Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales

Dautin, Nathalie; Silva, Célia Regina Sousa da; Constantinesco-Becker, Florence; Labarre, C; Oberto, Jacques; Sierra-Gallay, I. Li de la; Dietrich, Christiane; Issa, Hanane; Houssin, Christine; Bayan, Nicolas

doi:10.1016/j.bbagen.2016.06.020

Cited by 39 publications

(37 citation statements)

References 106 publications

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“…As no straightforward enzymatic test exists to specifically characterize the MM-bound proteins, we checked the fractions by immunodetection methods for the presence of very well characterized antigenic proteins known to be localized in the CW, namely, the mycoloyl transferases (the so-called antigen 85, Ag85) 23 . These enzymes catalyze the transfer of a mycoloyl residue from the trehalose monomycolate (TMM) to another TMM molecule to form trehalose dimycolate (TDM) 24 , or to arabinosyl residues of AG to form mycoloylated arabinosyl extremities 25 – 27 . Using anti-Ag85 polyclonal antibodies 28 , we specifically identified the Ag85 protein family in the MMCW, without residual signal in the PM, of both Mau and Msm (Fig.…”

Section: Resultsmentioning

confidence: 99%

Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane

Chiaradia

Lefebvre

Parra

et al. 2017

Sci Rep

204

183

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The mycobacterial envelope is unique, containing the so-called mycomembrane (MM) composed of very-long chain fatty acids, mycolic acids (MA). Presently, the molecular composition of the MM remains unproven, due to the diversity of methods used for determining its composition. The plasma membranes (PM) and the native MM-containing cell walls (MMCW) of two rapid-growing mycobacterial species, Mycobacterium aurum and M. smegmatis, were isolated from their cell lysates by differential ultracentrifugation. Transmission electron microscopy and biochemical analyses demonstrated that the two membranes were virtually pure. Bottom-up quantitative proteomics study indicated a different distribution of more than 2,100 proteins between the PM and MMCW. Among these, the mannosyltransferase PimB, galactofuranosyltransferase GlfT2, Cytochrome p450 and ABC transporter YjfF, were most abundant in the PM, which also contain lipoglycans, phospholipids, including phosphatidylinositol mannosides, and only a tiny amount of other glycolipids. Antigen85 complex proteins, porins and the putative transporters MCE protein family were mostly found in MMCW fraction that contains MA esterifying arabinogalactan, constituting the inner leaflet of MM. Glycolipids, phospholipids and lipoglycans, together with proteins, presumably composed the outer leaflet of the MM, a lipid composition that differs from that deduced from the widely used extraction method of mycobacterial cells with dioctylsulfosuccinate sodium.

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Section: Resultsmentioning

confidence: 99%

Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane

Chiaradia

Lefebvre

Parra

et al. 2017

Sci Rep

204

183

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“…Next, we sought to test whether incorporation of the new TMM reporters 4 – 6 into mycomembrane‐containing bacteria was dependent on mycoloyltransferase activity. This is a challenging task because all members of the Corynebacterineae have multiple mycoloyltransferase isoforms, which are collectively essential for viability and exhibit high functional redundancy . Not only does this make the knockout of all mycoloyltransferases impossible due to lethality, but experiments involving partial blockage of mycoloyltransferase activity (e.g., by chemical inhibition or partial knockout) are complicated due to isoform redundancy and the resulting perturbation of cell envelope structure and permeability, which presumably can impact probe uptake and thus labeling efficiency.…”

