Metabolic Profiling of Lung Granuloma in Mycobacterium tuberculosis Infected Guinea Pigs: Ex vivo ¹H Magic Angle Spinning NMR Studies

Abstract

A crucial and distinctive feature of tuberculosis infection is that Mycobacterium tuberculosis (Mtb) resides in granulomatous lesion at various stages of disease development and necrosis, an aspect that is little understood. We used a novel approach, applying high resolution magic angle spinning nuclear magnetic resonance spectroscopy (HRMAS NMR) directly to infected tissues, allowing us to study the development of tuberculosis granulomas in guinea pigs in an untargeted manner. Significant up-regulation of lactate, alanine, acetate, glutamate, oxidized and the reduced form of glutathione, aspartate, creatine, phosphocholine, glycerophosphocholine, betaine, trimethylamine N-oxide, myo-inositol, scyllo-inositol, and dihydroxyacetone was clearly visualized and was identified as the infection progressed. Concomitantly, phosphatidylcholine was down-regulated. Principal component analysis of NMR data revealed clear group separation between infected and uninfected tissues. These metabolites are suggestive of utilization of alternate energy sources by the infiltrating cells that generate much of the metabolites in the increasingly necrotic and hypoxic developing granuloma through the glycolytic, pentose phosphate, and tricarboxylic acid pathways. The most relevant changes seen are, surprisingly, very similar to metabolic changes seen in cancer during tumor development.

A Duplicated ESAT-6 Region of ESX-5 Is Involved in Protein Export and Virulence of Mycobacteria

“…This protein was shown to partially depend on the ESX-5 secretion system for export. It would be interesting to see if secreted ALD has a complementary role to play, particularly because the relative quantities of certain amino acids, such as alanine, are found to increase in granulomatous lesions (63,64). Altogether, our findings point toward an important function of the three replicate ESAT-6 regions of ESX-5 in protein secretion and virulence of mycobacteria.…”

Acetate Dissimilation and Assimilation in Mycobacterium tuberculosis Depend on Carbon Availability

“…Inside the phagosome of the macrophage or inside granulomas, the access to high glucose concentrations is restricted. Data about the metabolic composition of the M. tuberculosis-containing vacuoles inside macrophages are lacking, but mass spectrometry analysis determining the metabolic pool in the granulomas of guinea pigs have been performed (16). The detected levels of glucose or intermediates of the glycolysis were rather low, while free amino acids, organic acids, and lipid components accumulated during infection.…”

“…When pyruvate was offered as a carbon substrate to growing M. tuberculosis cells, high levels of acetate were detected in the supernatant of the culture. As the pyruvate concentration in macrophages and inside granulomas is considered rather low (15,16) and is most likely not a physiological carbon source for M. tuberculosis, we analyzed whether growth on fatty acids would also result in acetate secretion. In principle, acetyl-CoA and to a lesser extent propionylCoA are generated in the course of ␤-oxidation of fatty acids, turning fatty acids into a potential source for acetate formation.…”

“…Metabolic analysis of the granulomas of guinea pigs revealed that besides lipid components, different amino acids and organic acids, such as acetate, accumulate during an M. tuberculosis infection (16). Although it is generally believed that most of the detected metabolites are derived from the metabolism of the host, they might also be produced by the pathogen itself.…”

Broadening Our View About the Role of Mycobacterium tuberculosis Cell Envelope Components During Infection: A Battle for Survival

“…Thus, studies using scanning electron and atomic force microscopy revealed that the M.tb cell wall from MDRand XDR-strains differs from susceptible strains (Velayati et al, 2009a(Velayati et al, , 2009b(Velayati et al, , 2010. Experiments analyzing infected granulomas by using high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR) showed that we are capable of analyzing the cell wall of M.tb w h e n i n s i d e o f t h e g r a n u l o m a w i t h o u t f u r t h e r manipulation (Somashekar et al, 2011). The introduction of novel reporters that can be used for selective labeling of the cell wall of M.tb during infection in vitro and in vivo is already allowing us to see how the cell wall of M.tb is remodeling during infection .…”