Insights Into Histoplasma capsulatum Behavior on Zinc Deprivation

Abstract

Histoplasma capsulatum is a thermodimorphic fungus that causes histoplasmosis, a mycosis of global incidence. The disease is prevalent in temperate and tropical regions such as North America, South America, Europe, and Asia. It is known that during infection macrophages restrict Zn availability to H. capsulatum as a microbicidal mechanism. In this way the present work aimed to study the response of H. capsulatum to zinc deprivation. In silico analyses showed that H. capsulatum has eight genes related to zinc homeostasis ranging from transcription factors to CDF and ZIP family transporters. The transcriptional levels of ZAP1, ZRT1, and ZRT2 were induced under zinc-limiting conditions. The decrease in Zn availability increases fungicidal macrophage activity. Proteomics analysis during zinc deprivation at 24 and 48 h showed 265 proteins differentially expressed at 24 h and 68 at 48 h. Proteins related to energy production pathways, oxidative stress, and cell wall remodeling were regulated. The data also suggested that low metal availability increases the chitin and glycan content in fungal cell wall that results in smoother cell surface. Metal restriction also induces oxidative stress triggered, at least in part, by reduction in pyridoxin synthesis.

Keywords: Zn and cell wall remodeling; Zn uptake; fungal pathogenesis; proteomics; zinc homeostasis.

Figure 1

Influence of Zn availability on H. capsulatum growth and viability. (A) Growth in solid MMcM medium during 5 days with 20 μM of ZnSO₄ or 100 μM DPTA. (B) Growth of H. capsulatum in liquid medium was measured by optical density at 595 nm. (C) Viability of H. capsulatum in liquid medium was assessed by trypan blue dye. Growth and viability experiments were carried out in biological triplicates.

Figure 2

Transcript levels of Zn homeostasis genes during Zn deprivation. ZAP1: transcription factor that regulates the expression of zinc transporters. ZRT11, ZRT2, and ZRT3: Zinc carriers of the ZIP family. ZRC1 and ZRC2: Zinc transporters of the CDF family. The DTPA transcript levels were normalized against the transcript levels of control condition. Transcriptional levels of Zn-related genes were measured in biological triplicates. *:p-value ≤ 0.05.

Figure 3

Influence of Zn availability in H. capsulatum survival in macrophages. (A) Relative expression of Zap1 and Zrt1 in H. capsulatum during infection overtime. (B) Fungal burden of macrophages treated with (Hc + MØ), ZnSO4 (Hc + MØ + ZnSO4), and DTPA (Hc + MØ + DTPA) conditions. Control: H. capsulatum incubated in macrophage medium; Infection: macrophages infected with H. capsulatum yeasts. (C) Viability of macrophages in RPMI medium was measured by trypan blue dye. Statistical analysis was performed using t-test, one-way ANOVA, and the Tukey multiple comparison test with *p ≤ 0.05. All experiments were carried out in triplicates.

Figure 4

Functional categorization of proteins identified during zinc deprivation at 24 h (A) and 48 h (B). Up, upregulated proteins during zinc deprivation. Down, downregulated proteins during zinc deprivation.

Figure 5

Metabolic profile of H. capsulatum yeasts during zinc deprivation. Red: downregulated proteins; Blue: upregulated proteins.

Figure 6

Influence of zinc scarcity glucose metabolism of H. capsulatum. (A) Enzymatic activity of ADH during zinc deprivation in 24 h. (B) Glucose dosage in culture supernatants of cells grown in Zn replete or Zn depleted conditions. All experiments were carried out in biological triplicates. Student t test with *p ≤ 0.05.

Figure 7

Effect of Zn availability on H. capsulatum cell wall carbohydrates. (A) Dosage of total carbohydrate in H. capsulatum cells during zinc deprivation. (B) Fluorescence microscopy H. capsulatum grown under control and DTPA conditions. Calcofluor white and aniline blue were used to dosage chitin and glycan, respectively. (C) Measurement of glycan and chitin contents using fluorescence intensity. (D) Scanning electron microscopy of H. capsulatum during zinc deprivation. All experiments were carried out in biological triplicates. The comparisons were made using the Student t test with *p ≤ 0.05.

Figure 8

Influence of pyridoxine on H. capsulatum growth and oxidative stress level. (A) Growth curve of H. capsulatum during zinc deprivation at different concentrations of pyridoxine. (B) ROS detection of H. capsulatum labeled with DCFH-DA. Ctrl, fungal cells grown in zinc; DTPA, fungal cells grown in DTPA. All experiments were carried out in biological triplicates. The comparison was made using the Student t test with *p ≤ 0.05.

Figure 9

Hypothetical model of H. capsulatum response to zinc deprivation.