Bartonella quintana deploys host and vector temperature-specific transcriptomes

Abstract

The bacterial pathogen Bartonella quintana is passed between humans by body lice. B. quintana has adapted to both the human host and body louse vector niches, producing persistent infection with high titer bacterial loads in both the host (up to 10(5) colony-forming units [CFU]/ml) and vector (more than 10(8) CFU/ml). Using a novel custom microarray platform, we analyzed bacterial transcription at temperatures corresponding to the host (37°C) and vector (28°C), to probe for temperature-specific and growth phase-specific transcriptomes. We observed that transcription of 7% (93 genes) of the B. quintana genome is modified in response to change in growth phase, and that 5% (68 genes) of the genome is temperature-responsive. Among these transcriptional changes in response to temperature shift and growth phase was the induction of known B. quintana virulence genes and several previously unannotated genes. Hemin binding proteins, secretion systems, response regulators, and genes for invasion and cell attachment were prominent among the differentially-regulated B. quintana genes. This study represents the first analysis of global transcriptional responses by B. quintana. In addition, the in vivo experiments provide novel insight into the B. quintana transcriptional program within the body louse environment. These data and approaches will facilitate study of the adaptation mechanisms employed by Bartonella during the transition between human host and arthropod vector.

Figure 1. B. quintana were enumerated to select time points for microarray analysis of growth stage-regulated genes.

(A) The diagram depicts the experimental design utilized in cultivation of B. quintana for in vitro transcriptome profiling at early vs. late stage growth. B. quintana were plated on chocolate agar and grown at 37°C for 2 days, at which point half of the cultures were shifted to 28°C. B. quintana were harvested for RNA extraction and colony-forming unit (CFU) enumeration on the days highlighted in green. (B) For each experiment, B. quintana growth was analyzed by enumerating CFU per plate after 3 to 11 total days of growth. CFU enumeration was done to determine the growth stage of the B. quintana cultures. Based on the data shown in 1B, the days highlighted in green in 1A and 1B were selected for B. quintana transcriptional profiling. CFU data from a single representative experiment are shown, and error bars represent the standard deviation of the mean CFU per plate from three replicates.

Figure 2. Growth stage-responsive genes comprise two large clusters and include a large proportion of the genome.

The heat map depicts unsupervised clustering of data from expression arrays from two independent time courses of B. quintana grown at either 28°C or 37°C for 7–9 days, as outlined in Figure 1. The arrays are depicted in columns, and the rows represent the probes on the array. The dendrogram at the left describes the similarity of the gene clusters. Regardless of the temperature at which the B. quintana were grown, there is a clear division into two distinct transcriptional programs (genes turned on then off, and off then on, over the duration of the time course). The inset legend shows the range of log₂-fold changes related to the range of colors in the heatmap. Genes of interest are noted along the right-hand side of the heatmap, in their cluster position.

Figure 3. B. quintana were enumerated to select time points for microarray analysis of temperature-regulated genes.

(A) The diagram summarizes the experimental design utilized in cultivation of B. quintana for in vitro transcriptome profiling at 37°C vs. 28°C. B. quintana were plated on chocolate agar and grown at 37°C for 2 days, at which point half of the cultures were shifted to 28°C. B. quintana were harvested for RNA extraction and colony-forming unit (CFU) enumeration on the days highlighted in green. (B) For each experiment, bacterial growth was analyzed by enumerating CFU per plate from 3 to 7 total days post plating. CFU enumeration was done to ensure that B. quintana cultures selected for global transcriptional profiling were in log phase growth at the respective temperatures. The days subsequently selected for in vitro transcriptional analysis of B. quintana are highlighted in green. CFU data from a single representative experiment are shown, and error bars indicate the standard deviation of the mean CFU per plate from three replicates.

Figure 4. RT-qPCR quantification of B. quintana transcription corroborates microarray data for temperature-regulated genes.

Transcription of select genes up-regulated at 28°C by microarray analysis was analyzed by RT-qPCR at 37°C (black) and 28°C (gray) to validate the microarray results. Transcript level was normalized to the B. quintana reference gene, purA. Error bars indicate standard errors of the mean. *, P≤0.05; **, P≤0.01 by Student's t test, comparing the relative level of transcription at 37°C and 28°C for each gene.

Figure 5. B. quintana genes up-regulated at 28°C are overrepresented in several COG functional categories.

The graph shows the COG classification of each gene that was significantly up- or down-regulated in B. quintana grown at 28°C, from microarray analysis (Table 1). Genes with increased transcription at 28°C are represented by black bars; genes with decreased transcription at 28°C are represented by gray bars. Of the categories with attributable function, there is an overrepresentation of up-regulated genes in the transcription, signal transduction, intracellular trafficking/secretion/vesicular transport, and defense mechanisms in B. quintana grown in vitro on agar, at the arthropod vector temperature of 28°C. The greatest number of genes down-regulated at 28°C are in the inorganic ion transport and metabolism category.

Figure 6. MEME searching identifies an overrepresented, purine-rich motif upstream of B. quintana genes up-regulated at 28°C (A).

Sequence logo of the top scoring MEME result for the top 11 regulated genes, by SAM score; and (B) position and scoring of motif sites (p-value threshold <1e-3) in upstream sequences. The motif is present in upstream sequences for 8 of the top 11 genes, often with multiple instances, as shown by the blue block diagram depicting motif position within upstream sequences.

Figure 7. The number of B. quintana per body louse increases over time during in vivo infection.

The number of B. quintana bacteria per louse was determined by real-time PCR analysis of DNA isolated from infected body lice. At 1 day post-infection (dpi), there were approximately 1.42×10⁴ ±2.83×10³ B. quintana per louse; at 5 dpi, 3.82×10⁴ ±1.02×10⁴ B. quintana per louse; and at 9 dpi, 1.36×10⁵ ±4.00×10⁴ B. quintana per louse. These findings corroborate the quantification of B. quintana in experimentally infected body lice reported by Seki et al., 2007. The average of data from three separate experiments is shown; error bars represent the standard errors of the mean.

Figure 8. Transcription of hbpC and BQ10280 in vivo corroborates transcription results in vitro at 28°C.

In vivo transcription of hbpC and BQ10280, genes determined to be highly expressed in vitro at 28°C by microarray, was analyzed in B. quintana-infected body lice (white bars) at 1, 5, and 9 days post-infection (dpi) by RT-qPCR. The in vitro transcription of hbpC and BQ10280 in B. quintana grown in vitro on chocolate agar at 28°C (gray bars) or 37°C (black bars) also was evaluated by RT-qPCR. Transcript level was normalized to B. quintana 16S rRNA. The relative level of hbpC and BQ10280 transcript in infected body lice was similar to that observed during in vitro growth of B. quintana at 28°C. The average of data from three separate experiments is shown; error bars represent the standard errors of the mean.