Figure 1.
O. tsutsugamushi replication within monocytes.
Monocytes and L929 cells were infected with O. tsutsugamushi (two viable bacteria per cell) for different periods of time. A. The number of bacterial DNA copies was determined using qRT-PCR. The data are expressed as the mean ± SD of two independent experiments performed in triplicate. B. Monocytes were infected with O. tsutsugamushi, and the bacteria were detected using indirect immunofluorescence. C. Monocytes infected with O. tsutsugamushi for 5 days were labeled with bodipy phallacidin to detect filamentous actin and bacteria were detected using indirect immunofluorescence (in red). One representative micrograph performed in confocal microscopy is shown.
Figure 2.
GO analysis of differentially expressed genes.
Monocytes were stimulated with O. tsutsugamushi or mock stimulated for 8 hours, and the modulation of genes was analyzed using microarrays and GO term tools. The upregulated (A) and downregulated (B) genes were classified based on the major biological processes in which they are involved. The total number of genes present in each biological process and the number of differentially expressed genes are indicated. The results are expressed as the percentage of the upregulated or downregulated genes.
Figure 3.
Quantitative RT-PCR of selected genes in stimulated monocytes.
Monocytes were stimulated with or without O. tsutsugamushi for 8 (A) and 24 (B) hours. RNA was extracted, and qRT-PCR was performed on 16 genes involved in the immune response that were differentially expressing in the microarray experiments. The results, expressed as the log2 ratio of fold changes, are presented as the mean ± SEM of three experiments performed in triplicate.
Table 1.
Modulated genes in the “response to virus” GO term.
Figure 4.
Bacterial viability and monocyte responses.
Monocytes were stimulated with live or heat-killed O. tsutsugamushi for 8 (A) or 24 (B, C) hours. A. RNA was extracted, and qRT-PCR was performed to detect several genes that were differentially expressed in the microarray experiments. The results, expressed as the log2 ratio of fold changes, are presented as the mean ± SEM of two experiments performed in triplicate. B and C. Culture supernatants were analyzed for the presence of TNF (B) and IL-1β (C) using ELISAs. The results are expressed in ng/ml and are presented as the mean ± SD of two experiments performed in duplicate. *p<0.05.
Figure 5.
Hierarchical clustering of differentially expressed genes in stimulated monocytes.
Monocytes were stimulated with O. tsutsugamushi or IFN-γ (500 UI/ml) for 8 hours, and genome-wide expression studies were performed using microarrays from Agilent Technologies. A hierarchical clustering consisting of 300 highly altered genes is shown.
Table 2.
O. tsutsugamushi-induced apoptosis.
Figure 6.
Hierarchical clustering in patients with scrub typhus.
RNA was isolated from PBMCs from healthy controls and patients with different infectious diseases, and microarray studies were performed using Illumina Human-6 v2 BeadChips. The unsupervised hierarchical clustering of 22 patients and 2 RNA pools from healthy controls was performed based on the expression of 65 genes specific to scrub typhus (typ). The normalized expression level in each sample was baseline-adjusted to the mean expression level of the healthy control group and color-scaled, with red indicating increased expression and blue indicating decreased expression.
Figure 7.
Reduced transcriptional signature of scrub typhus.
The microarrays performed on RNA isolated from healthy controls or patients with infectious diseases suggested that five genes can be used as a specific signature of scrub typhus. A. The unsupervised hierarchical clustering of 22 patients and 2 pools of RNA from healthy controls was performed based on the expression of these five genes. The normalized expression level in each sample was baseline-adjusted to the mean expression level of the healthy control group and color-scaled, with red indicating increased expression and blue indicating decreased expression. B. The expression of the five genes was assessed using qRT-PCR. The results, expressed as the log2 ratio of fold changes, are presented as the mean ± SEM of all the patients in each group performed in duplicate.
Table 3.
Enriched biological processes in scrub typhus.
Figure 8.
Comparison between patient blood samples and in vitro-infected monocytes.
Monocytes from healthy donors were stimulated with O. tsutsugamushi for 8 hours, and Agilent microarrays were used to detect the differential expression of 2,015 genes that corresponded to 1,606 genes in the Illumina microarrays. Among these genes, 184 (250 probes) were altered in patients with scrub typhus with a p value less than 0.01. The hierarchical clustering of these genes demonstrates that the resulting transcriptional signature was specific to scrub typhus.