1 Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstr 5, D-91058 Erlangen, Germany
2 Molekulare Infektiologie, Forschungszentrum Borstel, Parkallee 22, D-23845 Borstel, Germany
3 Max-Planck-Institut für Biochemie, Abteilung Molekulare Strukturbiologie, Am Klopferspitz 18, D-82152 Martinsried, Germany
4 Lehrstuhl für Umweltmesstechnik, Universität Karlsruhe, Engler-Bunte-Ring 1, D-76128 Karlsruhe, Germany
Correspondence
Michael Niederweis
mnieder{at}biologie.uni-erlangen.de
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ABSTRACT |
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INTRODUCTION |
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It has long been suggested that the extremely low permeability of the unusual mycobacterial cell wall renders mycobacteria intrinsically resistant to many antibiotics such as -lactams, macrolides, tetracyclines, novobiocin and chloramphenicol (Nikaido & Jarlier, 1991
). Based on X-ray diffraction experiments of purified mycobacterial cell walls, which showed that the mycolic acids are oriented perpendicular to the cell surface (Nikaido et al., 1993
), it was proposed that the mycolic acids form the inner leaflet, and extractable lipids the outer leaflet, of an outer membrane (OM)-like structure (Brennan & Nikaido, 1995
). It is assumed that at least two general diffusion pathways across the mycobacterial OM exist: 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 (Niederweis, 2003
). Nikaido and co-workers showed that the fluidity of the OM of Mycobacterium chelonae is very low and strongly depends on the nature of the mycolic acids (Liu et al., 1995
, 1996
). They proposed that the unique structure and composition of the outer lipid bilayer make mycobacteria exceptionally impermeable to lipophilic solutes, but quantitative data are lacking so far. The uptake pathways for cephaloridine of Mycobacterium smegmatis and M. tuberculosis (Chambers et al., 1995
; Trias & Benz, 1994
) and of M. chelonae (Jarlier & Nikaido, 1990
) were shown to be 100-fold and 1000-fold, respectively, less efficient than that of Escherichia coli (Nikaido, 1986
). We provided evidence that permeation of cephaloridine across the OM of M. smegmatis is mainly mediated by the porin MspA (Stahl et al., 2001
). Furthermore, the 45-fold lower number of pores and the 2·5-fold longer pore channels compared to E. coli were identified as two determinants of the low efficiency of the porin pathway in M. smegmatis (Engelhardt et al., 2002
). Similar causes for low OM permeability for hydrophilic solutes are likely to exist for all mycobacteria (Niederweis, 2003
). These results implied that the low porin permeability of M. tuberculosis may limit (i) the efficiency of hydrophilic drugs in TB chemotherapy as suggested by many authors (Brennan & Nikaido, 1995
; Draper, 1998
; Jarlier & Nikaido, 1994
; Lambert, 2002
) and (ii) the growth rate of mycobacteria due to restricted uptake of polar nutrients (Jarlier & Nikaido, 1990
). Considering the importance of M. tuberculosis as a bacterial pathogen, and the need to understand how nutrients and drugs are transported inside the cell, it is surprising that the importance of porins for these processes has not been experimentally examined yet. For example, it is not known which proteins mediate the diffusion of hydrophilic solutes across the OM of M. tuberculosis. OmpATb displays a low channel activity in vitro (Senaratne et al., 1998
) and has transport activity for serine at low pH in vivo, but is unlikely to be a major general porin of M. tuberculosis, because the uptake rates for serine and glycine at pH 7·2 were not greatly affected by deletion of the ompATb gene (Raynaud et al., 2002
). The existence of other porins of M. tuberculosis has been demonstrated (Kartmann et al., 1999
), but these proteins still await identification. It is also unknown which antitubercular drugs are capable of diffusion through mycobacterial porins.
Since porin-negative mutants of M. tuberculosis or M. bovis BCG are lacking, we used a different approach to examine the importance of porins for the OM permeability of slow-growing mycobacteria. To this end, the porin MspA of M. smegmatis was expressed in the OMs of M. bovis BCG and M. tuberculosis and the OM permeability for glucose, the sensitivity to antibiotics and the growth rate of the recombinant strains were analysed.
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METHODS |
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Bacterial strains and growth conditions.
