Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum

Abstract

The Escherichia coli hygromycin phosphotransferase (hph) gene, which confers hygromycin resistance, is commonly used as a dominant selectable marker in genetically modified bacteria, fungi, plants, insects, and mammalian cells. Expression of the hph gene has rarely been reported to induce effects other than those expected. Hygromycin B is the most common dominant selectable marker used in the molecular manipulation of Histoplasma capsulatum in the generation of knockout strains of H. capsulatum or as a marker in mutant strains. hph-expressing organisms appear to have no defect in long-term in vitro growth and survival and have been successfully used to exploit host-parasite interaction in short-term cell culture systems and animal experiments. We introduced the hph gene as a selectable marker together with the gene encoding green fluorescent protein into wild-type strains of H. capsulatum. Infection of mice with hph-expressing H. capsulatum yeast cells at sublethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes or to the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or an overwhelming fungal burden. The lethality was dependent on the kinase activity of hygromycin phosphotransferase. These results should raise awareness of the potential detrimental effects of the hph gene.

FIG. 1.

Expression of hph results in lethality among mice infected with Histoplasma capsulatum. Groups of mice were infected intranasally with 2 × 10⁴ to 2 × 10⁷ yeast cells of H. capsulatum strain G217B (n = 8), strain G186AR (n = 8), the hygromycin-resistant G217B derivative UC1 (n = 16), or the hygromycin-resistant G186AR derivative UC2 (n = 8). Survival curves demonstrate the loss of animals infected with >2 × 10⁵ hygromycin-resistant H. capsulatum cells between days 9 and 20.

FIG. 2.

Tissue fungal burden following Histoplasma capsulatum infection. Groups of mice were infected intranasally with 2 × 10⁶ yeast cells of H. capsulatum strain G217B or the hygromycin-resistant G217B derivative UC1. CFU in lungs (A) and spleens (B) were counted on day 7 (n = 13) or day 9 (n = 10) after infection. Data are means ± standard errors. *, P < 0 0.05; **, P < 0.0001.

FIG. 3.

Lung cell phenotype in Histoplasma capsulatum-infected animals. Mice were infected with H. capsulatum strain G217B or the hygromycin-resistant G217B derivative UC1, and on day 7 following infection lung homogenates were analyzed for CD4⁺ T cells, CD8⁺ T cells, dendritic cells, neutrophils, macrophages, and B cells. Data are means ± standard errors for four mice.

FIG. 4.

Cytokine profiles of splenic homegenates from Histoplasma capsulatum-infected animals. Mice were infected with H. capsulatum strain G217B or the hygromycin-resistant G217B derivative UC1, and lungs and spleens were harvested on day 7 and day 9 following infection. Levels of TNF-α, IFN-γ, IL-4, IL-10, and IL-12 were measured in the lung and spleen homegenates. Data are means ± standard errors for four mice.

FIG. 5.

Expression of hph results in lethality among mice infected with Histoplasma capsulatum. Groups of mice were intranasally infected with 2 × 10⁶ yeast cells of H. capsulatum strains UC16, expressing zeocin resistance; UC17, expressing hygromycin resistance; UC24 and UC25, expressing zeocin and hygromycin resistance; or UC18 and UC19, expressing zeocin resistance and a nonfunctional hygromycin phosphotransferase (n = 6 to 12 mice per strain). Survival curves demonstrate the loss of animals infected with hygromycin-resistant H. capsulatum between days 10 and 15.