Mycobacteria contain an outer membrane of unusually low permeability which contributes to their intrinsic resistance to many agents. It is assumed that small and hydrophilic antibiotics cross the outer membrane via porins, whereas hydrophobic antibiotics may diffuse through the membrane directly. A mutant of Mycobacterium smegmatis lacking the major porin MspA was used to examine the role of the porin pathway in antibiotic sensitivity. Deletion of the mspA gene caused high-level resistance of M. smegmatis to 256 g of ampicillin/ml by increasing the MIC 16-fold. The permeation of cephaloridine in the mspA mutant was reduced ninefold, and the resistance increased eightfold. This established a clear relationship between the activity and the outer membrane permeation of cephaloridine. Surprisingly, the MICs of the large and/or hydrophobic antibiotics vancomycin, erythromycin, and rifampin for the mspA mutant were increased 2-to 10-fold. This is in contrast to those for Escherichia coli, whose sensitivity to these agents was not affected by deletion of porin genes. Uptake of the very hydrophobic steroid chenodeoxycholate by the mspA mutant was retarded threefold, which supports the hypothesis that loss of MspA indirectly reduces the permeability by the lipid pathway. The multidrug resistance of the mspA mutant highlights the prominent role of outer membrane permeability for the sensitivity of M. smegmatis to antibiotics. An understanding of the pathways across the outer membrane is essential to the successful design of chemotherapeutic agents with activities against mycobacteria.The prevalence and spread of antibiotic resistance are increasingly serious problems that hamper the effective treatment of infectious diseases (26). The search for new antibiotics is mainly based on novel bacterial targets and high-throughput screening assays (10). However, many lead compounds discovered in vitro may fail because they do not reach their targets at sufficiently high concentrations in vivo (7). This is true in particular for gram-negative bacteria, which, in contrast to grampositive bacteria, are protected from the toxic actions of certain antibiotics, dyes, and detergents and host defense factors such as lysozyme by an additional outer membrane (OM) (49). The OM can be crossed by at least two general pathways: the hydrophobic (or lipid) pathway, which is characterized by the nature and the interactions of the membrane lipids, and the hydrophilic (or porin) pathway, whose properties are determined by water-filled channel proteins, the porins, which span the OM of gram-negative bacteria (49). It has been shown by the pioneering work of Nikaido and collaborators (21, 46) that Escherichia coli and Salmonella porins play a major role in the transport of ␤-lactam antibiotics. Subsequent studies showed that porin-deficient mutants of gram-negative bacteria were also more resistant to quinolones, tetracyclines, chloramphenicol, nalidixic acid, and trimethoprim (6,15,25,52). These data suggest that porins are involved in the transport...

The impermeability of the outer membrane in combination with drug efflux are major determinants of the natural drug resistance of mycobacteria. ␤-Lactams are the most widely used antibiotics for treatment of bacterial infections. However, it is unknown how ␤-lactams enter Mycobacterium tuberculosis and whether efflux pumps exist that can export these drugs out of the cell. To identify the molecular mechanisms of M. tuberculosis resistance to ␤-lactams, a library of 7,500 transposon mutants was generated in the model organism Mycobacterium bovis BCG. Thirty-three unique insertion sites were determined that conferred medium or high-level (>2,000 g/ml) resistance to ampicillin. Three mutants in sulfolipid synthesis or transport were highly resistant to ampicillin, indicating an indirect effect of the lipid composition on the outer membrane permeability of M. bovis BCG to ampicillin. Mutants with insertions in genes encoding surface molecules such as PPE proteins or lipoarabinomannan were also completely resistant to ampicillin, thus suggesting a lack of transport across the outer membrane. Insertion of the transposon in front of bcg0231 increased transcription of the gene and concomitantly the resistance of M. bovis BCG to ampicillin, streptomycin, and chloramphenicol by 32-to 64-fold. Resistance to vancomycin and tetracycline was increased four-to eightfold. Bcg0231 and Rv0194 are almost identical ATP-binding cassette transporters. Expression of rv0194 significantly reduced accumulation of ethidium bromide and conferred multidrug resistance to Mycobacterium smegmatis. Both effects were abrogated in the presence of the efflux pump inhibitor reserpine. These results demonstrate that Rv0194 is a novel multidrug efflux pump of M. tuberculosis. Tuberculosis (TB) is caused by Mycobacterium tuberculosisand kills approximately 2 million people each year (22). Due to the intrinsic resistance of M. tuberculosis to many antibiotics, chemotherapy of TB is restricted to a very limited number of drugs, which have to be used in combination for at least 6 months. Infections with multidrug-resistant M. tuberculosis strains require a prolonged treatment with second-line drugs for up to 2 years, which increases the cost of treatment drastically (56). Even more alarming are the extensively drug-resistant strains of M. tuberculosis, which are resistant not only to isoniazid and rifampin but also to at least two second-line anti-TB drugs (38). A better understanding of resistance mechanisms to known antibiotics may be one way to overcome these problems. A major determinant of the intrinsic resistance of M. tuberculosis is its unique outer membrane, which presents an effective permeability barrier to both hydrophobic and hydrophilic solutes (7). However, the mycobacterial outer membrane can only slow down the influx of drugs and is required to act in synergy with alternative mechanisms to effectively decrease the intracellular concentration of drugs. Recent evidence suggests that multidrug resistance of M. tuberculosis is associated wit...

Porins mediate the diffusion of hydrophilic solutes across the outer membrane of mycobacteria, but the efficiency of this pathway is very low compared to Gram-negative bacteria. To examine the importance of porins in slow-growing mycobacteria, the major porin MspA of Mycobacterium smegmatis was expressed in Mycobacterium tuberculosis and Mycobacterium bovis. Approximately 20 and 35 MspA molecules per mm 2 cell wall were observed in M. tuberculosis and M. bovis BCG, respectively, by electron microscopy and quantitative immunoblot experiments. Surface accessibility of MspA in M. tuberculosis was demonstrated by flow cytometry. Glucose uptake was twofold faster, indicating that the outer membrane permeability of M. bovis BCG to small and hydrophilic solutes was increased by MspA. This significantly accelerated the growth of M. bovis BCG, identifying very slow nutrient uptake as one of the determinants of slow growth in mycobacteria. The susceptibility of both M. bovis BCG and M. tuberculosis to zwitterionic b-lactam antibiotics was substantially enhanced by MspA, decreasing the minimal inhibitory concentration up to 16-fold. Furthermore, M. tuberculosis became significantly more susceptible to isoniazid, ethambutol and streptomycin. Fluorescence with the nucleic acid binding dye SYTO 9 was 10-fold increased upon expression of mspA. These results indicated that MspA not only enhanced the efficiency of the porin pathway, but also that of pathways mediating access to large and/or hydrophobic agents. This study provides the first experimental evidence that porins are important for drug susceptibility of M. tuberculosis.

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...