scholarly journals Structures of the mycobacterial membrane protein MmpL3 reveal its mechanism of lipid transport

PLoS Biology ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. e3001370
Author(s):  
Chih-Chia Su ◽  
Philip A. Klenotic ◽  
Meng Cui ◽  
Meinan Lyu ◽  
Christopher E. Morgan ◽  
...  
Keyword(s):  
Cell Wall ◽  
Membrane Protein ◽  
Md Simulations ◽  
Mycolic Acid ◽  
Lipid Transport ◽  
X Ray Crystallography ◽  
Trehalose Dimycolate ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall ◽  
Trehalose Monomycolate

The mycobacterial membrane protein large 3 (MmpL3) transporter is essential and required for shuttling the lipid trehalose monomycolate (TMM), a precursor of mycolic acid (MA)-containing trehalose dimycolate (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), in Mycobacterium species, including Mycobacterium tuberculosis and Mycobacterium smegmatis. However, the mechanism that MmpL3 uses to facilitate the transport of fatty acids and lipidic elements to the mycobacterial cell wall remains elusive. Here, we report 7 structures of the M. smegmatis MmpL3 transporter in its unbound state and in complex with trehalose 6-decanoate (T6D) or TMM using single-particle cryo-electron microscopy (cryo-EM) and X-ray crystallography. Combined with calculated results from molecular dynamics (MD) and target MD simulations, we reveal a lipid transport mechanism that involves a coupled movement of the periplasmic domain and transmembrane helices of the MmpL3 transporter that facilitates the shuttling of lipids to the mycobacterial cell wall.

scholarly journals MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

2019 ◽  
Vol 116 (23) ◽  
pp. 11241-11246 ◽  
Author(s):  
Chih-Chia Su ◽  
Philip A. Klenotic ◽  
Jani Reddy Bolla ◽  
Georgiana E. Purdy ◽  
Carol V. Robinson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Native Mass Spectrometry ◽  
Mycolic Acids ◽  
Mycobacterial Cell ◽  
Exact Function ◽  
Species Specific ◽  
Mycobacterial Cell Wall ◽  
Trehalose Monomycolate ◽  
Lipid Transporter

The cell envelope ofMycobacterium tuberculosisis notable for the abundance of mycolic acids (MAs), essential to mycobacterial viability, and of other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, which contributes to virulence and antibiotic resistance. However, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell-wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA-containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan, but the exact function of MmpL3 remains elusive. Here, we report a crystal structure ofMycobacterium smegmatisMmpL3 at a resolution of 2.59 Å, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

scholarly journals TREM2 is a receptor for non-glycosylated mycolic acids of mycobacteria that limits anti-mycobacterial macrophage activation

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Ei’ichi Iizasa ◽  
Yasushi Chuma ◽  
Takayuki Uematsu ◽  
Mio Kubota ◽  
Hiroaki Kawaguchi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Macrophage Activation ◽  
Mycolic Acid ◽  
Dependent Manner ◽  
Independent Manner ◽  
Lectin Receptors ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

AbstractMycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, a mechanism of action is unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages respond to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages respond to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhances Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerates the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

scholarly journals Mycobacteriophage Ms6 LysB specifically targets the outer membrane of Mycobacterium smegmatis

Microbiology ◽  
2010 ◽  
Vol 156 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Filipa Gil ◽  
Anna E. Grzegorzewicz ◽  
Maria João Catalão ◽  
João Vital ◽  
Michael R. McNeil ◽  
...  
Keyword(s):  
Cell Wall ◽  
Outer Membrane ◽  
Mycobacterium Smegmatis ◽  
Mycolic Acid ◽  
Head Group ◽  
Lipolytic Enzyme ◽  
Ester Bond ◽  
Mycolic Acids ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

LysB, a mycobacteriophage Ms6-encoded protein, was previously identified as a lipolytic enzyme able to hydrolyse the ester bond in lipase and esterase substrates. In the present work, we show that LysB can hydrolyse lipids containing mycolic acids from the outer membrane of the mycobacterial cell wall. LysB was shown to hydrolyse the mycolic acids from the mycolyl-arabinogalactan–peptidoglycan complex where the mycolates of the inner leaflet of the outer membrane are covalently attached to an arabinosyl head group. In addition, treatment of the extractable lipids from Mycobacterium smegmatis, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra with LysB showed that trehalose 6,6′-dimycolate (TDM), a trehalose diester of two mycolic acid molecules, was hydrolysed by the enzyme. We have also determined the structures of the mycolic acid molecules that form the M. smegmatis TDM. The identification of a phage-encoded enzyme that targets the outer membrane of the mycobacterial cell wall enhances our understanding of the mechanism of mycobacteriophage lysis.

scholarly journals FbpA-Dependent Biosynthesis of Trehalose Dimycolate Is Required for the Intrinsic Multidrug Resistance, Cell Wall Structure, and Colonial Morphology of Mycobacterium smegmatis

2005 ◽  
Vol 187 (19) ◽  
pp. 6603-6611 ◽  
Author(s):  
Liem Nguyen ◽  
Satheesh Chinnapapagari ◽  
Charles J. Thompson
Keyword(s):  
Cell Wall ◽  
Antibiotic Resistance ◽  
Mycobacterium Smegmatis ◽  
Mycolic Acid ◽  
Wall Structure ◽  
Single Mutation ◽  
Mycolic Acids ◽  
Trehalose Dimycolate ◽  
Colonial Morphology ◽  
Mycobacterial Cell Wall

