MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis

Pacheco, Sophia A.; Hsu, Fong‐Fu; Powers, Katelyn M.; Purdy, Georgiana E.

doi:10.1074/jbc.m113.473371

Cited by 94 publications

(95 citation statements)

References 43 publications

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“…3B). Alkaline hydrolysis of these apolar lipids did not result in liberation of MAs, ruling out their being mycolyl glycerols (16,17). These apolar lipids remain unidentified at present.…”

Section: Selectivity and Completeness Of Rms Extraction Procedures Inmentioning

confidence: 88%

Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides

Bansal‐Mutalik

Nikaido

2014

Proc. Natl. Acad. Sci. U.S.A.

218

232

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Mycobacterium species, including the human pathogen Mycobacterium tuberculosis, are unique among Gram-positive bacteria in producing a complex cell wall that contains unusual lipids and functions as a permeability barrier. Lipids in the cell wall were hypothesized to form a bilayer or outer membrane that would prevent the entry of chemotherapeutic agents, but this could not be tested because of the difficulty in extracting only the cell-wall lipids. We used reverse micellar extraction to achieve this goal and carried out a quantitative analysis of both the cell wall and the inner membrane lipids of Mycobacterium smegmatis. We found that the outer leaflet of the outer membrane contains a similar number of hydrocarbon chains as the inner leaflet composed of mycolic acids covalently linked to cell-wall arabinogalactan, thus validating the outer membrane model. Furthermore, we found that preliminary extraction with reverse micelles permitted the subsequent complete extraction of inner membrane lipids with chloroform-methanol-water, revealing that one-half of hydrocarbon chains in this membrane are contributed by an unusual lipid, diacyl phosphatidylinositol dimannoside. The inner leaflet of this membrane likely is composed nearly entirely of this lipid. Because it contains four fatty acyl chains within a single molecule, it may produce a bilayer environment of unusually low fluidity and may slow the influx of drugs, contributing to the general drug resistance phenotype of mycobacteria.Mycobacterium smegmatis | mycolic acid | phosphoinositides I t is now generally accepted that the organisms belonging to the Corynebacteria-Mycobacteria-Nocardia group are covered by a complex cell envelope containing the inner plasma membrane (IM), the peptidoglycan-arabinogalactan complex, and the outer membrane (OM) that is covalently linked to the arabinogalactan (1, 2). However, it has been nearly impossible to isolate the OM without contamination from IM components, and this makes the previous published studies on OM composition (3) and the models of OM (4, 5) less than convincing.The major limitation here has been the lack of a reliable technique capable of separating the IM and OM of mycobacteria. Although methods such as shaking with glass beads have been used in the past (3), these are random mechanical methods and an absolute selectivity cannot be achieved. Recently we used the technique of reverse micellar solution (RMS) extraction for Corynebacterium glutamicum cells and found that complete and selective removal of lipids from the OM was possible (6). Through the quantification of lipids we proved that a member of the Corynebacterineae had enough fatty acid chains beyond the peptidoglycan to form a complete OM bilayer barrier around the cell. In the process, we could assign the location of different lipid species to the OM and IM of the corynebacterial envelope. We now extend this approach to Mycobacterium smegmatis, which is commonly used as a model species of mycobacteria, a group including Mycobacterium tuberculosis tha...

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“…3B). Alkaline hydrolysis of these apolar lipids did not result in liberation of MAs, ruling out their being mycolyl glycerols (16,17). These apolar lipids remain unidentified at present.…”

Section: Selectivity and Completeness Of Rms Extraction Procedures Inmentioning

confidence: 88%

Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides

Bansal‐Mutalik

Nikaido

2014

Proc. Natl. Acad. Sci. U.S.A.

218

232

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“…Similarly, farE is divergently transcribed from farR and encodes an 822-amino-acid protein that is annotated as a drug exporter of the resistance-nodulation-division (RND) superfamily (30), to which AcrB and orthologous efflux pumps are also assigned (42). Genome annotation also assigns FarE to the MMPL (mycobacterial membrane proteins, large) family of proteins, on the basis of homology to large membrane proteins of Mycobacterium tuberculosis that transport mycolic acids to the cell surface (43). Using protein structural modeling programs HHPRED and PHYRE2 (44,45), FarR was predicted with greater than 99% confidence to resemble known AcrR family regulators, including PfmR and FadR of Thermus thermophilus, which control expression of genes involved in fatty acid synthesis and metabolism (46,47), and MtrR, an efflux pump regulator of Neisseria gonorrhoeae (41,48).…”

