The MspA porin promotes growth and increases antibiotic susceptibility of both Mycobacterium bovis BCG and Mycobacterium tuberculosis

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 853-864 ◽  
Author(s):  
Claudia Mailaender ◽  
Norbert Reiling ◽  
Harald Engelhardt ◽  
Stefan Bossmann ◽  
Stefan Ehlers ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Outer Membrane ◽  
Mycobacterium Bovis ◽  
Mycobacterium Smegmatis ◽  
Drug Susceptibility ◽  
Nucleic Acid Binding ◽  
Outer Membrane Permeability ◽  
Syto 9 ◽  
Slow Growing ◽  
Hydrophilic Solutes

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 μm2 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 β-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.

scholarly journals The Mycobacterium tuberculosis Outer Membrane Channel Protein CpnT Confers Susceptibility to Toxic Molecules

2015 ◽  
Vol 59 (4) ◽  
pp. 2328-2336 ◽  
Author(s):  
Olga Danilchanka ◽  
David Pires ◽  
Elsa Anes ◽  
Michael Niederweis
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Membrane Permeability ◽  
Nutrient Uptake ◽  
Outer Membrane ◽  
Membrane Channel ◽  
Content Type ◽  
Channel Protein ◽  
Outer Membrane Channel ◽  
Outer Membrane Permeability ◽  
Slow Growing

ABSTRACTMycobacterium tuberculosis, the causative agent of tuberculosis, is protected from toxic solutes by an effective outer membrane permeability barrier. Recently, we showed that the outer membrane channel protein CpnT is required for efficient nutrient uptake byM. tuberculosisandMycobacterium bovisBCG. In this study, we found that thecpnTmutant ofM. bovisBCG is more resistant than the wild type to a large number of drugs and antibiotics, including rifampin, ethambutol, clarithromycin, tetracycline, and ampicillin, by 8- to 32-fold. Furthermore, thecpnTmutant ofM. bovisBCG was 100-fold more resistant to nitric oxide, a major bactericidal agent required to controlM. tuberculosisinfections in mice. Thus, CpnT constitutes the first outer membrane susceptibility factor in slow-growing mycobacteria. The dual functions of CpnT in uptake of nutrients and mediating susceptibility to toxic molecules are reflected in macrophage infection experiments: while loss of CpnT was detrimental forM. bovisBCG in macrophages that enable bacterial replication, presumably due to inadequate nutrient uptake, it conferred a survival advantage in macrophages that mount a strong bactericidal response. Importantly, thecpnTgene showed a significantly higher density of nonsynonymous mutations in drug-resistant clinicalM. tuberculosisstrains, indicating that CpnT is under selective pressure in human tuberculosis and/or during chemotherapy. Our results indicate that the CpnT channel constitutes an outer membrane gateway controlling the influx of nutrients and toxic molecules into slow-growing mycobacteria. This study revealed that reducing protein-mediated outer membrane permeability might constitute a new drug resistance mechanism in slow-growing mycobacteria.

scholarly journals Comparison of the Construction of Unmarked Deletion Mutations in Mycobacterium smegmatis, Mycobacterium bovis Bacillus Calmette-Guérin, and Mycobacterium tuberculosis H37Rv by Allelic Exchange

1999 ◽  
Vol 181 (16) ◽  
pp. 4780-4789 ◽  
Author(s):  
Martin S. Pavelka ◽  
William R. Jacobs
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Mycobacterium Smegmatis ◽  
Tween 80 ◽  
Defined Medium ◽  
Lysine Biosynthesis ◽  
Rich Medium ◽  
Deletion Mutations ◽  
Allelic Exchange ◽  
Slow Growing

ABSTRACT Until recently, genetic analysis of Mycobacterium tuberculosis, the causative agent of tuberculosis, was hindered by a lack of methods for gene disruptions and allelic exchange. Several groups have described different methods for disrupting genes marked with antibiotic resistance determinants in the slow-growing organismsMycobacterium bovis bacillus Calmette-Guérin (BCG) and M. tuberculosis. In this study, we described the first report of using a mycobacterial suicidal plasmid bearing the counterselectable marker sacB for the allelic exchange of unmarked deletion mutations in the chromosomes of two substrains ofM. bovis BCG and M. tuberculosis H37Rv. In addition, our comparison of the recombination frequencies in these two slow-growing species and that of the fast-growing organismMycobacterium smegmatis suggests that the homologous recombination machinery of the three species is equally efficient. The mutants constructed here have deletions in the lysA gene, encoding meso-diaminopimelate decarboxylase, an enzyme catalyzing the last step in lysine biosynthesis. We observed striking differences in the lysine auxotrophic phenotypes of these three species of mycobacteria. The M. smegmatis mutant can grow on lysine-supplemented defined medium or complex rich medium, while the BCG mutants grow only on lysine-supplemented defined medium and are unable to form colonies on complex rich medium. The M. tuberculosis lysine auxotroph requires 25-fold more lysine on defined medium than do the other mutants and is dependent upon the detergent Tween 80. The mutants described in this work are potential vaccine candidates and can also be used for studies of cell wall biosynthesis and amino acid metabolism.

scholarly journals Multiple small RNAs identified in Mycobacterium bovis BCG are also expressed in Mycobacterium tuberculosis and Mycobacterium smegmatis

2010 ◽  
Vol 38 (12) ◽  
pp. 4067-4078 ◽  
Author(s):  
J. M. DiChiara ◽  
L. M. Contreras-Martinez ◽  
J. Livny ◽  
D. Smith ◽  
K. A. McDonough ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Mycobacterium Smegmatis ◽  
Small Rnas ◽  
Mycobacterium Bovis Bcg

scholarly journals Protein-DNA Complexes in Mycobacteriophage L5 Integrative Recombination