ABSTRACT Ligation of mycolic acids to structural components of the mycobacterial cell wall generates a hydrophobic, impermeable barrier that provides resistance to toxic compounds such as antibiotics. Secreted proteins FbpA, FbpB, and FbpC attach mycolic acids to arabinogalactan, generating mycolic acid methyl esters (MAME) or trehalose, generating α,α′-trehalose dimycolate (TDM; also called cord factor). Our studies of Mycobacterium smegmatis showed that disruption of fbpA did not affect MAME levels but resulted in a 45% reduction of TDM. The fbpA mutant displayed increased sensitivity to both front-line tuberculosis-targeted drugs as well as other broad-spectrum antibiotics widely used for antibacterial chemotherapy. The irregular, hydrophobic surface of wild-type M. smegmatis colonies became hydrophilic and smooth in the mutant. While expression of M. smegmatis fbpA restored defects of the mutant, heterologous expression of the Mycobacterium tuberculosis fbpA gene was less effective. A single mutation in the M. smegmatis FbpA esterase domain inactivated its ability to provide antibiotic resistance. These data show that production of TDM by FbpA is essential for the intrinsic antibiotic resistance and normal colonial morphology of some mycobacteria and support the concept that FbpA-specific inhibitors, alone or in combination with other antibiotics, could provide an effective treatment to tuberculosis and other mycobacterial diseases.

Contrasting roles of the innate receptors TREM2 versus Mincle in the recognition and response of macrophages to mycolic acid-containing lipids in mycobacterial cell walls

2020 ◽  
Author(s):  
Ei'ichi Iizasa ◽  
Yasushi Chuma ◽  
Takayuki Uematsu ◽  
Mio Kubota ◽  
Hiroaki Kawaguchi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterial Infection ◽  
Mycolic Acid ◽  
Dependent Manner ◽  
Independent Manner ◽  
Lectin Receptors ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall

Abstract Mycobacterial cell-wall glycolipids elicit an anti-mycobacterial immune response via FcRγ-associated C-type lectin receptors, including Mincle, and caspase-recruitment domain family member 9 (CARD9). Additionally, mycobacteria harbor immuno-evasive cell-wall lipids associated with virulence and latency; however, their mechanism of action remains unclear. Here, we show that the DAP12-associated triggering receptor expressed on myeloid cells 2 (TREM2) recognizes mycobacterial cell-wall mycolic acid (MA)-containing lipids and suggest a mechanism by which mycobacteria control host immunity via TREM2. Macrophages responded to glycosylated MA-containing lipids in a Mincle/FcRγ/CARD9-dependent manner to produce inflammatory cytokines and recruit inducible nitric oxide synthase (iNOS)-positive mycobactericidal macrophages. Conversely, macrophages responded to non-glycosylated MAs in a TREM2/DAP12-dependent but CARD9-independent manner to recruit iNOS-negative mycobacterium-permissive macrophages. Furthermore, TREM2 deletion enhanced Mincle-induced macrophage activation in vitro and inflammation in vivo and accelerated the elimination of mycobacterial infection, suggesting that TREM2-DAP12 signaling counteracts Mincle-FcRγ-CARD9-mediated anti-mycobacterial immunity. Mycobacteria, therefore, harness TREM2 for immune evasion.

scholarly journals Mycolic Acid Structure Determines the Fluidity of the Mycobacterial Cell Wall

1996 ◽  
Vol 271 (47) ◽  
pp. 29545-29551 ◽  
Author(s):  
Jun Liu ◽  
Clifton E. Barry ◽  
Gurdyal S. Besra ◽  
Hiroshi Nikaido
Keyword(s):  
Cell Wall ◽  
Mycolic Acid ◽  
Mycobacterial Cell ◽  
Mycobacterial Cell Wall ◽  
Acid Structure

scholarly journals MmpL3 is a lipid transporter that binds trehalose monomycolate and phosphatidylethanolamine

2019 ◽  
Author(s):  
Chih-Chia Su ◽  
Philip Klenotic ◽  
Jani Reddy Bolla ◽  
Georgiana Purdy ◽  
Carol Robinson ◽  
...  
Keyword(s):  
Cell Wall ◽  
Cell Envelope ◽  
Native Mass Spectrometry ◽  
Mycolic Acids ◽  
Mycobacterial Cell ◽  
Exact Function ◽  
Species Specific ◽  
Mycobacterial Cell Wall ◽  
Trehalose Monomycolate ◽  
Lipid Transporter

The cell envelope of Mycobacterium tuberculosis is notable for the abundance of mycolic acids (MAs), which are essential to mycobacterial viability, and other species-specific lipids. The mycobacterial cell envelope is extremely hydrophobic, contributes to virulence and antibiotic resistance. Yet, exactly how fatty acids and lipidic elements are transported across the cell envelope for cell wall biosynthesis is unclear. Mycobacterial membrane protein Large 3 (MmpL3) is essential and required for transport of trehalose monomycolates (TMMs), precursors of MA containing trehalose dimycolates (TDM) and mycolyl arabinogalactan peptidoglycan (mAGP), but the exact function of MmpL3 remains elusive. Here, we report a high-resolution crystal structure of M. smegmatis MmpL3, revealing a monomeric molecule that is structurally distinct from all known bacterial membrane proteins. A previously unknown MmpL3 ligand, phosphatidylethanolamine (PE), was discovered inside this transporter. We also show, via native mass spectrometry, that MmpL3 specifically binds both TMM and PE, but not TDM, in the micromolar range. These observations provide insight into the function of MmpL3 and suggest a possible role for this protein in shuttling a variety of lipids to strengthen the mycobacterial cell wall.

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