Section: Resultsmentioning

confidence: 99%

Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids

et al. 2015

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Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions, and in abscesses, a specific mechanism of inducible resistance to this important facet of innate immunity has not been identified. Here, we have sequenced the genome of S. aureus USA300 variants selected for their ability to grow at an elevated concentration of linoleic acid. The fatty acid-resistant clone FAR7 had a single nucleotide polymorphism resulting in an H 121 Y substitution in an uncharacterized transcriptional regulator belonging to the AcrR family, which was divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multidrug efflux pumps. We named these genes farR and farE, for regulator and effector of fatty acid resistance, respectively. Several lines of evidence indicated that FarE promotes efflux of antimicrobial fatty acids and is regulated by FarR. First, expression of farE was strongly induced by arachidonic and linoleic acids in an farRdependent manner. Second, an H 121 Y substitution in FarR resulted in increased expression of farE and was alone sufficient to promote increased resistance of S. aureus to linoleic acid. Third, inactivation of farE resulted in a significant reduction in the inducible resistance of S. aureus to the bactericidal activity of 100 M linoleic acid, increased accumulation of [ 14 C]linoleic acid by growing cells, and severely impaired growth in the presence of nonbactericidal concentrations of linoleic acid. Cumulatively, these findings represent the first description of a specific mechanism of inducible resistance to antimicrobial fatty acids in a Gram-positive pathogen. IMPORTANCEStaphylococcus aureus colonizes approximately 25% of humans and is a leading cause of human infectious morbidity and mortality. To persist on human hosts, S. aureus must have intrinsic defense mechanisms to cope with antimicrobial fatty acids, which comprise an important component of human innate defense mechanisms. We have identified a novel pair of genes, farR and farE, that constitute a dedicated regulator and effector of S. aureus resistance to linoleic and arachidonic acids, which are major fatty acids in human membrane phospholipid. Expression of farE, which encodes an efflux pump, is induced in an farRdependent mechanism, in response to these antimicrobial fatty acids that would be encountered in a tissue abscess. Staphylococcus aureus has a dichotomous relation with human hosts, being able to establish an asymptomatic commensal relationship, but is also historically known as a leading cause of human infectious morbidity and mortality. Significantly, death attributed to S. aureus in the United States is now comparable to mortality rates for AIDS, tuberculosis, and viral hepatitis (1-3). Not surprisingly, therefore, S. aureus has been the subject of intensive research on mechanisms of pathogenesis and acquisition and transfer of antibiotic resistance and of efforts to identify potential vaccine antigens (4-6). Until the late ...

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“…3). The helices of Rv0302 are designated numerically from the N-terminus as a1 (residues [14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29], a2 (residues 37-44), a3 (residues 48-55), a4 (residues 58-76), a5 (residues 88-104), ). The red circles corresponding to the Rv0302 transcription factor are placed at the putative binding sites.…”

Section: Resultsmentioning

confidence: 99%

“…Cell debris was removed by centrifugation for 45 min at 48C and 20,000 rev/min. The crude lysate was filtered through a 0.2 lm membrane and loaded onto a 5 mL Hi-Trap Ni 21 -chelating column (GE Healthcare Biosciences, Pittsburgh, PA) pre-equilibrated with 20 mM Na-HEPES (pH 7.2) and 250 mM NaCl. To remove unbound proteins and impurities, the column was first washed with eight column volumes of buffer containing 20 mM imidazole, 250 mM NaCl, and 20 mM Na-HEPES (pH 7.2), and then five column volumes of buffer containing 50 mM imidazole, 250 mM NaCl, and 20 mM Na-HEPES (pH 7.2).…”

Section: Expression and Purification Of Rv0302mentioning

confidence: 99%

“…9 Instead, there is strong evidence that these MmpL transporters and their MmpS accessory proteins are responsible for shuttling fatty acid and lipid components of the cell wall, such as trehalose monomycolate, sulfolipids, phthiocerol dimycocerosate, diacyltrehalose, monomeromycolyl diacylglycerol, and mycolate wax ester. 3,9,11,12,[19][20][21][22][23] The regulation of MmpL protein expression and the role of MmpLs in cell wall remodeling in different environmental conditions has not been explored. Thus we capitalized on data made available by the TB Systems Biology Consortium to begin an in-depth analysis of how mmpL and mmpS genes are regulated.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of the Mycobacterium tuberculosis transcriptional regulator Rv0302

et al. 2015

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Mycobacterium tuberculosis is a pathogenic bacterial species, which is neither Gram positive nor Gram negative. It has a unique cell wall, making it difficult to kill and conferring resistance to antibiotics that disrupt cell wall biosynthesis. Thus, the mycobacterial cell wall is critical to the virulence of these pathogens. Recent work shows that the mycobacterial membrane protein large (MmpL) family of transporters contributes to cell wall biosynthesis by exporting fatty acids and lipidic elements of the cell wall. The expression of the Mycobacterium tuberculosis MmpL proteins is controlled by a complicated regulatory network system. Here we report crystallographic structures of two forms of the TetR-family transcriptional regulator Rv0302, which participates in regulating the expression of MmpL proteins. The structures reveal a dimeric, two-domain molecule with architecture consistent with the TetR family of regulators. Comparison of the two Rv0302 crystal structures suggests that the conformational changes leading to derepression may be due to a rigid body rotational motion within the dimer interface of the regulator. Using fluorescence polarization and electrophoretic mobility shift assays, we demonstrate the recognition of promoter and intragenic regions of multiple mmpL genes by this protein. In addition, our isothermal titration calorimetry and electrophoretic mobility shift experiments indicate that fatty acids may be the natural ligand of this regulator. Taken together, these experiments provide new perspectives on the regulation of the MmpL family of transporters.

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MmpL11 Protein Transports Mycolic Acid-containing Lipids to the Mycobacterial Cell Wall and Contributes to Biofilm Formation in Mycobacterium smegmatis

Cited by 94 publications

References 43 publications

Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides

Mycobacterial outer membrane is a lipid bilayer and the inner membrane is unusually rich in diacyl phosphatidylinositol dimannosides

Inducible Expression of a Resistance-Nodulation-Division-Type Efflux Pump in Staphylococcus aureus Provides Resistance to Linoleic and Arachidonic Acids

Crystal structure of the Mycobacterium tuberculosis transcriptional regulator Rv0302

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