Metabolomics Studies To Decipher Stress Responses in Mycobacterium smegmatis Point to a Putative Pathway of Methylated Amine Biosynthesis

Abstract

Mycobacterium smegmatis, the saprophytic soil mycobacterium, is routinely used as a surrogate system to study the human pathogen Mycobacterium tuberculosis. It has also been reported as an opportunistic pathogen in immunocompromised hosts. In addition, it can exist in several ecological setups, thereby suggesting its capacity to adapt to a variety of environmental cues. In this study, we employed untargeted proton nuclear magnetic resonance ( 1 H-NMR)-based metabolomics to identify metabolites and metabolic pathways critical for early adaptive responses to acidic stress, oxidative stress, and nutrient starvation in Mycobacterium smegmatis. We identified 31, 20, and 46 metabolites that showed significant changes in levels in response to acidic, oxidative, and nutrient starvation stresses, respectively. Pathway analyses showed significant perturbations in purine-pyrimidine, amino-acid, nicotinate-nicotinamide, and energy metabolism pathways. Besides these, differential levels of intermediary metabolites involved in ␣-glucan biosynthesis pathway were observed. We also detected high levels of organic osmolytes, methylamine, and betaine during nutrient starvation and oxidative stress. Further, tracing the differential levels of these osmolytes through computational search tools, gene expression studies (using reverse transcription-PCR [RT-PCR]), and enzyme assays, we detected the presence of a putative pathway of biosynthesis of betaine, methylamine, and dimethylamine previously unreported in Mycobacterium smegmatis. IMPORTANCE Alterations in metabolite levels provide fast and direct means to regulate enzymatic reactions and, therefore, metabolic pathways. This study documents, for the first time, the metabolic changes that occur in Mycobacterium smegmatis as a response to three stresses, namely, acidic stress, oxidative stress, and nutrient starvation. These stresses are also faced by intracellular mycobacteria during infection and therefore may be extended to frame therapeutic interventions for pathogenic mycobacteria. In addition to the purine-pyrimidine, amino acid, nicotinate-nicotinamide, and energy metabolism pathways that were found to be affected in response to different stresses, a novel putative methylamine biosynthesis pathway was identified to be present in Mycobacterium smegmatis.

Rewiring of Metabolic Network in Mycobacterium tuberculosis During Adaptation to Different Stresses

“…It is known that nitrogen metabolism pathway helps in alleviating acidic stress by producing ammonia (Gouzy et al, 2014). We make a similar observation in one of our earlier studies involving M. smegmatis , where several of these pathways were perturbed during acidic stress (Rizvi et al, 2019). Similarly, nutrient starvation can alter nitrogen assimilation and use efficiency to adjust to nutrient limiting conditions for survival.…”

“…Primarily M.tb cultures were grown in 7H9 media [consisting 0.4% glycerol (v/v), 0.05% tyloxapol (v/v) and supplemented with 10% Middlebrook Oleic Albumin Dextrose Catalase (OADC) growth supplement (v/v)] at 37°C with shaking at 180 rpm till the absorbance at OD 600 nm reached to 0.6–0.7. Cultures were then harvested, washed, and resuspended in 10 ml of respective stress media for 36 h. The study was performed by using well-established in vitro models mimicking stress conditions such as acidic stress (pH 5.5), oxidative stress, iron deprivation (no iron supplemented) in Sauton’s minimal medium (Vemula et al, 2016; Balakrishnan et al, 2017; Rizvi et al, 2019). For nutrient starvation stress, 1xPBS was used as described by Loebel et al (1933) and Rizvi et al (2019).…”

“…Cultures were then harvested, washed, and resuspended in 10 ml of respective stress media for 36 h. The study was performed by using well-established in vitro models mimicking stress conditions such as acidic stress (pH 5.5), oxidative stress, iron deprivation (no iron supplemented) in Sauton’s minimal medium (Vemula et al, 2016; Balakrishnan et al, 2017; Rizvi et al, 2019). For nutrient starvation stress, 1xPBS was used as described by Loebel et al (1933) and Rizvi et al (2019). Any possible contamination in the culture was ruled out by using ZN staining before and after stress.…”

“…RNA extraction was performed by using the Trizol method as describe (Rizvi et al, 2019). After growing the cells under respective stress conditions, the samples were harvested and resuspended in trizol along with 0.1 mm glass beads.…”

Elucidating the antimycobacterial mechanism of action of ciprofloxacin using metabolomics

“…Furthermore, it is well known, that PPP metabolism is fueled by glucose and the glucogenic amino acids; valine, aspartic acid and glutamic acid [109], all of which were specifically and significantly reduced in the ciprofloxacin-treated Mtb, in addition to their degradation products; β-aminoisobutanoic acid [110], 5-oxoproline [111] and N-acetyl-lysine [112], respectively (Figure 4). Furthermore, the reduced levels of glutamic acid and aspartic acid in our investigation, support an oxidative state in the ciprofloxacin treated group [113]. The above-mentioned amino acids also serve as precursors of cell wall related intermediates.…”