Mycobacterium bovis BCG, strain Institut Pasteur, was obtained from the American Type Culture Collection (ATCC 27291). M. tuberculosis H37Rv was kindly provided by Dr Peter Sander (Institute of Medical Microbiology, University of Zurich, Switzerland). Unless otherwise noted, M. bovis BCG and M. tuberculosis H37Rv were grown in Middlebrook 7H9 broth (Difco) or on 7H10 agar plates supplemented with 0·05 % Tween 80 (Sigma), 0·2 % glycerol and ADS (0·5 % bovine serum albumin fraction V, 0·2 % glucose and 14 mM NaCl) enrichment at 37 °C. All experiments with live M. tuberculosis were carried out under biosafety level 3 conditions. E. coli DH5 was used for all cloning experiments and was routinely grown in LB medium. Hygromycin B was used when required at the following concentrations: 200 µg ml1 for E. coli and 75 µg ml1 for mycobacteria.
Construction of plasmids.
The hsp60 promoter was amplified from pSTM3 by PCR (kindly provided by Dr Sabine Ehrt, Cornell University, New York, USA) with the oligonucleotides hsp60_fwd (5'-AAACGGTGACCACAACGACGCGCCC-3'), which has a half-side of the PmeI site and hsp60_rev (5'-GCTCTAGATTAATTAACTCACCGGTCGCGAGTGCCAACG-3'), which introduced a PacI site. The PCR fragment was digested with PacI, purified by preparative gel electrophoresis and ligated with PacI- and PmeI-digested pMS2 DNA to give pMS3. The mspA gene was isolated from pMN014 as a PacISwaI fragment and cloned via the same restriction sites into pMS3 to yield the mspA expression vector pMN066. All plasmid constructions were verified by restriction enzyme digestion and double-stranded DNA sequencing. The plasmids used in this study are summarized in Table 1.
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Electron microscopy.
Cell suspensions of M. bovis BCG (0·5 ml) were sedimented in a table top centrifuge, at low speed, to collect intact cells only. The pellet was resuspended in distilled water. The suspension was cooled in ice-water and sonified in a Branson Sonifier for two or three pulses (50 W). This was sufficient to break part of the cells and to obtain cell wall fragments of reasonable size. Five microlitres of this suspension was put on carbon-coated copper grids. The liquid was blotted after 20 seconds of adsorption and the sample was negatively stained with 2 % uranyl acetate. The specimens were inspected in the electron microscope (Philips EM420 or CM12) at a nominal magnification of x36 000 and recorded using Agfa film material.
Transport measurements.
Glucose uptake experiments were carried out as described previously (Bardou et al., 1998) with minor modifications. M. bovis BCG, with the control plasmid (pMN006) and with the mspA expression vector (pMN013), were grown as 10 ml precultures for 1015 days in Middlebrook 7H9 medium containing 0·05 % Tween 80 and ADS enrichment. The cells were passed through a filter with a pore size of 5 µm (Sartorius) to remove cell aggregates. After filtration more than 95 % of all bacteria were single, viable cells as demonstrated by staining with the LIVE/DEAD kit (Molecular Probes) and fluorescence microscopy (Axioskop2, Zeiss). The preculture was grown to an OD600 of 0·60·8 and then diluted into 100 ml fresh medium. After the culture reached an OD600 of 0·81, the bacteria were harvested, washed with 10 mM HEPES buffer (pH 7·2), resuspended in the same buffer and adjusted to a concentration of approximately 20 mg dry weight ml1. [14C]Glucose [specific activity 311 mCi mmol1 (11·5 GBq mmol1), Amersham) was added to 1 ml cell suspension to obtain a final concentration of 6·4 µM (dilution 1 : 100). The mixture was incubated at 37 °C and 100 µl samples were removed at times ranging from 1 to 32 min. The bacteria were separated from the liquid by filtration through a 0·45 µm pore size filter (Sartorius), washed with 0·1 M LiCl and the radioactivity was determined in a liquid scintillation counter. Five independent experiments were carried out in triplicate and uptake of glucose was expressed as pmol (mg dry weight cells)1. The condition of the cells after the transport measurements was checked by fluorescence microscopy using the LIVE/DEAD kit. In all experiments more than 97 % of all bacteria were single, live cells.
Cephaloridine hydrolysis assay.
The hydrolysis of cephaloridine by -lactamases of M. bovis BCG was measured spectrophotometrically using the method of Zimmermann and Rosselet as described previously (Stahl et al., 2001
).
