Insights into the Physiology and Metabolism of a Mycobacterial Cell in an Energy-Compromised State

Abstract

Mycobacterium tuberculosis, a bacterium that causes tuberculosis, poses a serious threat, especially due to the emergence of drug-resistant strains. M. tuberculosis and other mycobacterial species, such as M. smegmatis, are known to generate an inadequate amount of energy by substrate-level phosphorylation and mandatorily require oxidative phosphorylation (OXPHOS) for their growth and metabolism. Hence, antibacterial drugs, such as bedaquiline, targeting the multisubunit ATP synthase complex, which is required for OXPHOS, have been developed with the aim of eliminating pathogenic mycobacteria. Here, we explored the influence of suboptimal OXPHOS on the physiology and metabolism of M. smegmatis. M. smegmatis harbors two identical copies of atpD, which codes for the β subunit of ATP synthase. We show that upon deletion of one copy of atpD (M. smegmatis ΔatpD), M. smegmatis synthesizes smaller amounts of ATP and enters into an energy-compromised state. The mutant displays remarkable phenotypic and physiological differences from the wild type, such as respiratory slowdown, reduced biofilm formation, lesser amounts of cell envelope polar lipids, and increased antibiotic sensitivity compared to the wild type. Additionally, M. smegmatis ΔatpD overexpresses genes belonging to the dormancy operon, the β-oxidation pathway, and the glyoxylate shunt, suggesting that the mutant adapts to a low energy state by switching to alternative pathways to produce energy. Interestingly, M. smegmatis ΔatpD shows significant phenotypic, metabolic, and physiological similarities with bedaquiline-treated wild-type M. smegmatis. We believe that the identification and characterization of key metabolic pathways functioning during an energy-compromised state will enhance our understanding of bacterial adaptation and survival and will open newer avenues in the form of drug targets that may be used in the treatment of mycobacterial infections. IMPORTANCE M. smegmatis generates an inadequate amount of energy by substrate-level phosphorylation and mandatorily requires oxidative phosphorylation (OXPHOS) for its growth and metabolism. Here, we explored the influence of suboptimal OXPHOS on M. smegmatis physiology and metabolism. M. smegmatis harbors two identical copies of the atpD gene, which codes for the ATP synthase β subunit. Here, we carried out the deletion of only one copy of atpD in M. smegmatis to understand the bacterial survival response in an energy-deprived state. M. smegmatis ΔatpD shows remarkable phenotypic, metabolic, and physiological differences from the wild type. Our study thus establishes M. smegmatis ΔatpD as an energy-compromised mycobacterial strain, highlights the importance of ATP synthase in mycobacterial physiology, and further paves the way for the identification of novel antimycobacterial drug targets.

Deciphering the Role of Holin in Mycobacteriophage D29 Physiology

“…M. smegmatis log phase culture was infected with either D29WT or D29 gp11 phage at MOI of 1, and the viability of cells after phage infection was measured using AlamarBlue (BIO-RAD) cell viability reagent essentially as described previously (Warrier et al, 2012;Joshi et al, 2019;Patil and Jain, 2019) at different time points post-infection. Phage-induced bacterial cell lysis was also monitored by measuring the optical density of the culture at 600 nm (OD 600 ) at specified time points on a spectrophotometer (Jenway, Bibby scientific).…”

“…Phage-induced bacterial cell lysis was also monitored by measuring the optical density of the culture at 600 nm (OD 600 ) at specified time points on a spectrophotometer (Jenway, Bibby scientific). The amount of ATP released into the culture supernatant upon cell lysis was determined by BacTiter-Glo Microbial Cell Viability Assay kit (Promega) as described previously (Joshi et al, 2019;Patil and Jain, 2019).…”

“…Luminescence data thus obtained showed surge in ATP amounts in the culture supernatant at 90 min post-infection of M. smegmatis with D29WT phage, whereas no significant increase in the ATP level was detected in case of culture infected with D29 gp11 at the same time point; in the latter, the ATP amount increased in the culture supernatant only after 120 min of infection ( Figure 3B), clearly indicating a delay in the host cell lysis by the mutant phage. We also assessed the host cell viability during phage infection by following the alamarBlue assay (Warrier et al, 2012; Joshi et al, 2019;Patil and Jain, 2019). The assay was performed at different time points after infecting M. smegmatis with either D29WT or D29 gp11 phage.…”

Deciphering the mechanism of action of antitubercular compounds with metabolomics

“…Bedaquiline is an inhibitor of the ATP synthase complex that results in lower ATP levels and a compromised energy state for the bacterium. This mechanism is possible because Mycobacterium must use oxidative phosphorylation to generate ATP from non-fermentable lipids, the preferred carbon source for intracellular pathogenic mycobacteria [108] , [109] . As substrate-level phosphorylation is inadequate to supply sufficient ATP, ATP synthase becomes essential for in vivo survival of both replicating and dormant mycobacteria as both states require to a greater or lesser extent a level of ATP for survival.…”

Construction of E. coli—Mycobacterium shuttle vectors with a variety of expression systems and polypeptide tags for gene expression in mycobacteria

“…We had previously reported the construction of an acetamide-inducible plasmid, pMVAcet, which was used for the expression of various genes in M. smegmatis [14][15][16]. Here, we cloned various polypeptide tag-coding sequences in pMVAcet vector and further replaced the promoter with others inducible systems to prepare our plasmids ( Table 2).…”