Section: Resultsmentioning

confidence: 99%

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

et al. 2019

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Mycobacteria and related organisms in the Corynebacterineae suborder are characterized by a distinctive outer membrane referred to as the mycomembrane. Biosynthesis of the mycomembrane occurs through an essential process called mycoloylation, which involves antigen 85 (Ag85)‐catalyzed transfer of mycolic acids from the mycoloyl donor trehalose monomycolate (TMM) to acceptor carbohydrates and, in some organisms, proteins. We recently described an alkyne‐modified TMM analogue (O‐AlkTMM‐C7) which, in conjunction with click chemistry, acted as a chemical reporter for mycoloylation in intact cells and allowed metabolic labeling of mycoloylated components of the mycomembrane. Here, we describe the synthesis and evaluation of a toolbox of TMM‐based reporters bearing alkyne, azide, trans‐cyclooctene, and fluorescent tags. These compounds gave further insight into the substrate tolerance of mycoloyltransferases (e.g., Ag85s) in a cellular context and they provide significantly expanded experimental versatility by allowing one‐ or two‐step cell labeling, live cell labeling, and rapid cell labeling via tetrazine ligation. Such capabilities will facilitate research on mycomembrane composition, biosynthesis, and dynamics. Moreover, because TMM is exclusively metabolized by Corynebacterineae, the described probes may be valuable for the specific detection and cell‐surface engineering of Mycobacterium tuberculosis and related pathogens. We also performed experiments to establish the dependence of probe incorporation on mycoloyltransferase activity, results from which suggested that cellular labeling is a function not only of metabolic incorporation (and likely removal) pathway(s), but also accessibility across the envelope. Thus, whole‐cell labeling experiments with TMM reporters should be carefully designed and interpreted when envelope permeability may be compromised. On the other hand, this property of TMM reporters can potentially be exploited as a convenient way to probe changes in envelope integrity and permeability, facilitating drug development studies.

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“…First, we decided to test whether metabolic-labeling and click-chemistry allow detection of the two known mycoloylated proteins, namely PorA and PorH. The accumulation of trehalose monomycolate (TMM) in the Δ mytC :: Km strain and the use of TMM as substrate by other mycoloyltransferases, suggests that the mycolate donor for protein mycoloylation could be TMM [ 29 , 30 ]. Although clickable analogues of trehalose can be used to generate azido-trehalose monomycolates (Az-TMM) in vivo , [ 40 ], these probes cannot be used here since the clickable group, located on the trehalose moiety of Az-TMM, will not be transfered onto mycoloylated proteins.…”

Section: Resultsmentioning

confidence: 99%

“…C . glutamicum encodes six of these "mycoloyltransferases" (Myts) [ 29 ]. Among these six, only MytC was found to be necessary for protein mycoloylation.…”

Section: Introductionmentioning

confidence: 99%

Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

et al. 2017

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Protein mycoloylation is a recently identified, new form of protein acylation. This post-translational modification consists in the covalent attachment of mycolic acids residues to serine. Mycolic acids are long chain, α-branched, β-hydroxylated fatty acids that are exclusively found in the cell envelope of Corynebacteriales, a bacterial order that includes important genera such as Mycobacterium, Nocardia or Corynebacterium. So far, only 3 mycoloylated proteins have been identified: PorA, PorH and ProtX from C. glutamicum. Whereas the identity and function of ProtX is unknown, PorH and PorA associate to form a membrane channel, the activity of which is dependent upon PorA mycoloylation. However, the exact role of mycoloylation and the generality of this phenomenon are still unknown. In particular, the identity of other mycoloylated proteins, if any, needs to be determined together with establishing whether such modification occurs in Corynebacteriales genera other than Corynebacterium. Here, we tested whether a metabolic labeling and click-chemistry approach could be used to detect mycoloylated proteins. Using a fatty acid alkyne analogue, we could indeed label PorA, PorH and ProtX and determine ProtX mycoloylation site. Importantly, we also show that two other porins from C. glutamicum, PorB and PorC are mycoloylated.

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Mycoloyltransferases: A large and major family of enzymes shaping the cell envelope of Corynebacteriales

Cited by 39 publications

References 106 publications

Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane

Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane

Engineering the Mycomembrane of Live Mycobacteria with an Expanded Set of Trehalose Monomycolate Analogues

Click-chemistry approach to study mycoloylated proteins: Evidence for PorB and PorC porins mycoloylation in Corynebacterium glutamicum

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