Major differential gene regulation in Coxiella burnetii between in vivo and in vitro cultivation models

Abstract

Background: Coxiella burnetii is the causative agent of the zoonotic disease Q fever. As it is an intracellular pathogen, infection by C. burnetii requires adaptation to its eukaryotic host and intracellular environment. The recently developed cell-free medium also allows the bacteria to propagate without host cells, maintaining its infection potential. The adaptation to different hosts or extracellular environments has been assumed to involve genome-wide modulation of C. burnetii gene expression. However, little is currently known about these adaptation events which are critical for understanding the intracellular survival of C. burnetii.

Results: We studied C. burnetii genome-wide transcriptional patterns in vivo (mice spleen) and in cell and cell-free in vitro culture models to examine its metabolic pathways and virulence associated gene expression patterns that are required to colonize and persist in different environments. Within each model, the gene expression profiles of the Dutch C. burnetii outbreak strain (602) and NM reference strains were largely similar. In contrast, modulation of gene-expression was strongly influenced by the cultivation method, indicating adaptation of the bacterium to available components. Genome-wide expression profiles of C. burnetii from in vitro cell culture were more similar to those seen for in vivo conditions, while gene expression profiles of cell-free culture were more distant to in vivo. Under in vivo conditions, significant alterations of genes involved in metabolism and virulence were identified. We observed that C. burnetii under in vivo conditions predominantly uses glucose as a carbon source (mostly for biosynthetic processes) and fatty acids for energy generation. C. burnetii experienced nutrient limitation and anaerobiosis as major stressors, while phosphate limitation was identified as an important signal for intracellular growth inside eukaryotic host cells. Finally, the in vivo environment significantly induced expression of several virulence genes, including those implicated in LPS synthesis, colonization, host component modulation and DNA repair mechanisms.

Conclusion: Our study shows that C. burnetii, with its relative small genome, requires only a subset of core gene functions to survive under in vitro conditions, but requires the induction of full repertoire of genes for successful pathogenesis and thriving in harsh environments in vivo.

Fig. 1

Principal component analysis on genome-wide expression profiles of C. burnetii strains 602 and NM obtained from various culture systems. Triplicate findings for each sample from different culture systems are clustered together indicative for experimental reproducibility. The plots of independent samples demonstrate also that based on their transcriptome profile, samples from different culture models can be differentiated

Fig. 2

Hierarchical clustering analysis on normalised signal intensities of probes of 602 and NM strains in different culture models. Data shows clustering of in vivo and in vitro models separately based on patterns of gene expression. Normalised signal intensity (log2) of probes (average of triplicates) for each condition are represented as a colour scale from red for high expressions to blue for lower expressions. In vivo (M = mice spleen), cell culture (cc), cell-free adapting phase passage 1 (p1) and passage 2 (p2), cell-free culture model (cf)

Fig. 3

Functional COG-categories of differentially regulated C. burnetii genes under in vivo and in vitro culture models. Functional categories of regulated genes of the 602 strain under in vivo and cell-free culture compared with cell culture model. The up-regulated and down-regulated genes are shown on the right and left side of the y-axis respectively. Largest group of regulated genes in all culture models belonged to the unknown function category (S). Large number of up-regulated genes under in vivo conditions belonged to the metabolism group such as coenzyme, carbohydrate, amino acid and lipid transport and metabolism (HGEI). Whereas, the largest number of up-regulated genes in cell-free culture compared to cell culture belonged to the protein synthesis group (O)

Fig. 4

Venn diagram of differentially expressed genes of 602 strain in different culture models. Comparison of differentially regulated genes under in vivo, cell-free adapting serial passages (p1 and p2) and fully adapted culture with respect to in vitro growth in cell culture

Fig. 5

Some metabolic pathways and pathways implicated in virulence and repair mechanisms induced in vivo compared to in vitro cultivation. The pathways were drawn according to KEGG database. Circles next to each gene display its regulation for each condition. a–i represent significantly up-regulated KEGG pathways under in vivo conditions: a Cardiolipin synthesis, b Mevalonate pathway, c Tryptophan synthesis, d Biotin synthesis, e Thiamine synthesis, f Fatty acid degradation, g Base excision repair, h Nucleotide excision repair, and i LPS synthesis. Reactivity of in vivo vs cell and cell-free cultures are always the same (except cbu0608, due to induction of mevalonate pathway in cell culture similar to in vivo, relative to cell-free culture system)