by immunofluorescent staining. f,g) IL‐10 and TGF‐β transcription level upon co‐culture of RAW cells with Sap2‐273L or Sap2‐273V for 3 and 6 h. Data are shown as means ± SD, and from one of three independent experiment. p values were analyzed by Student's t‐test. *p < 0.05, **p < 0.01.

Figure 7

The model of how disease associated V273L variation within Candida albicans Sap2 enhances fungal pathogenicity. Our study illustrated the V273L variation, by enhancing Sap2‐mediated C3 and C3b cleavage, efficiently blocked C3b/iC3b surface deposition and CR3‐mediated phagocytosis. This chain of events led to a more potent deficiency of the fast clearance of Sap2‐273L strain. Therefore, persistent chronic infection of Sap2‐273L strain with a higher fungal burden in the body accelerated the phenotype switch of macrophages from M0 to M2‐like type, which in turn up‐regulates CD11b expression level on the surface of M2‐like macrophages. A higher CD11b level, by binding to more β‐glucan present on fungal surfaces, triggered M2‐like macrophages to potentially release TGF‐β. Such cytokine patterns further interfered with T cell responses and developed an immunosuppressed microenvironment characterized by more Tregs and exhausted T cell formation.