Global transcriptome analysis of Tropheryma whipplei in response to temperature stresses

Abstract

Tropheryma whipplei, the agent responsible for Whipple disease, is a poorly known pathogen suspected to have an environmental origin. The availability of the sequence of the 0.92-Mb genome of this organism made a global gene expression analysis in response to thermal stresses feasible, which resulted in unique transcription profiles. A few genes were differentially transcribed after 15 min of exposure at 43 degrees C. The effects observed included up-regulation of the dnaK regulon, which is composed of six genes and is likely to be under control of two HspR-associated inverted repeats (HAIR motifs) found in the 5' region. Putative virulence factors, like the RibC and IspDF proteins, were also overexpressed. While it was not affected much by heat shock, the T. whipplei transcriptome was strongly modified following cold shock at 4 degrees C. For the 149 genes that were differentially transcribed, eight regulons were identified, and one of them was composed of five genes exhibiting similarity with genes encoding ABC transporters. Up-regulation of these genes suggested that there was an increase in nutrient uptake when the bacterium was exposed to cold stress. As observed for other bacterial species, the major classes of differentially transcribed genes encode membrane proteins and enzymes involved in fatty acid biosynthesis, indicating that membrane modifications are critical. Paradoxically, the heat shock proteins GroEL2 and ClpP1 were up-regulated. Altogether, the data show that despite the lack of classical regulation pathways, T. whipplei exhibits an adaptive response to thermal stresses which is consistent with its specific environmental origin and could allow survival under cold conditions.

FIG. 1.

Relative normalized fluorescence intensities of DNA microarrays. (A) Comparison of cDNAs derived from the same culture of bacteria grown at 37°C. (B) Comparison of cDNAs derived from two independent cultures of bacteria grown at 37°C and at 4°C. (C) Comparison of cDNAs derived from two independent cultures of bacteria grown at 37°C and at 28°C. (D) Comparison of cDNAs derived from two independent cultures of bacteria grown at 37°C and at 43°C. The upper and lower dashed lines indicate 1.5-fold changes in the signal intensities.

FIG. 2.

Comparison of transcription measurements obtained by microarray and real-time RT-PCR assays. The relative transcriptional levels for 11 genes listed in Table 2 were determined by microarray analysis and real-time RT-PCR. The real-time RT-PCR log₂ values were plotted against the microarray log₂ values.

FIG. 3.

Genes differentially expressed at 4°C and 43°C compared to 37°C grouped by functional classification according to The Institute for Genome Research T. whipplei genome database (http://www.tigr.org/). The percentages of genes were calculated from the number of genes belonging to each functional category, as follows: column 1, amino acid biosynthesis; column 2, biosynthesis of cofactors, prosthetic groups, and carriers; column 3, cell envelope; column 4, cellular processes; column 5, central intermediary metabolism; column 6, DNA metabolism; column 7, energy metabolism; column 8, fatty acid and phospholipid metabolism; column 9, protein fate; column 10, protein synthesis; column 11, purines, pyrimidines, nucleosides, and nucleotides; column 12, regulatory functions; column 13, transcription; column 14, transport and binding proteins; column 15, unknown function.

FIG. 4.

Kinetics of dnaK and groEL transcription for T. whipplei exposed to heat shock. The relative levels of transcription of groEL2 (TWT441) and dnaK (TWT750) were determined by real-time RT-PCR using primers listed in Table 2. The data obtained were expressed as the ratio of the values obtained with bacteria exposed to heat shock to the values obtained with untreated cells grown at 37°C. The histograms are representative of two distinct experiments.

FIG. 5.

Evidence for two HAIR motifs upstream of the dnaK operon in T. whipplei. (A) Schematic representation of the dnaK operon, including six genes up-regulated with a 15-min heat shock at 43°C. Two homologues of the HAIR motif (18), designated TW_HAIR1 (5′-CATGAGTCGATATGACTCAAT-3′) and TW_HAIR2 (5′-CTTGAGTCATTACATGTCAAG-3′), were identified upstream of this region. (B) ClustalW alignment with the HAIR motif initially described for Mycobacterium tuberculosis H37Rv (53).

FIG. 6.

Circular representation of the T. whipplei Twist transcriptome. The outermost (first) circle indicates the nucleotide positions. The second and third circles indicate the ORF locations on the plus and minus strands, respectively. The fourth, fifth, and sixth circles indicate the microarray expression profiles for T. whipplei at 4°C versus 37°C, at 28°C versus 37°C, and at 43°C versus 37°C, respectively. The expression profiles for each gene, which are shown with a color-coded, base 2 logarithmic scale, were independently centered about zero. Green indicates decreased expression relative to the expression at 37°C. Red indicates increased expression relative to the expression at 37°C. Yellow indicates no change in expression relative to the expression at 37°C.