Rod-shaped mycobacteria expand from their poles, yet d-amino acid probes label cell wall peptidoglycan in this genus at both the poles and sidewall. We sought to clarify the metabolic fates of these probes. Monopeptide incorporation was decreased by antibiotics that block peptidoglycan synthesis or l,d-transpeptidation and in an l,d-transpeptidase mutant. Dipeptides complemented defects in d-alanine synthesis or ligation and were present in lipid-linked peptidoglycan precursors. Characterizing probe uptake pathways allowed us to localize peptidoglycan metabolism with precision: monopeptide-marked l,d-transpeptidase remodeling and dipeptide-marked synthesis were coincident with mycomembrane metabolism at the poles, septum and sidewall. Fluorescent pencillin-marked d,d-transpeptidation around the cell perimeter further suggested that the mycobacterial sidewall is a site of cell wall assembly. While polar peptidoglycan synthesis was associated with cell elongation, sidewall synthesis responded to cell wall damage. Peptidoglycan editing along the sidewall may support cell wall robustness in pole-growing mycobacteria.

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.

1 2 D-amino acid probes label cell wall peptidoglycan at both the poles and sidewall of pole-growing 3 mycobacteria. Since peptidoglycan assembly along the cell periphery could provide a rapid, 4 growth-independent means by which to edit the cell wall, we sought to clarify the precise 5 metabolic fates of these probes. D-amino acid monopeptides were incorporated into 6 peptidoglycan by L,D-transpeptidase remodeling enzymes to varying extents. Dipeptides were 7 incorporated into cytoplasmic precursors. While dipeptide-marked peptidoglycan synthesis at 8 the poles was associated with cell elongation, synthesis along the periphery was highly 9 responsive to cell wall damage. Our observations suggest a post-expansion role for 1 0 peptidoglycan assembly along the mycobacterial sidewall and provide a conceptual framework 1 1 for understanding cell wall robustness in the face of polar growth.

Novel therapeutics against multidrug-resistant Neisseria gonorrhoeae are urgently needed. Gonococcal lipooligosaccharide (LOS) often expresses lacto-N-neotetraose (LNnT), which becomes sialylated in vivo, enhancing factor H (FH) binding and contributing to the organism’s ability to resist killing by complement. We previously showed that FH domains 18–20 (with a D-to-G mutation at position 1119 in domain 19) fused to Fc (FHD1119G/Fc) displayed complement-dependent bactericidal activity in vitro and attenuated gonococcal vaginal colonization of mice. Gonococcal LOS phase-variation can result in loss of LNnT expression. Loss of sialylated LNnT, although associated with a considerable fitness cost, could decrease efficacy of FHD1119G/Fc. Similar to N. meningitidis, gonococci also bind FH domains 6 and 7 through Neisseria surface protein A (NspA). Here we show that a fusion protein comprising FH domains 6 and 7 fused to human IgG1 Fc (FH6,7/Fc) bound to 15 wild-type antimicrobial resistant isolates of N. gonorrhoeae and to each of six lgtA gonococcal deletion mutants. FH6,7/Fc mediated complement-dependent killing of 8 of the 15 wild-type gonococcal isolates and effectively reduced the duration and burden of vaginal colonization of three gonococcal strains tested in wild-type mice, including two strains that resisted complement-dependent killing, but on which FH6,7/Fc enhanced C3 deposition. FH/Fc lost efficacy when Fc was mutated to abrogate C1q binding and in C1q−/− mice, highlighting the requirement of the classical pathway for its activity. Targeting gonococci with FH6,7/Fc provides an additional immunotherapeutic approach against multidrug-resistant gonorrhea.

2The mycomembrane layer of the mycobacterial cell envelope is a barrier to 3 environmental, immune and antibiotic insults. We find that there is mycomembrane 4 remodeling along the periphery of nutrient-starved, non-replicating mycobacterial cells. 5Remodeling is supported by recycling of trehalose, a non-mammalian disaccharide that 6 shuttles long-chain mycolate lipids to the mycomembrane. In the absence of trehalose 7 recycling, mycomembrane synthesis continues but mycobacteria experience ATP 8 depletion, enhanced respiration and redox stress. Redox stress from depletion of the 9 trehalose pool is suppressed in a mutant that lacks the OtsAB de novo trehalose 10 synthesis pathway. Our data suggest that trehalose recycling alleviates the energetic 11 burden of mycomembrane remodeling. Loss of trehalose salvage is known to attenuate 12