A simple approach for estimating gene expression in Candida albicans directly from a systemic infection site.
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.
Andes
D
D
Department of Medicine, Section of Infectious Diseases, University of Wisconsin, Madison, USA. dra@medicine.wisc.edu
Lepak
A
A
Pitula
A
A
Marchillo
K
K
Clark
J
J
D000818:Animals; D002176:Candida albicans / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D002177:Candidiasis / Q000382:microbiology*; D002473:Cell Wall / Q000378:metabolism; D004271:DNA, Fungal / Q000096:biosynthesis / Q000235:genetics; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000382:microbiology; D051379:Mice; D008813:Mice, Inbred ICR; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis; D012331:RNA, Fungal / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases / Q000378:metabolism; D015854:Up-Regulation
D004271:DNA, Fungal; D012331:RNA, Fungal; D012260:Ribonucleases