Identification of an unusual pattern of global gene expression in group B Streptococcus grown in human blood

Abstract

Because passage of the bacterium to blood is a crucial step in the pathogenesis of many group B Streptococcus (GBS) invasive infections, we recently conducted a whole-genome transcriptome analysis during GBS incubation ex vivo with human blood. In the current work, we sought to analyze in detail the difference in GBS gene expression that occurred in one blood sample (donor A) relative to other blood samples. We incubated GBS strain NEM316 with fresh heparinized human blood obtained from healthy volunteers, and analyzed GBS genome expression and cytokine production. Principal component analysis identified extensive clustering of the transcriptome data among all samples at time 0. In striking contrast, the whole bacterial gene expression in the donor A blood sample was significantly different from the gene expression in all other blood samples studied, both after 30 and 90 min of incubation. More genes were up-regulated in donor A blood relative to the other samples, at 30 min and 90 min. Furthermore, there was significant variation in transcript levels between donor A blood and other blood samples. Notably, genes with the highest transcript levels in donor A blood were those involved in carbohydrate metabolism. We also discovered an unusual production of proinflammatory and immunomodulatory cytokines: MIF, tPAI-1 and IL-1beta were produced at higher levels in donor A blood relative to the other blood samples, whereas GM-CSF, TNF-alpha, IFN-gamma, IL-7 and IL-10 remained at lower levels in donor A blood. Potential reasons for our observations are that the immune response of donor A significantly influenced the bacterial transcriptome, or both GBS gene expression and immune response were influenced by the metabolic status of donor A.

Figure 1. Number of bacterial cells during incubation with human blood.

The number of CFU/ml in the donor A blood sample (marked by a cross) is similar to the number of CFU/ml in the seven other blood samples at time 0 and after 90 min of incubation at 37°C.

Figure 2. Principal component analysis (PCA) plot showing transcriptome differences between expression microarray data of GBS NEM316 strain incubated in human blood at 37°C.

Samples were analyzed at time 0, and after 30 min and 90 min of incubation. The PCA plot captures the variance in a dataset in terms of principal components and displays the most significant of these on the x, y, and z axes. The percentages of the total variation that are accounted for by the 1^st, 2^nd, and 3^rd principal components are shown on the x-, y- and z-axes labels. Plots are colored and shaped by time point. Plots for donor A blood sample are specifically marked (“” for time 0, “” for time 1, and “” for time 2).

Figure 3. Differential regulation of transcript expression in GBS strain NEM316 after incubation with human blood.

Genes were classified into 22 main functional categories. Bars indicate the numbers of genes whose expression was modified in the donor A blood sample and the average of the seven other donor blood samples at time 1 and time 2 relative to time 0. On the left, genes that were down-regulated relative to time 0. After 30 min of incubation, 62 transcripts were down-regulated in the donor A blood sample, while 658 transcripts were down-regulated in the other donor blood samples. After 90 min of incubation, 369 transcripts were down-regulated in the donor A blood sample, while 518 transcripts were down-regulated in the other donor blood samples. On the right, genes that were up-regulated relative to time 0. After 30 min of incubation, 160 transcripts were up-regulated in the donor A blood sample, while 134 transcripts were up-regulated in the other donor blood samples. After 90 min of incubation, 270 transcripts were up-regulated in the donor A blood sample, while 115 transcripts were up-regulated in the other donor blood samples.

Figure 4. Level of GBS strain NEM316 gene expression in donor A blood and other donor blood samples.

Levels of expression were categorized arbitrarily as: (i) expressed at very low level or not expressed, (ii) “normally” expressed, and (iii) highly expressed (form left to right, first column following five columns, and last 15 columns, respectively). After 30 min of incubation, 1,390, 588 and no ORFs were categorized as mentioned above in donor A blood, while there were 1,621, 305 and 6 ORFs in the other blood samples. After 90 min of incubation, 1,576, 395 and no ORFs were categorized as mentioned above in donor A blood, while there were 1,674, 294 and 6 ORFs in the other blood samples.

Figure 5. Differential expression of genes involved in carbohydrate metabolism in GBS strain NEM316.

All of the 14 red plots mark genes involved in carbohydrate metabolism expressed at higher levels in donor A blood (axis) relative to the other blood samples (ordinate) after 90 min incubation. For comparison, small figure on the top left represents gene expression after 30 min incubation.

Figure 6. Cytokine production in the eight blood donor samples.

Cytokine concentrations were measured using the xMAP technology combining a sandwich immunoassay with fluorescent bead-based technology. Cytokines were measured immediately before contact with GBS (left of each figure) and after 90 min of incubation with the bacteria (right of each figure). tPAI-1 was higher in donor A blood relative to the other blood samples at time 0, while MIF and IL-1β were higher in donor A blood than in the other blood samples after 90 min. Cytokine concentrations are expressed in pg/ml.