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1 September 2005

Volume 192, Number 5
The Journal of Infectious Diseases 2005;192:893–900
© 2005 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2005/19205-0022$15.00
DOI: 10.1086/432104
MAJOR ARTICLE

A Simple Approach for Estimating Gene Expression in Candida albicans Directly from a Systemic Infection Site

D. Andes,1,2

A. Lepak,1

A. Pitula,1

K. Marchillo,1 and

J. Clark1

1Department of Medicine, Section of Infectious Diseases, and 2Department of Medical Microbiology and Immunology, University of Wisconsin, Madison

Gene expression analysis after the host‐pathogen interaction is revolutionizing our understanding of the host response to infection. Numerous studies have utilized microarray analysis to follow host cell transcriptome alterations in response to interactions with infectious pathogens. However, similar analyses of pathogen transcriptional adaptation at the infection site have been limited. Understanding the nature of this interaction from the pathogen perspective at different sites and stages of infection is central to strategies for development of new anti‐infective therapies. Toward this end, we developed a protocol to analyze changes in gene expression for a eukaryotic pathogen, Candida albicans, during systemic infection in mice. The experimental approach takes advantage of the resistance of the cell wall of many fungal pathogens to cell lysis, relative to mammalian cells. After lysis of mammalian cells, the tissue mixture containing fungal cells is depleted of mammalian RNA by centrifugation, followed by enzymatic digestion. RNA‐digesting enzymes are then inhibited before eukaryotic cell lysis and RNA isolation. The protocol provides a reproducible quantity of RNA based on pathogen cell number. The quality of the RNA allowed reliable downstream transcriptional analysis using reverse‐transcription polymerase chain reaction and microarrays. The in vivo gene expression data confirmed involvement of several putative pathogenesis genes. More importantly, the results provided a wealth of biologically interesting hypotheses to direct future investigation.

Received 24 January 2005; accepted 23 March 2005; electronically published 25 July 2005.

Reprints or correspondence: Dr. David Andes, 600 Highland Ave, Room H4/572, Madison, WI 53792 ().

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  • Financial support: National Institutes of Health (grant K08 AI 001767‐04 to D.A.).

    Potential conflicts of interest: none reported.

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