1999 ◽  
Vol 181 (2) ◽  
pp. 454-461 ◽  
Author(s):  
Carol E. A. Peña ◽  
J. Michelle Kahlenberg ◽  
Graham F. Hatfull
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Bovis ◽  
Binding Sites ◽  
Mycobacterium Smegmatis ◽  
Recombination Event ◽  
Specific Protein ◽  
Integration Host Factor ◽  
Dna Complexes ◽  
Active Complex ◽  
Mycobacteriophage L5

ABSTRACT The temperate mycobacteriophage L5 integrates site specifically into the genomes of Mycobacterium smegmatis,Mycobacterium tuberculosis, and Mycobacterium bovis bacillus Calmette-Guérin. This integrative recombination event occurs between the phage L5 attP site and the mycobacterial attB site and requires the phage-encoded integrase and mycobacterial-encoded integration host factor mIHF. Here we show that attP, Int-L5, and mIHF assemble into a recombinationally active complex, the intasome, which is capable of attB capture and formation of products. The arm-type integrase binding sites within attP play specialized roles in the formation of specific protein-DNA architectures; the intasome is constructed by the formation of intramolecular integrase bridges between one pair of sites, P4-P5, and the attP core, while an additional pair of sites, P1-P2, is required for interaction with attB.

scholarly journals Multidrug Resistance of a Porin Deletion Mutant of Mycobacterium smegmatis

2004 ◽  
Vol 48 (11) ◽  
pp. 4163-4170 ◽  
Author(s):  
Joachim Stephan ◽  
Claudia Mailaender ◽  
Gilles Etienne ◽  
Mamadou Daffé ◽  
Michael Niederweis
Keyword(s):  
Multidrug Resistance ◽  
Outer Membrane ◽  
Mycobacterium Smegmatis ◽  
Antibiotic Sensitivity ◽  
Chemotherapeutic Agents ◽  
Intrinsic Resistance ◽  
Successful Design ◽  
High Level ◽  
Outer Membrane Permeability

ABSTRACT 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 μg 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.

scholarly journals Mutations in theembC-embAIntergenic Region Contribute to Mycobacterium tuberculosis Resistance to Ethambutol

2014 ◽  
Vol 58 (11) ◽  
pp. 6837-6843 ◽  
Author(s):  
Zhenling Cui ◽  
Yuanyuan Li ◽  
Song Cheng ◽  
Hua Yang ◽  
Junmei Lu ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycobacterium Smegmatis ◽  
Transcriptional Activity ◽  
Drug Susceptibility ◽  
Drug Susceptibility Testing ◽  
Mobility Shift ◽  
Content Type ◽  
Clinical Strains ◽  
Gel Mobility Shift

ABSTRACTThe rapid increase inMycobacterium tuberculosisresistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in theembC-embAintergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767M. tuberculosisclinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and theembC-embAIGRs of these strains were sequenced. The transcriptional activity of theembC-embAIGR mutations was examined by reporter gene assays in recombinantMycobacterium smegmatisstrains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator ofembAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that theembC-embAIGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level ofembAandembBmRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed intoM. smegmatisstrains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of theembC-embAIGR enhance the binding of EmbR to the promoter region ofembABand increase the expression ofembAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.

scholarly journals Role of Porins in Iron Uptake by Mycobacterium smegmatis

2010 ◽  
Vol 192 (24) ◽  
pp. 6411-6417 ◽  
Author(s):  
Christopher M. Jones ◽  
Michael Niederweis
Keyword(s):  
Outer Membrane ◽  
Mycobacterium Smegmatis ◽  
Iron Homeostasis ◽  
Iron Acquisition ◽  
Gram Negative Bacteria ◽  
Ferric Ions ◽  
Intracellular Iron ◽  
High Iron ◽  
Hydrophilic Solutes

ABSTRACT Many bacteria rely on siderophores to extract iron from the environment. However, acquisition of iron-loaded siderophores is dependent on high-affinity uptake systems that are not produced under high-iron conditions. The fact that bacteria are able to maintain iron homeostasis in the absence of siderophores indicates that alternative iron acquisition systems exist. It has been speculated that such low-affinity uptake of iron in Gram-negative bacteria includes diffusion of iron ions or chelates across the outer membrane through porins. The outer membrane of the saprophytic Mycobacterium smegmatis contains the Msp family of porins, which enable the diffusion of small and hydrophilic solutes, such as monosaccharides, amino acids, and phosphate. However, it is unknown how cations cross the outer membrane of mycobacteria. Here, we show that the Msp porins of M. smegmatis are involved in the acquisition of soluble iron under high-iron conditions. Uptake of ferric ions by a triple porin mutant was reduced compared to wild-type (wt) M. smegmatis. An intracellular iron reporter indicated that derepression of iron-responsive genes occurs at higher iron concentrations in the porin mutant. This was consistent with the finding that the porin mutant produced more siderophores under low-iron conditions than wt M. smegmatis. In contrast, uptake of the exochelin MS, the main siderophore of M. smegmatis, was not affected by the lack of porins, indicating that a specific outer membrane siderophore receptor exists. These results provide, to our knowledge, the first experimental evidence that general porins are indeed the outer membrane conduit of low-affinity iron acquisition systems in bacteria.

Sign in / Sign up

Export Citation Format

Share Document