Abstract
Mycobacteria share with other actinomycetes the ability to produce large quantities of triacylglycerol (TAG), which accumulate as intracytoplasmic lipid inclusions (ILI) also known as lipid droplets (LD). Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis, acquires fatty acids from the human host which are utilized to synthesize TAG, subsequently stored in the form of ILI to meet the carbon and nutrient requirements of the bacterium during long periods of persistence. However, environmental factors governing mycobacterial ILI formation and degradation remain poorly understood. Herein, we demonstrated that in the absence of host cells, carbon excess and nitrogen starvation promote TAG accumulation in the form of ILI in M. smegmatis and M. abscessus, used as surrogate species of M. tb. Based on these findings, we developed a simple and reversible in vitro model to regulate ILI biosynthesis and hydrolysis in mycobacteria. We also showed that TAG formation is tgs1 dependent and that lipolytic enzymes mediate TAG breakdown. Moreover, we confirmed that the nitrogen-deprived and ILI-rich phenotype was associated with an increased tolerance towards several drugs used for treating mycobacterial infections. Importantly, we showed that the presence of ILI substantially enhanced the bacterial burden and granuloma abundance in zebrafish embryos infected with lipid-rich M. abscessus as compared to embryos infected with lipid-poor M. abscessus, suggesting that ILI are actively contributing to mycobacterial virulence and pathogenesis.
Highlights
Upon infection with Mycobacterium tuberculosis (M. tb), the causative agent of TB, less than 5% of infected people will progress towards active infection, whereas 95% remain classified as latently infected without any symptoms or signs of illness[1]
To investigate the effect of nitrogen requirements for TAG biosynthesis, M. smegmatis was grown in well-defined Minimal Mineral Salt Medium supplemented with either high nitrogen (MSM; containing 1 g/L NH4+) or low nitrogen (MSM NL; containing 0.05 g/L NH4+) concentrations, and 1% Gly as the sole carbon source
In this study, using two mycobacterial species with distinct lifestyles, we demonstrated that simple modulation of nutrient availability rapidly triggers the formation/consumption of ILI
Summary
Upon infection with Mycobacterium tuberculosis (M. tb), the causative agent of TB, less than 5% of infected people will progress towards active infection, whereas 95% remain classified as latently infected without any symptoms or signs of illness[1]. Growth of slow-growing mycobacteria under hypoxic conditions, as described in the Wayne and Hayes dormancy model, triggers upregulation of the major TAG synthase, tgs[1], in a DosR regulon dependent-manner[29,30,31,32] These early observations led to the development of more complex systems, such as an in vitro multiple stress model combining hypoxia, low pH and exposure to nitric oxide, inducing TAG production and accumulation, loss of acid-fastness and tolerance to drugs[17]. To better characterize the environmental conditions modulating TAG formation/consumption processes, the non-pathogenic strain M. smegmatis was initially used to demonstrate that nitrogen and carbon availability are two key metabolic factors governing TAG formation and accumulation in the form of ILI in mycobacteria These physiological processes are highly conserved in the opportunistic pathogen M. abscessus, a fast-growing non-tuberculous mycobacterium responsible for chronic pseudo-tubercular infection[36]. By using the well-established M. abscessus/zebrafish infection model[37,38], we have demonstrated that ILI formation confers significant advantages to the bacteria in the establishment and progress of the infection in vivo
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