Porin MspA Research Articles

Evolution toward extremely high imipenem resistance in Mycobacterium abscessus outbreak strains.

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem). M. abscessus produces a β-lactamase (BlaMab) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a cystic fibrosis center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC > 512 µg/mL). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased blaMab mRNA level. Concurrent increase in transcription of the preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 µg/mL). A frameshift mutation in msp2 responsible for the rough colony morphology and a SNP in ATP-dependent helicase hrpA cooccurred with the second increase in MIC (from 32 to 256 µg/mL). Increased BlaMab expression and enzymatic activity may have been due to altered regulation of the ppiA-blaMab operon by the mutated HrpA alone or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating M. abscessus, particularly imipenem-resistant strains.

Evolution towards extremely high β-lactam resistance in Mycobacterium abscessus outbreak strains.

Treatment of Mycobacterium abscessus pulmonary disease requires multiple antibiotics including intravenous β-lactams (e.g., imipenem, meropenem). M. abscessus produces a β-lactamase (BlaMab) that inactivates β-lactam drugs but less efficiently carbapenems. Due to intrinsic and acquired resistance in M. abscessus and poor clinical outcomes, it is critical to understand the development of antibiotic resistance both within the host and in the setting of outbreaks. We compared serial longitudinally collected M. abscessus subsp. massiliense isolates from the index case of a CF center outbreak and four outbreak-related strains. We found strikingly high imipenem resistance in the later patient isolates, including the outbreak strain (MIC >512 μg/ml). The phenomenon was recapitulated upon exposure of intracellular bacteria to imipenem. Addition of the β-lactamase inhibitor avibactam abrogated the resistant phenotype. Imipenem resistance was caused by an increase in β-lactamase activity and increased bla Mab mRNA level. Concurrent increase in transcription of preceding ppiA gene indicated upregulation of the entire operon in the resistant strains. Deletion of the porin mspA coincided with the first increase in MIC (from 8 to 32 μg/ml). A frameshift mutation in msp2 responsible for the rough colony morphology, and a SNP in ATP-dependent helicase hrpA co-occurred with the second increase in MIC (from 32 to 256 μg/ml). Increased BlaMab expression and enzymatic activity may have been due to altered regulation of the ppiA-bla Mab operon by the mutated HrpA alone, or in combination with other genes described above. This work supports using carbapenem/β-lactamase inhibitor combinations for treating M. abscessus, particularly imipenem resistant strains.

Photoactivatable Glycolipid Probes for Identifying Mycolate-Protein Interactions in Live Mycobacteria.

Mycobacteria have a distinctive glycolipid-rich outer membrane, the mycomembrane, which is a critical target for tuberculosis drug development. However, proteins that associate with the mycomembrane, or that are involved in its metabolism and host interactions, are not well-characterized. To facilitate the study of mycomembrane-related proteins, we developed photoactivatable trehalose monomycolate analogues that metabolically incorporate into the mycomembrane in live mycobacteria, enabling in vivo photo-cross-linking and click-chemistry-mediated analysis of mycolate-interacting proteins. When deployed in Mycobacterium smegmatis with quantitative proteomics, this strategy enriched over 100 proteins, including the mycomembrane porin (MspA), several proteins with known mycomembrane synthesis or remodeling functions (CmrA, MmpL3, Ag85, Tdmh), and numerous candidate mycolate-interacting proteins. Our approach is highly versatile, as it (i) enlists click chemistry for flexible protein functionalization; (ii) in principle can be applied to any mycobacterial species to identify endogenous bacterial proteins or host proteins that interact with mycolates; and (iii) can potentially be expanded to investigate protein interactions with other mycobacterial lipids. This tool is expected to help elucidate fundamental physiological and pathological processes related to the mycomembrane and may reveal novel diagnostic and therapeutic targets.

α-Synuclein Interaction with and Translocation by the MspA Porin

We have recently reported (Hoogerheide et al., Biophysical Journal 2018, 114, 772-776) the use of “selectivity tags” to track the translocation of a non-uniformly charged polypeptide through the mitochondrial VDAC nanopore on the single molecule level. We now demonstrate the generality of the selectivity tag technique with another widely used, robust β-barrel nanopore, the mycobacterial porin MspA. First, we show that α-Synuclein (α-Syn), a naturally occurring, intrinsically disordered polypeptide associated with Parkinson's disease pathology and mitochondrial bioenergetics, can interact with the bilayer-reconstituted D90N/D91N/D93N/D118R/E139K/D134R MspA pore (“MspA”). α-Syn causes a discrete ∼80% blockage of the MspA current, with the blockage on-rate rising exponentially with the applied field. The residence time, on the other hand, displays a crossover behavior, indicating that at voltages >50 mV the entire α-Syn polymer passes through the pore. Second, threading α-Syn, which has a “diblock copolymer” structure with regions of opposite uniform charges at its ends, through MspA strongly modulates the electrostatics and ionic selectivity of MspA's constriction, with an increased sensitivity compared to the original findings reported for the VDAC nanopore (Hoogerheide et al., 2018). Under conditions of 0.2/1 M KCl gradient across the membrane, the α-Syn interactions with MspA produce readily detectable current sub-levels, which correspond to differently charged regions of α-Syn polypeptide as it interacts and threads through the pore and allow for direct single-molecule detection of α-Syn translocation success. Third, we employ the selectivity tag technique to observe the effect of various co-solvents (e.g. salt species) on the process of α-Syn-MspA translocation.

