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Eukaryotic Cell, November 2007, p. 2066-2071, Vol. 6, No. 11
1535-9778/07/$08.00+0     doi:10.1128/EC.00139-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Expression of Hygromycin Phosphotransferase Alters Virulence of Histoplasma capsulatum{triangledown}

A. George Smulian,1,2* Reta S. Gibbons,1 Jeffery A. Demland,1 Deborah T. Spaulding,1 and George S. Deepe Jr.1,2

Infectious Disease Division, University of Cincinnati College of Medicine,1 Cincinnati VA Medical Center, Cincinnati, Ohio2

Received 24 April 2007/ Accepted 2 September 2007

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.

* Corresponding author. Mailing address: 231 Albert Sabin Way, Cincinnati, OH 45267-0560. Phone: (513) 861-3100, ext. 4425. Fax: (513) 475-6415. E-mail: Alan.Smulian{at}uc.edu

{triangledown} Published ahead of print on 14 September 2007.

Eukaryotic Cell, November 2007, p. 2066-2071, Vol. 6, No. 11
1535-9778/07/$08.00+0     doi:10.1128/EC.00139-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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