Growth experiments.
M. bovis BCG/pMN006 (control strain) and the mspA-expressing strain BCG/pMN013 were grown as 10 ml precultures for 15 days in Middlebrook 7H9 broth supplemented with 0·05 % Tween 80 and ADS enrichment. The cells were passed through a filter with a pore size of 5 µm (Sartorius) to remove cell clumps. The new 10 ml cultures, containing more than 97 % live, single bacteria, were grown until an OD600 of 1 was reached. The bacteria were harvested and diluted in 100 ml of fresh 7H9 broth. Growth rates were determined in three independent cultures by OD600 measurements. Condition of precultures and cultures was followed by staining of cell aliquots with the LIVE/DEAD kit and fluorescence microscopy.
Antibiotic sensitivity experiments.
Minimal inhibititory concentrations (MICs) were determined for the control strain M. tuberculosis H37Rv/pMN006 and the mspA-expressing strain H37Rv/pMN013, as well as for M. bovis BCG/pMN006 and BCG/pMN013 by agar dilution experiments. Each strain was grown as 10 ml preculture for 15 days and then passed through a filter with a pore size of 5 µm to remove cell clumps. The filtrate, containing only single bacteria, was grown until an OD600 of 0·60·8 was achieved. A reference curve was constructed with a correlation between the number of colony-forming units (c.f.u.) and OD600. Using this curve, dilutions of each strain were made to obtain a final concentration of 5000 c.f.u. ml1. Five hundred colony-forming units were streaked out on plates with rising antibiotic concentrations. The MIC was defined as the lowest drug concentration inhibiting the visible growth of 99 % of all cells after 25 days of incubation at 37 °C.
Flow cytometry.
Aliquots of M. tuberculosis suspensions were thawed and centrifuged for 10 min at 1000 g. Bacteria were resuspended in phosphate-buffered saline (PBS) and several times passed through a syringe with a 26 gauge needle to disrupt aggregates. Aliquots of 2x106 bacteria of the MspA-expressing M. tuberculosis strain, containing the plasmid pMN013, and of the control strain, containing the plasmid pMN006 with a promoterless mspA gene, were incubated with a murine monoclonal antibody directed against MspA (mAb A15) for 30 min at 4 °C. Cells were washed, and binding of the primary antibodies was detected by the use of Cy5-labelled goat anti-mouse antiserum (GaMCy5) (Dianova) for 30 min. In experiments using the nucleic acid staining SYTO dyes (Molecular Probes), cells were incubated with 25 µM SYTO 9 and SYTO 12 for the times indicated. After staining the cells were washed, resuspended and fixed in PBS containing 1·5 % paraformaldehyde until analysis in a FACSCalibur flow cytometer (BD Bioscience) using CellQuest Pro software (BD Bioscience).
Apparent n-octanol/water partition coefficients Pow.
Apparent partition coefficients of SYTO 9 and SYTO 12 were determined by the shake flask method (OECD, Paris, 1981, Test Guideline 107; http://www.oecd.org/dataoecd/17/35/1948169.pdf). Solutions of the dyes were diluted 50-fold in n-octanol, saturated with water, to a final concentration of 10 µM. Reference UV spectra of each compound were recorded (Novaspec II spectrophotometer, Pharmacia). Five hundred mictolitres of the 10 µM solution in n-octanol was mixed with the same volume of water, saturated with n-octanol, and shaken at 25 °C for 3 h to achieve equilibrium. The phases were separated by centrifugation (8500 r.p.m., 25 °C, 30 min) and UV absorption spectra of both phases were measured. To determine the concentrations of the compounds, the absorption maxima of the UV spectra (473 nm for SYTO 9 and 492 nm for SYTO 12, in water) were used. The concentration of the compound in the aqueous phase was calculated as the difference of the total amount in the original 10 µM solution and the amount dissolved in the n-octanol phase after extraction with n-octanol. All experiments were done in triplicate; the values were averaged and used to calculate the partition coefficient using the formula POW=coctanol/cwater (c, concentration).
Fluorescence analysis of M. bovis BCG stained with SYTO 9 and SYTO 12.
To determine the masses of the hydrophobic fluorescent dyes SYTO 9 and SYTO 12, 2 µl of samples with concentrations of 48 µM and 500 µM, respectively, were loaded on a cross-linked polyethylene glycol capillary column (HP-INNOWAX, 1·20 m length) of a gas chromatograph coupled with a mass selective infrared detector (HP 5971A MSD, HP 5965B ID). Data analysis was performed using the HP 5965B/Chemstation software using a NIST/2001 database.
