Media component bovine serum albumin facilitates the formation of mycobacterial biofilms in response to reductive stress.

BACKGROUND: Mycobacterium tuberculosis (Mtb) forms physiologically relevant biofilms harboring drug-tolerant bacteria. This observation has brought the study of mycobacterial biofilms to the forefront of tuberculosis research. We established earlier that dithiothreitol (DTT) mediated reductive stress induces cellulose-rich biofilm formation in Mtb cultures. The molecular events associated with the DTT-induced biofilm formation are not known. Furthermore, there are only limited tools for monitoring the presence of cellulose in biofilms. RESULTS: To decipher the molecular events associated with DTT-induced biofilm formation, we used Mtb and non-pathogenic, fast-growing Mycobacterium smegmatis (Msm). We observed that DTT induces biofilm formation in Msm cultures. We explored whether media components facilitate biofilm formation in mycobacteria upon exposure to DTT. We observed that media component bovine serum albumin promotes mycobacterial biofilm formation in response to DTT. Furthermore, we analyzed the composition of extracellular polymeric substances of Msm biofilms. We found that, like Mtb biofilms, Msm biofilms are also rich in polysaccharides and proteins. We also developed a novel protein-based molecular probe for imaging cellulose by utilizing the cellulose-binding domain of cellulase CenA from Cellulomonas fimi and fusing it to fluorescent reporter mCherry. Characterization of this new probe revealed that it has a high affinity for cellulose and could be used for visualizing cellulose biosynthesis during the development of Agrobacterium biofilms. Furthermore, we have demonstrated that biological macromolecule cellulose is present in the extracellular polymeric substances of Msm biofilms using this novel probe. CONCLUSIONS: This study indicates that DTT-mediated reduction of media component BSA leads to the formation of nucleating foci. These nucleating foci are critical for subsequent attachment of bacterial cells and induction of EPS production. Furthermore, this new tool, IMT-CBD-mC, could be used for monitoring cellulose incorporation in plant cells, understanding cellulose biosynthesis dynamics during biofilm formation, etc.

Reductive Stress: New Insights in Physiology and Drug Tolerance of Mycobacterium.

Significance:Mycobacterium tuberculosis (Mtb) encounters reductive stress during its infection cycle. Notably, host-generated protective responses, such as acidic pH inside phagosomes and lysosomes, exposure to glutathione in alveolar hypophase (i.e., a thin liquid lining consisting of surfactant and proteins in the alveolus), and hypoxic environments inside granulomas are associated with the accumulation of reduced cofactors, such as nicotinamide adenine dinucleotide (reduced form), nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide (reduced form), and nonprotein thiols (e.g., mycothiol), leading to reductive stress in Mtb cells. Dissipation of this reductive stress is important for survival of the bacterium. If reductive stress is not dissipated, it leads to generation of reactive oxygen species, which may be fatal for the cells. Recent Advances: This review focuses on mechanisms utilized by mycobacteria to sense and respond to reductive stress. Importantly, exposure of Mtb cells to reductive stress leads to growth inhibition, altered metabolism, modulation of virulence, and drug tolerance. Mtb is equipped with thiol buffering systems of mycothiol and ergothioneine to protect itself from various redox stresses. These systems are complemented by thioredoxin and thioredoxin reductase (TR) systems for maintaining cellular redox homeostasis. A diverse array of sensors is used by Mycobacterium for monitoring its intracellular redox status. Upon sensing reductive stress, Mtb uses a flexible and robust metabolic system for its dissipation. Branched electron transport chain allows Mycobacterium to function with different terminal electron acceptors and modulate proton motive force to fulfill energy requirements under diverse scenarios. Interestingly, Mtb utilizes variations in the tricarboxylic cycle and a number of dehydrogenases to dissipate reductive stress. Upon prolonged exposure to reductive stress, Mtb utilizes biosynthesis of storage and virulence lipids as a dissipative mechanism. Critical Issues: The mechanisms utilized by Mycobacterium for sensing and tackling reductive stress are not well characterized. Future Directions: The precise role of thiol buffering and TR systems in neutralizing reductive stress is not well defined. Genetic systems that respond to metabolic reductive stress and thiol reductive stress need to be mapped. Genetic screens could aid in identification of such systems. Given that management of reductive stress is critical for both actively replicating and persister mycobacteria, an improved understanding of the mechanisms used by mycobacteria for dissipation of reductive stress may lead to identification of vulnerable choke points that could be targeted for killing Mtb in vivo.

Thiol reductive stress induces cellulose-anchored biofilm formation in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) forms biofilms harbouring antibiotic-tolerant bacilli in vitro, but the factors that induce biofilm formation and the nature of the extracellular material that holds the cells together are poorly understood. Here we show that intracellular thiol reductive stress (TRS) induces formation of Mtb biofilms in vitro, which harbour drug-tolerant but metabolically active bacteria with unchanged levels of ATP/ADP, NAD(+)/NADH and NADP(+)/NADPH. The development of these biofilms requires DNA, RNA and protein synthesis. Transcriptional analysis suggests that Mtb modulates only ∼7% of its genes for survival in biofilms. In addition to proteins, lipids and DNA, the extracellular material in these biofilms is primarily composed of polysaccharides, with cellulose being a key component. Our results contribute to a better understanding of the mechanisms underlying Mtb biofilm formation, although the clinical relevance of Mtb biofilms in human tuberculosis remains unclear.