SummaryPorins form channels in the mycolic acid layer of mycobacteria and thereby control access of hydrophilic molecules to the cell. We puri®ed a 100 kDa protein from Mycobacterium smegmatis and demonstrated its channel-forming activity by reconstitution in planar lipid bilayers. The mspA gene encodes a mature protein of 184 amino acids and an N-terminal signal sequence. MALDI mass spectrometry of the puri®ed porin revealed a mass of 19 406 Da, in agreement with the predicted mass of mature MspA. Dissociation of the porin by boiling in 80% dimethyl sulphoxide yielded the MspA monomer, which did not form channels any more. Escherichia coli cells expressing the mspA gene produced the MspA monomer and a 100 kDa protein, which had the same channel-forming activity as whole-cell extracts of M. smegmatis with organic solvents. These proteins were speci®cally detected by a polyclonal antiserum that was raised to puri®ed MspA of M. smegmatis. These results demonstrate that the mspA gene encodes a protein of M. smegmatis, which assembles to an extremely stable oligomer with high channel-forming activity. Database searches did not reveal signi®cant similarities to any other known protein. Southern blots showed that the chromosomes of fast-growing mycobacterial species contain homologous sequences to mspA, whereas no hybridization could be detected with DNA from slow growing mycobacteria. These results suggest that MspA is the prototype of a new class of channel-forming proteins.
Mycobacteria protect themselves with an outer lipid bilayer, which is the thickest biological membrane hitherto known and has an exceptionally low permeability rendering mycobacteria intrinsically resistant against many antibiotics. Pore proteins mediate the diffusion of hydrophilic nutrients across this membrane. Electron microscopy revealed that the outer membrane of Mycobacterium smegmatis contained about 1000 protein pores per m 2 , which are about 50-fold fewer pores per m 2 than in Gram-negative bacteria. The projection structure of the major porin MspA of M. smegmatis was determined at 17 Å resolution. MspA forms a cone-like tetrameric complex of 10 nm in length with a single central pore. Thus, MspA is drastically different from the trimeric porins of Gram-negative bacteria and represents a new class of channel proteins. The formation of MspA micelles indicated that the ends of MspA have different hydrophobicities. Oriented insertion of MspA into membranes was demonstrated in lipid bilayer experiments, which revealed a strongly asymmetrical voltage gating of MspA channels at -30 mV. The length of MspA is sufficient to span the outer membrane and contributes in combination with the tapering end of the pore and the low number of pores to the low permeability of the cell wall of M. smegmatis for hydrophilic compounds. About two million people die each year from tuberculosis (TB).1 This number qualifies TB as the leading agent of death due to a single infectious disease (1). Treatment of TB caused by non-resistant strains of Mycobacterium tuberculosis is effective but is based on a regimen of up to four drugs over a period of six months (2). The major problem in TB therapy is the slow uptake of drugs across the mycobacterial cell wall, which mounts a formidable permeability barrier toward diffusion of hydrophobic and hydrophilic compounds (3). The diffusion of hydrophilic compounds across the mycobacterial cell wall is mediated by water-filled channel proteins (4). The transport function of these channel proteins is similar to that of the porins of Gram-negative bacteria, but the porin pathway of mycobacteria is at least 1000-fold less efficient than that of Escherichia coli (5,6). It is assumed that the combination of low cell wall permeability with the action of detoxifying proteins such as degrading enzymes or efflux pumps is responsible for the intrinsic resistance of mycobacteria to many antibiotics such as penicillins, cephalosporins, and tetracyclines (7). However, it is not known why the porin pathway in mycobacteria is so inefficient.Two integral proteins with transport properties have been identified in mycobacterial cell walls: OmpATb from M. tuberculosis, which has channel activity in vitro but unexplored physiological functions (8), and MspA, which was first discovered in chloroform-methanol extracts from Mycobacterium smegmatis as an oligomeric channel protein composed of 20 kDa subunits (9). Enzyme-linked immunosorbent assays and immunogold labeling experiments demonstrated that MspA is locali...
MspA is the major porin of Mycobacterium smegmatis mediating the exchange of hydrophilic solutes across the cell wall and is the prototype of a new family of tetrameric porins with a single central pore of 10 nm in length. Infrared and circular dichroism spectroscopy revealed that MspA consists mainly of antiparallel -strands organized in a coherent domain. Heating to 92 and 112°C was required to dissociate the MspA tetramer and to unfold the -sheet domain in the monomer, respectively. The stability of the MspA tetramer exceeded the remarkable stability of the porins of Gram-negative bacteria for every condition tested and was not reduced in the presence of 2% SDS and at any pH from 2 to 14. These results indicated that the interactions between the MspA subunits are different from those in the porins of Gram-negative bacteria and are discussed in the light of a channel-forming -barrel as a core structure of MspA. Surprisingly, the channel activity of MspA in 2% SDS and 7.6 M urea at 50°C was reduced 13-and 30-fold, respectively, although the MspA tetramer and the -sheet domain were stable under those conditions. Channel closure by conformational changes of extracellular loops under those conditions is discussed to explain these observations. This study presents the first experimental evidence that outer membrane proteins not only from Gram-negative bacteria but also from mycobacteria are -sheet proteins and demonstrates that MspA constitutes the most stable transmembrane channel protein known so far. Thus, MspA may be of special interest for biotechnological applications.
Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 ؎ 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acidcontaining outer membranes.Mycobacteria are classified as Gram-positive bacteria but have evolved a complex cell wall, comprising a peptidoglycanarabinogalactan polymer with covalently bound mycolic acids of considerable length (up to 90 carbon atoms) and a large variety of extractable lipids (1, 2). Most of these lipids are constituents of the cell envelope that provides an extraordinarily efficient permeability barrier and is an essential part of the intrinsic resistance of mycobacteria to many toxic compounds and antibiotics (3). To account for these observations, Minnikin (4) proposed a model in which the mycolic acids form the inner leaflet of an asymmetrical bilayer. Mutants and treatments affecting mycolic acid biosynthesis and the production of extractable lipids showed an increase in cell wall permeability and a drastic decrease in virulence, underlining the importance of the cell wall integrity for intracellular survival of Mycobacterium tuberculosis (1). Cryoelectron tomography revealed the native organization of the Mycobacterium smegmatis cell envelope. Further, the three-dimensional data and the investigation of ultrathin frozen-hydrated cryosections of M. smegmatis, M. bovis BCG, and Corynebacterium glutamicum identified the outermost layer as a lipid bilayer. Mycolic acids were shown to be essential components of this bilayer, therefore providing the first visualization of mycobacterial outer membranes in their native state (5).These findings raise the question of how the mycobacterial outer membrane is functionalized fo...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.