Fluorescence analysis of SYTO 9 and SYTO 12.
The fluorescence properties and the binding specificity of the dyes SYTO 9 and 12 were examined using a fluorimeter (Fluorolog-3, Jasco) equipped with two monochromators at an excitation and emission bandpass of 2 nm. One millilitre samples of 10 µM of the SYTO dyes were measured in the presence of increasing amounts of DNA ranging from 1 to 2000 ng, or 5 and 10 µg purified MspA. SYTO 9 was excited at 470 nm and the fluorescence emission was recorded between 500 and 700 nm. SYTO 12 was excited at 499 nm and the fluorescence emission was recorded between 510 and 700 nm. All experiments were done at a sample temperature of 25 °C.
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RESULTS |
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Streptomycin is an aminocyclitol glycoside antibiotic and is highly efficient against M. tuberculosis (Kremer & Besra, 2002b). Streptomycin is a large, polar molecule and was thought to be too large to diffuse via mycobacterial porins (Senaratne et al., 1998
). Surprisingly, expression of MspA in the OM significantly increased the sensitivity of M. tuberculosis to streptomycin (Fig. 7D
).
MspA specifically increases the uptake of a fluorescent dye by M. tuberculosis
Fluorescent stains are widely used in microscopy and flow cytometry to visualize bacteria and to report on their viability and other cellular parameters (Joux & Lebaron, 2000; Novo et al., 2000
). A comprehensive study revealed that mycobacteria are stained equally well by the membrane-permeant SYTO stains, whose fluorescence is approximately 40-fold enhanced upon binding to nucleic acids (Molecular Probes, www.probes.com). Quantitative staining experiments with different SYTO dyes were performed to analyse whether the presence of MspA had an influence on uptake of these dyes. The mspA-expressing strain of M. tuberculosis showed a 10-fold increased fluorescence compared to the control when stained with 25 µM SYTO 9 for 30 min at 25 °C and analysed by flow cytometry (Fig. 8
A). By contrast, staining of M. tuberculosis with SYTO 12 was not dependent on MspA (Fig. 8B
). The kinetics of the staining of M. tuberculosis with SYTO 9 and 12 was determined to examine whether staining was saturated for both dyes. These experiments revealed that M. tuberculosis is stained at the same rate by both dyes at concentrations of 25 µM and that staining was already saturated after 5 min (Fig. 8C
). Strikingly, staining of M. tuberculosis by SYTO 9 was clearly faster in the presence of MspA, whereas staining with SYTO 12 did not depend on MspA. Similar results, at reduced staining rates, were obtained with lower dye concentrations of 2·5 and 0·25 µM (data not shown). Although nucleic acids are the only molecules known to enhance the fluorescence of the SYTO stains, we wanted to exclude the possibility that the fluorescence enhancement of M. tuberculosis was caused by a direct binding of SYTO 9 to MspA. The fluorescence intensity of SYTO 9 at 500700 nm, when excited at the absorption maximum at 470 nm, did not increase upon addition of MspA, indicating that MspA increased the permeability of SYTO 9 across the OM of M. tuberculosis and thereby the access of SYTO 9 to nucleic acids inside M. tuberculosis (data not shown). A possible explanation for the apparently increased permeability for SYTO 9 compared to SYTO 12 is that the former might be significantly smaller and more hydrophilic than the latter. Therefore, we measured both the hydrophobicities of the dye molecules and their masses. The octanol/water partition coefficients Pow were 8·5±0·9 and 13·2±1·5 for SYTO 9 and SYTO 12, respectively. Mass spectroscopy revealed one mass of 355 (m/z) for SYTO 9 and several masses between 330430 (m/z) for SYTO 12. Thus, the gross physico-chemical properties of both dyes are similar and do not explain the drastic permeability differences in the mspA-expressing strain of M. tuberculosis.
