Commonalities of Mycobacterium tuberculosis Transcriptomes in Response to Defined Persisting Macrophage Stresses

Abstract

As the goal of a bacterium is to become bacteria, evolution has imposed continued selections for gene expression. The intracellular pathogen Mycobacterium tuberculosis, the causative agent of tuberculosis, has adopted a fine-tuned response to survive its host's methods to aggressively eradicate invaders. The development of microarrays and later RNA sequencing has led to a better understanding of biological processes controlling the relationship between host and pathogens. In this study, RNA-seq was performed to detail the transcriptomes of M. tuberculosis grown in various conditions related to stresses endured by M. tuberculosis during host infection and to delineate a general stress response incurring during persisting macrophage stresses. M. tuberculosis was subjected to long-term growth, nutrient starvation, hypoxic and acidic environments. The commonalities between these stresses point to M. tuberculosis maneuvering to exploit propionate metabolism for lipid synthesis or to withstand propionate toxicity whilst in the intracellular environment. While nearly all stresses led to a general shutdown of most biological processes, up-regulation of pathways involved in the synthesis of amino acids, cofactors, and lipids were observed only in hypoxic M. tuberculosis. This data reveals genes and gene cohorts that are specifically or exclusively induced during all of these persisting stresses. Such knowledge could be used to design novel drug targets or to define possible M. tuberculosis vulnerabilities for vaccine development. Furthermore, the disruption of specific functions from this gene set will enhance our understanding of the evolutionary forces that have caused the tubercle bacillus to be a highly successful pathogen.

Keywords: RNA-seq; dormancy; hypoxia; pH; starvation; tuberculosis.

Figure 1

(A) Heat map showing the expression level of 4014 genes using Z-score for normalized FPKM value. Red represents high expression level, while blue represents low expression level. The two-way hierarchical clustering is based on euclidean distance, showing similarity between different samples. (B, C) Heat map of the genes up- (B) and down-regulated (C) in all stress conditions. The log2 fold change is indicated in each cell for the specific gene/stress condition combination: hypoxic, 1%O₂; nutrient starvation, PBS; acidic, pH; stationary phase, StPh. Functional categories are as follows: 0 virulence, detoxification, adaptation; 1 lipid metabolism; 2 information pathways; 3 cell wall and cell processes; 5 insertion seqs and phages; 6 PE/PPE; 7 intermediary metabolism and respiration; 9 regulatory proteins; 10 conserved hypotheticals. *Genes part of the enduring hypoxic response.

Figure 2

Heat map of genes differentially expressed in stress conditions (A) or in the dormancy regulon (B). The log2 fold change for each gene and condition is indicated in the cell.

Figure 3

Heat map of genes differentially transcribed in (A) electron transfer chain, (B) glycolysis and tricarboxylic acid cycle (TCA), (C) nitrogen metabolism, (D) propionate metabolism (MCC, methylcitrate cycle; MMP, methylmalonate pathway). The log2 fold change for each gene and condition is indicated in the cell.

Figure 4

Heat map of genes differentially expressed in (A) amino acid, (B) vitamins and cofactor biosynthesis and metabolism. The log2 fold change for each gene and condition is indicated in the cell.

Figure 5

Heat map of genes differentially expressed in lipid biosynthesis and metabolism: (A) fatty acid and mycolic acid biosynthesis, (B) peptidoglycan and arabinogalactan synthesis, (C) glycerolipid metabolism (tgs, triacylglycerol synthase), (D) methyl-branched lipid biosynthesis [sulfolipids (SL), acylated trehaloses (AT), PDIMs]. The log2 fold change for each gene and condition is indicated in the cell.

Figure 6

Heat map of genes expression associated with the ESX systems (A) and the mammalian cell entry (mce) system (B). The log2 fold change for each gene and condition is indicated in the cell.

Figure 7

Fragment per kilobase million (FPKM) of genes in the 95^th percentile. The five M. tuberculosis growth conditions were: exponential, hypoxia (1% O₂), nutrient starvation (PBS), acidic environment (pH) and stationary phase (StPh).