Identification and isolation by DDRT-PCR of genes differentially expressed byHistoplasma capsulatumduring macrophages infection

Abstract

Establishment of infection and disease implies modifications in the genetic programmes of the cell systems that are involved and the differential expression of genes in both parasite and host. In order to identify and isolate relevant genes of the fungus,Histoplasma capsulatum, in which expression is specifically induced during its interaction with murine macrophages (Mф), we performed a comparative analysis of the pattern of gene expression of the fungus before and after exposure to, and internalization into Mф by using differential display reverse transcriptase-PCR (DDRT-PCR). Using a limited set of primer combinations, six cDNA fragments ofH. capsulatumwere identified and isolated; five representing fungal genes in which expressions were enhanced during Mф infection, whereas one mRNA fragment was down-regulated. Slot blots followed by Northern blot analyses confirmed that the transcripts detected with cDNA clones were over expressed after 1 h of Mф infection, whereas no transcripts were detected with mRNA purified fromH. capsulatumbefore infection. Sequence analyses and database searches revealed no significant homology to any known sequence for five of these clones. One of the clones showed homology to the rat p105 kD protein, and to the p100 kD co-activator proteins of human andCaenorhabditis elegans. To our knowledge, this is the first experimental evidence that specific genes are differentially expressed by a fungal pathogen when it is exposed to, and phagocytosed by Mф. Furthermore, these results show that the DDRT-PCR procedure has adequate sensitivity to detect fungal genes induced during parasite–host interaction to identify potential new targets that can be used to develop new antifungal drugs.