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DISCUSSION |
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Glucose was taken up by M. bovis BCG at a rate of approximately 0·7 pmol min1 (mg dry weight cells)1 at a concentration of 6·4 µM. This is 1430-fold slower than the rate of 1 nmol min1 (mg dry weight cells)1 measured for M. smegmatis under identical conditions (J. Stephan & M. Niederweis, unpublished) and 140-fold slower than that calculated for M. chelonae using the published Vmax and Km values (Jarlier & Nikaido, 1990). Glucose uptake kinetics for M. bovis BCG have been published previously (Yuan et al., 1998
). An uptake rate of 0·04 pmol min1 (mg dry weight cells)1 was calculated for this experiment assuming the same specific activity as in our experiments. Uptake of 6·5 µM glycerol by M. tuberculosis was also very slow with a rate of 0·1 pmol min1 (mg dry weight cells)1 (Jackson et al., 1999
). In conclusion, these results consistently indicated that the OM permeability of both M. bovis BCG and M. tuberculosis for small nutrient molecules is orders of magnitude lower than that of fast-growing mycobacteria. These data contrast with the observation that the OM permeability of M. tuberculosis for cephaloridine was similar to that of M. smegmatis (Chambers et al., 1995
) and one order of magnitude higher than that of M. chelonae (Jarlier & Nikaido, 1990
). This discrepancy might be caused by the different solutes, but usually both methods yielded consistent results: e.g. the permeability to cephaloridine decreased in the order E. coli, Pseudomonas aeruginosa and M. chelonae by almost four orders of magnitude as did the permeability to glucose (Jarlier & Nikaido, 1990
). The observation that uptake of glucose was about twofold faster upon expression of mspA demonstrates that diffusion through the endogenous porins and not low-affinity transport proteins in the inner membrane, limit the rate of glucose utilization by M. bovis BCG. Furthermore, electron microscopy analysis of cell wall fragments of M. bovis BCG did not reveal MspA-like pores, consistent with the apparent lack of MspA homologues in M. bovis BCG and M. tuberculosis (Niederweis, 2003
), and corroborating the assumption that the endogenous porins of M. bovis BCG are substantially different from the M. smegmatis porins. Less efficient porins and/or a lower number of porins would explain the lower permeability to hydrophilic solutes of slow- compared to fast-growing mycobacteria and might have resulted from adaptations to the very different habitats of these bacteria as discussed recently (Niederweis, 2003
). The antibiotic sensitivity experiments and the growth-promoting effect of MspA provided further support for the extremely low OM permeability of M. tuberculosis and M. bovis BCG compared to M. smegmatis. It is very unlikely that the same small increase of 2035 porins per µm2 cell wall as was observed for the mspA-expressing strains of M. tuberculosis and M. bovis BCG, respectively, would have led to any observable change of phenotype in M. smegmatis, which has 1000 MspA-like porins per µm2 cell wall (Engelhardt et al., 2002
). Thus, the permeability through the endogenous porins of M. tuberculosis and M. bovis BCG must be considerably lower compared to M. smegmatis. Certainly, a systematic and quantitative analysis of the OM permeability and identification of the major porins of slow-growing mycobacteria are needed to solve this puzzle.
The questions why the strictly pathogenic members of the genus Mycobacterium such as M. tuberculosis and M. leprae grow much more slowly than the non-pathogenic saprophytes such as M. smegmatis and M. phlei (generation times: >15 h vs <5 h; Rastogi et al., 2001) and whether slow growth is beneficial or even necessary for pathogenicity have puzzled generations of scientists. Thus, it is not surprising that many factors have been invoked to explain the slow growth of M. tuberculosis: (i) slow RNA synthesis (Harshey & Ramakrishnan, 1977
), (ii) slow DNA elongation (Hiriyanna & Ramakrishnan, 1986
), (iii) slow protein synthesis due to the lack of multiple copies of rRNA operons (Bercovier et al., 1986
), (iv) slow porin-mediated uptake of nutrients (Jarlier & Nikaido, 1990
) and (v) presence of the DNA-binding protein MDBP1 (Matsumoto et al., 2000
). So far, experimental evidence demonstrating that any of these factors really limits the growth rate of slow-growing mycobacteria is lacking. In this study, we showed that even the presence of the very low number of about 35 MspA porins per µm2 cell wall significantly accelerated the growth rate of M. bovis BCG. However, it is not clear whether increased nutrient influx directly affected the growth rate or whether this effect reflected regulatory events in the cell. In both cases, our results indicate that low porin permeability is probably one of multiple factors contributing to the slow growth of M. bovis BCG.
