Identification of Early Response Genes in Human Peripheral Leukocytes Infected with Orientia tsutsugamushi: The Emergent of a Unique Gene Expression Profile for Diagnosis of O. tsutsugamush Infection

Abstract

Scrub typhus, caused by infection with Orientia tsutsugamushi, is one of the most common rickettsial diseases in the Asia-Pacific region. The disease can cause up to 35% mortality if left untreated. In order to get a better understanding of the host responses to O. tsutsugamushi infection, freshly isolated peripheral blood mononuclear cells (PBMC) were infected with O. tsutsugamushi. The infected cells were collected at 1 h, 4 h, 8 h or 18 h post infection. The gene expression profiles were monitored by cDNA microarray. Among the 7,489 genes, 658 genes were up or down regulated by 2-fold upon infection. ANOVA t-test revealed 432 genes with statistically significant fold change (p < 0.05). Semi-quantitative PCR using specific primers that flank the mRNA splicing sites of these 658 genes was carried out to verify the microarray results. More than 9000 semi-quantitative PCR reactions were performed using glyceraldehyde 3- phosphate dehydrogenase (GAPDH) as the reference gene. Of these reactions, 22 genes were confirmed to exhibit up or down regulation. Quantitative PCR using 18S rRNA as the reference further confirmed two genes (NM_001547 and NM_006187) that exhibited significant increase of expression. Analysis of these 22 genes and the specific increase of both NM_001547 and NM_006187 suggest that the 22 genes are unique to O. tsutsugamushi infection and have the potential use for differentiating infections caused by virus, bacteria, parasites and other pathogens.