Trehalose Recycling Promotes Energy-Efficient Biosynthesis of the Mycobacterial Cell Envelope

Abstract

The mycomembrane layer of the mycobacterial cell envelope is a barrier to environmental, immune, and antibiotic insults. There is considerable evidence of mycomembrane plasticity during infection and in response to host-mimicking stresses. Since mycobacteria are resource and energy limited under these conditions, it is likely that remodeling has distinct requirements from those of the well-characterized biosynthetic program that operates during unrestricted growth. Unexpectedly, we found that mycomembrane remodeling in nutrient-starved, nonreplicating mycobacteria includes synthesis in addition to turnover. Mycomembrane synthesis under these conditions occurs along the cell periphery, in contrast to the polar assembly of actively growing cells, and both liberates and relies on the nonmammalian disaccharide trehalose. In the absence of trehalose recycling, de novo trehalose synthesis fuels mycomembrane remodeling. However, mycobacteria experience ATP depletion, enhanced respiration, and redox stress, hallmarks of futile cycling and the collateral dysfunction elicited by some bactericidal antibiotics. Inefficient energy metabolism compromises the survival of trehalose recycling mutants in macrophages. Our data suggest that trehalose recycling alleviates the energetic burden of mycomembrane remodeling under stress. Cell envelope recycling pathways are emerging targets for sensitizing resource-limited bacterial pathogens to host and antibiotic pressure. IMPORTANCE The glucose-based disaccharide trehalose is a stress protectant and carbon source in many nonmammalian cells. Mycobacteria are relatively unique in that they use trehalose for an additional, extracytoplasmic purpose: to build their outer “myco” membrane. In these organisms, trehalose connects mycomembrane biosynthesis and turnover to central carbon metabolism. Key to this connection is the retrograde transporter LpqY-SugABC. Unexpectedly, we found that nongrowing mycobacteria synthesize mycomembrane under carbon limitation but do not require LpqY-SugABC. In the absence of trehalose recycling, compensatory anabolism allows mycomembrane biosynthesis to continue. However, this workaround comes at a cost, namely, ATP consumption, increased respiration, and oxidative stress. Strikingly, these phenotypes resemble those elicited by futile cycles and some bactericidal antibiotics. We demonstrate that inefficient energy metabolism attenuates trehalose recycling mutant Mycobacterium tuberculosis in macrophages. Energy-expensive macromolecule biosynthesis triggered in the absence of recycling may be a new paradigm for boosting host activity against bacterial pathogens.

Surface-Shaving Proteomics of Mycobacterium marinum Identifies Biofilm Subtype-Specific Changes Affecting Virulence, Tolerance, and Persistence

“…This is in line with the temporally increased production of diacylglycerol O -acyltransferase (Tgs1) in PBFs, which can promote the accumulation of triacylglycerols (TAGs), a process that has been considered a hallmark feature of persisting Mtb/latent TB and a long-term energy source for Mtb and has been found in substantial amounts in the mycobacterial cell wall ( 67 , 68 ). The detection of trehalase as significantly more abundant in 4- to 12-week-old PBFs strengthens the idea that cells within this biofilm subtype suffer from nutrient stress and activate trehalose salvage/recycling to promote redox and energy homeostasis, as seen under carbon limitations in Mtb ( 69 ). These findings may also explain the detection of proteases, chaperones, and assisting stress proteins in high numbers in the biofilm ECMs, including, e.g., the proteases Clp/Lon and the cold shock protein CpsD, with known implications in stringent response, persistence, and/or postantibiotic recovery ( 70 – 72 ).…”

Interplay between central carbon metabolism and metal homeostasis in mycobacteria and other human pathogens

“…The weak point of this conclusion is that transcriptomics and proteomics data generated using ironstarved M. tuberculosis [4,[26][27][28] do not show upregulation of any of the genes encoding trehalose synthetic pathways (OtsA-OtsB, TreY-TreZ, TreS). In contrast, growth-arrested M. tuberculosis exposed to hypoxia, carbon limitation, or antibiotic stress does show a clear overexpression of the trehalose synthetic pathways OtsA-OtsB and TreS [43][44][45]. The different experimental conditions and methodology used, in particular the lower sensitivity of the microarray method used in iron starvation [26,27] compared to the RT-qPCR approach used for the other type of stress [43][44][45], may be partly responsible for the observed differences in expression.…”

“…In contrast, growth-arrested M. tuberculosis exposed to hypoxia, carbon limitation, or antibiotic stress does show a clear overexpression of the trehalose synthetic pathways OtsA-OtsB and TreS [43–45]. The different experimental conditions and methodology used, in particular the lower sensitivity of the microarray method used in iron starvation [26, 27] compared to the RT-qPCR approach used for the other type of stress [43–45], may be partly responsible for the observed differences in expression.…”

“…Three other studies assign the role of internal carbon source to trehalose in growth-arrested M. tuberculosis, significantly contributing to remodelling of the cell envelope composition [43][44][45]. These studies demonstrated that preexisting trehalose (TMM and TDM) is degraded (via the TreS pathway) to provide carbon for glycolysis, the pentose phosphate pathway, and amino-and nucleotide-sugar biosynthesis in growth-arrested cells exposed to hypoxia [43], antibiotic stress [44], and carbon limitation [45]. In light of these recent findings, we hypothesise that growth-arrested iron-starved M. tuberculosis up-regulates pgi, tpi, and pfkA to produce fructose 1,6-biphosphate and activate a fructose 1,6-biphosphate-dependent pyrophosphorylase.…”

“…Of course, anaplerotic PckA activity would require a source of PEP. The recent studies assigning a carbon source role to trehalose during exposure to several stressors [43][44][45] allow us to speculate that pre-existing trehalose (TDM and TMM) might be degraded via glycolysis to supply the PEP pool.…”