The intrinsic resistance of mycobacteria to most hydrophilic antibiotics and chemotherapeutic agents is believed to result from a low-efficiency porin pathway in synergy with other resistance mechanisms such as enzymic inactivation or active efflux of the drugs (Brennan & Nikaido, 1995; Jarlier & Nikaido, 1994
). It is also assumed that porins are necessary for the uptake of the first line TB drugs isoniazid and ethambutol, since they are small and hydrophilic molecules (Jarlier & Nikaido, 1994
; Lambert, 2002
). MspA increased the susceptibility of both M. bovis BCG and M. tuberculosis to
-lactam antibiotics. The MspA-mediated sensitivity was still significant but smaller for the TB drugs isoniazid and ethambutol, consistent with the finding that, in general, differences in porin permeabilities have less pronounced effects for smaller solutes (Nikaido & Rosenberg, 1983
). These results also suggested that
-lactam antibiotics and both isoniazid and ethambutol use the MspA porin pathway to enter the recombinant M. tuberculosis and M. bovis BCG strains. This assumption is is consistent with the observation that the mspA deletion mutant of M. smegmatis, which has a threefold reduced porin density, showed the reverse phenotype: It was 8- and 16-fold more resistant to cephaloridine and ampicillin, respectively, and slightly more resistant to ethambutol (J. Stephan, C. Mailaender, G. Etienne, M. Daffé & M. Niederweis, unpublished). The clear correlation of porin-mediated OM permeability and sensitivity indicated that cephaloridine, and most likely other zwitterionic
-lactam antibiotics, use the porin pathway for cell entry in M. smegmatis (J. Stephan, C. Mailaender, G. Etienne, M. Daffé & M. Niederweis, unpublished). The interpretation is less clear for isoniazid, which is also able to diffuse directly through lipid membranes, as shown by liposome-swelling experiments (Jackson et al., 1999
; Raynaud et al., 1999
). However, it is likely that the diffusion rate of isoniazid through the mycolic acid layer of mycobacteria is much lower compared to bilayers from conventional C16-phosphatidylcholine lipids (Jackson et al., 1999
). Therefore, further experiments are necessary to evaluate the relative importance of the porin and the lipid pathway for uptake of isoniazid by mycobacteria. The effect of MspA for all drugs tested was smaller in M. tuberculosis, consistent with the almost twofold lower amount of MspA compared to M. bovis BCG. These results provide the proof of principle that porins are essential for susceptibility of M. tuberculosis to drugs currently used in TB chemotherapy. However, it should be noted that these data do not allow us to clearly assign a particular OM pathway for the drugs examined in this study.
MspA also increased the sensitivity of M. tuberculosis to streptomycin, although aminoglycosides were believed to be too large to diffuse through porin channels (Senaratne et al., 1998). This assumption is supported by the observation that even disaccharides such as sucrose and maltose did not produce a significant swelling of multilamellar proteoliposomes containing MspA (C. Heinz & M. Niederweis, unpublished results). This suggested an indirect effect of MspA on the OM permeability of M. tuberculosis for streptomycin. Indeed, such an effect was observed for the mspA deletion mutant of M. smegmatis, which was hyper-resistant not only to
-lactam antibiotics, but also to very large antibiotics such kanamycin and vancomycin, and to hydrophobic antibiotics such as rifampicin (J. Stephan, C. Mailaender, G. Etienne, M. Daffé & M. Niederweis, unpublished). This may be explained by rearrangement and stronger interactions of OM lipids due to the loss of MspA in M. smegmatis. Local interruptions of lipidlipid interactions, which are not fully replaced by lipidprotein interactions, would explain the higher permeability of the OM of M. tuberculosis to large and hydrophobic compounds upon expression of mspA. Evidence that this model might be correct was provided by the observation that MspA drastically increased the fluorescence of M. tuberculosis upon staining with SYTO 9. Binding of SYTO 9 to DNA was demonstrated in fluorescence experiments, but binding to MspA and to other compounds of the cell wall of M. tuberculosis was excluded in vitro and in vivo, respectively. The hydrophobicity of SYTO 9 makes it rather unlikely that it can diffuse directly through water-filled protein channels at significant rates (Nikaido et al., 1983
). These results suggested that the presence of MspA indirectly increased the permeability of the OM of M. tuberculosis to streptomycin.
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ACKNOWLEDGEMENTS |
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Received 11 November 2003;
revised 12 December 2003;
accepted 16 December 2003.
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