RNA interference-mediated silencing of the YPS3 gene of Histoplasma capsulatum reveals virulence defects

Abstract

The YPS3 gene of Histoplasma capsulatum encodes a protein that is both surface localized in the cell wall of H. capsulatum and released into the culture medium. This protein is produced only during the pathogenic yeast phase of infection and is also expressed differentially in H. capsulatum strains of different virulence levels. In this study, we silenced the YPS3 transcript by using an interfering-RNA strategy and examined the silenced mutants for phenotypic differences in vitro and during infection. The mutants showed no growth defect during in vitro culture in a defined medium at 37 degrees C and appeared to have normal virulence in a RAW 264.7 murine macrophage-like cell line. In a C57BL/6 mouse model of infection, however, the mutants caused significantly decreased fungal burdens, particularly in the peripheral phagocyte-rich tissues of livers and spleens. This defect in organ colonization was evident within 3 days of infection; however, it appeared to be exacerbated at later time points.

FIG. 1.

YPS3-silencing plasmids. These plasmids contain 517 bp of the predicted YPS3 cDNA sequence cloned in an inverted orientation and driven by the CBP1 (pYPS610) or H2B (pYPS620) promoter. They include inverted telomeres and the Podospora anserina URA5 gene for selection in H. capsulatum.

FIG. 2.

RNAi-silenced YPS3 mutants show a reduction in the levels of secreted Yps3. A SYPRO ruby-stained polyacrylamide gel with filtered concentrated supernatants (top panel) reveals the disappearance of a band consistent with Yps3 protein (Yps3p) in three independent RNAi transformants compared to the empty-vector control transformant. A Western blot with anti-Yps3 antibody (bottom panel) shows the reduction in the levels of secreted Yps3. Molecular size standards (in kilodaltons) are indicated.

FIG. 3.

(A) Yps3 RNAi mutants are not defective during in vitro yeast-phase growth in rich defined medium HMM. Growth was measured by using culture turbidity. (B) Yps3 RNAi mutants do not have reduced virulence in the murine macrophage-like cell line RAW 264.7. The rate of survival of the RAW 264.7 host cell monolayer after infection was measured by BrdU uptake and expressed as the percentage of the value for uninfected control cells. MOI, multiplicity of infection; EV, G217B ura5-23 strain transformed with pWU45, an empty vector. RNAi 4 and RNAi 11 are two independently generated G217B ura5-23 YPS3-silenced mutants. The assay was performed in triplicate, and results from a representative experiment are shown. Error bars indicate standard deviations.

FIG. 4.

Silencing of YPS3 reduces fungal burdens in lungs, livers, and spleens in C57BL/6 mice following intranasal infection. Graphs represent numbers of CFU (expressed as log₁₀ values) recovered from homogenates of organ tissues 7 days after intranasal infection with 2 × 10⁶ CFU of H. capsulatum. Data are the pooled results of two separate experiments, each with three to four mice per fungal strain. Error bars indicate standard deviations. P values represent significant differences in comparison to the control empty-vector strain (EV).

FIG. 5.

Silencing of YPS3 reduces the colonization of livers and spleens in C57BL/6 mice following intraperitoneal infection. Graphs represent numbers of CFU (expressed as log₁₀ values) recovered from homogenates of organs 14 days after intraperitoneal infection with 6 × 10⁷ CFU of Histoplasma capsulatum. Four mice per fungal strain were used, and error bars indicate standard deviations. P values represent significant differences in comparison to the control empty-vector strain (EV).

FIG. 6.

The YPS3-silenced mutants show an initial colonization defect in lungs, livers, and spleens. This defect is amplified at the later time points in the peripheral tissues of livers and spleens. Graphs represent numbers of CFU (expressed as log₁₀ values) recovered from homogenates of organ tissues 3, 10, or 14 days after intranasal infection with 2 × 10⁶ CFU of H. capsulatum. Each time point is represented by the mean of results for four to five mice, and error bars indicate standard deviations. EV, empty-vector control strain.