HcZrt2, a zinc responsive gene, is indispensable for the survival of Histoplasma capsulatum in vivo

Abstract

Histoplasma capsulatum (Hc) exists in the soil and is capable of adapting to the shift in environment during infection to ensure survival. Yeast encounter a restrictive host environment low in nutrients such as zinc. In this study we functionally analyzed a putative zinc regulated transporter, HcZrt2, in zinc limiting conditions by complementation of HcZrt2 and gene knockdown through RNA interference (RNAi). Complementation analysis demonstrated HcZrt2's ability to functionally replace the characterized Saccharomyces cerevisiae zinc plasma membrane transporters Zrt1 and Zrt2 in zinc deficient medium. Gene silencing revealed that HcZrt2 is essential for growth in zinc deficient medium and plays a role in zinc accumulation. Fungal burden was reduced in mice infected with HcZrt2 silenced strains compared to a control strain. Sixty-seven percent of mice infected with a lethal dose of HcZrt2-RNAi#1 survived, and 100% of mice infected with HcZrt2-RNAi#2 withstood lethal infection. Our data suggest that HcZrt2 is a vital part of zinc homeostasis and essential for the pathogenesis of histoplasmosis.

Keywords: Histoplasma capsulatum; RNAi; TPEN; zinc deficiency.

Figure 1.

A) Topology model of H. capsulatum Zrt2 using HMMTOP http://www.enzim.hu/hmmtop/ (Prediction of transmembrane helices and topology of proteins) Version 2. Multiple alignment of well conserved regions, transmembrane regions (shaded in gray) of the S. cerevisiae (Sc), A. fumigatus (Af), and H. capsulatum (Hc) zinc regulated transporters of the ZIP family using DNAman. Area highlighted in yellow indicate the putative metal ion binding sequence motif found in some ZIP family proteins. Conserved histidyl and glycyl residues are highlighted in green. B) Phylogenetic analysis demonstrating the relationship of HcZrt2 to other ZIP family proteins. (HcZrt2, H. capsulatum predicted zinc transporter 1 & 2- HistoBase microarray spot unique IDs- G217Borfs1.15b8, G217Borfs1.5d12, G217Borfs35o1, respectively). This Figure is reproduced in color in the online version of Medical Mycology.

Figure 1

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Figure 2.

Quantitative real time RT-PCR of HcZrt2 mRNA expression. Yeasts were cultured in A) HMM + 10 μM TPEN alone, with respective metals added or B) HMM + 1 mM of Deferoxamine for 2 hours at 37°C. Transcript levels were normalized to Ct values obtained from control samples (HMM only) and GAPDH gene. Data are shown as the mean with SEM. **P ≤ .01 and ***P ≤ .001. Statistical analyses performed using two-way ANOVA.

Figure 3.

Analysis of HcZrt2 for growth in zinc limiting conditions. Complementation analysis of growth in zinc deficient media by the HcZrt2 gene in S. cerevisiae ZHY3 strain. Transformants with the HcZrt2 and ScZrt1 genes under the control of the S. cerevisiae Zrt1 promoter were grown in medium with either A) 10 μM TPEN or B) 1 mM EDTA. Optical density readings were recorded after 24 hours of growth at 37°C. Growth compared between media without zinc or zinc added in the presence of TPEN or EDTA. Data shown as the mean with SEM ***P ≤ .001. Statistical analyses performed using two-way ANOVA and Bonferroni post hoc test. Empty Vector compared to ScZrt1 and HcZrt2 in 4 independent experiments for each. C) Optical density readings of H. capsulatum yeasts (Wild-type, silenced vector control and RNAi-HcZrt2) grown in Histoplasma Macrophage Media only or treated with 2 μM TPEN (zinc deficient media). Statistical analyses performed using two-way ANOVA and Bonferroni post tests. Silenced strains compared to Empty Vector controls in four independent experiments. This Figure is reproduced in color in the online version of Medical Mycology

Figure 4.

Total concentration of Zn, Fe, Cu, and Mn by ICP-MS in vector control and HcZrt2-RNAi silenced strains after culture in zinc deficient medium (2 μM TPEN) and compared to total concentrations in zinc replete medium. Zinc content represented as parts per billion (ppb) indicating ng of metal per gram and calculated based on mass of yeasts in sample. Statistical analyses performed using two-way ANOVA and Bonferroni post test. P < .05.

Figure 5.

A) Kinetics of mouse survival and loss of body weight as a percentage after infection with a lethal dose (1 × 10⁷) of Histoplasma yeasts. Six mice per group in two independent experiments. Total n = 12. Statistical analyses were performed using the log-rank test. B) Fungal burden analysis of mice infected with vector control strains and HcZrt2-RNAi strains at day 3. Limit of detection 5 × 10² (lung) 2 × 10² (spleen). Data represented as mean with SEM. Statistical analyses performed using one-way ANOVA and Dunnett's multiple comparison test P < .05. C–F) Fungal burden analysis of mice infected with vector control strains and HcZrt2-RNAi strains at day 5 for sublethal and lethal inocula. Limit of detection 5 × 10² (lung) 2 × 10² (spleen). Data represented as mean with SEM. Statistical analyses performed using one-way ANOVA and Dunnett's multiple comparison test. P < .05. This Figure is reproduced in color in the online version of Medical Mycology

Figure 6.

Colony-forming units of yeasts from infected bone marrow derived macrophages. Macrophages were lysed with water at 2, 24, and 48 hours and plated on HMM plates. Data represented as mean with SEM. P < .05.