Eukaryotic Cell doi:10.1128/EC.00139-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.
Expression of hygromycin phosphotransferase alters virulence of Histoplasma capsulatum
A. George Smulian*,
Reta S. Gibbons,
Jeffery A. Demland,
Deborah T. Spaulding,
and
George S. Deepe Jr.
Infectious Disease Division, University of Cincinnati College of Medicine, and Cincinnati VA Medical Center, Cincinnati Ohio
* To whom correspondence should be addressed. Email:
Alan.Smulian{at}uc.edu.
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Abstract |
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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 H. 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 (GFP) into wild type strains of H. capsulatum. Infection of mice with hph expressing H. capsulatum yeast cells at sub-lethal doses resulted in lethality. The lethality was not attributable to the site of integration of the hph construct into the genomes, the method of integration and was not H. capsulatum strain related. Death of mice was not caused by altered cytokine profiles or 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.
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