High-Resolution Single-Molecule Analysis of UvrD Helicase using Nanopore Tweezers

Single-molecule methods reveal enzyme dynamics that cannot be observed with traditional bulk assays. We analyze UvrD helicase translocation on ssDNA using a new technique called Single-molecule Picometer Resolution Nanopore Tweezers (SPRNT), which has unprecedented spatiotemporal resolution. In SPRNT, a membrane porin MspA in a phospholipid bilayer forms an electrical connection between two salt solutions. A voltage applied across the pore causes ion current to flow through and draws negatively charged DNA bound to UvrD into the pore. UvrD is too large to fit through MspA, and comes to rest on the rim, arresting the DNA's motion. Different DNA nucleotides in the pore modulate the ion current. As UvrD walks along the DNA, it draws the DNA out of the pore and changes the ion current. Therefore, the durations of ion-current states are kinetic measurements of UvrD. Using SPRNT, we show that UvrD moves in single nucleotide steps along DNA and characterize its ATPase activity.

Expression and Purification of a Novel Mycobacterial Porin MspA Mutant in E. coli

Expression and Purification of a Novel Mycobacterial Porin MspA Mutant in <i>E. coli</i>

Investigating asymmetric salt profiles for nanopore DNA sequencing with biological porin MspA

Nanopore DNA sequencing is a promising single-molecule analysis technology. This technique relies on a DNA motor enzyme to control movement of DNA precisely through a nanopore. Specific experimental buffer conditions are required based on the preferred operating conditions of the DNA motor enzyme. While many DNA motor enzymes typically operate in salt concentrations under 100 mM, salt concentration simultaneously affects signal and noise magnitude as well as DNA capture rate in nanopore sequencing, limiting standard experimental conditions to salt concentrations greater than ~100 mM in order to maintain adequate resolution and experimental throughput. We evaluated the signal contribution from ions on both sides of the membrane (cis and trans) by varying cis and trans [KCl] independently during phi29 DNA Polymerase-controlled translocation of DNA through the biological porin MspA. Our studies reveal that during DNA translocation, the negatively charged DNA increases cation selectivity through MspA with the majority of current produced by the flow of K+ ions from trans to cis. Varying trans [K+] has dramatic effects on the signal magnitude, whereas changing cis [Cl-] produces only small effects. Good signal-to-noise can be maintained with cis [Cl-] as small as 20 mM, if the concentration of KCl on the trans side is kept high. These results demonstrate the potential of using salt-sensitive motor enzymes (helicases, polymerases, recombinases) in nanopore systems and offer a guide for selecting buffer conditions in future experiments to simultaneously optimize signal, throughput, and enzyme activity.

Hetero-oligomeric MspA pores in Mycobacterium smegmatis.

The porin MspA of Mycobacterium smegmatis is a biological nanopore used for DNA sequencing. The octameric MspA pore can be isolated from M. smegmatis in milligram quantities, is extremely stable against denaturation and rapidly inserts into lipid membranes. Here, we show that MspA pores composed of different Msp subunits are formed in M. smegmatis and that hetero-oligomers of different Msp monomers increase the heterogeneity of MspA pores designed for DNA sequencing. To improve the quality of preparations of mutant MspA proteins, all four msp genes were deleted from the M. smegmatis genome after insertion of an inducible porin gene from M. tuberculosis. In the msp quadruple mutant M. smegmatis ML712 no Msp porins were detected and mutant MspA proteins were produced at wild-type levels. Lipid bilayer experiments demonstrated that MspA pores isolated from ML712 formed functional channels and had a narrower conductance distribution than pores purified from M. smegmatis with background msp expression. Thus, the M. smegmatis msp quadruple mutant improves the homogeneity of MspA pores designed for DNA sequencing and might also facilitate the identification and functional characterization of other mycobacterial pore proteins.

Essential Role of the ESX-5 Secretion System in Outer Membrane Permeability of Pathogenic Mycobacteria.

Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identify crucial ESX-5 substrates confirmed that all detectable PE and PPE proteins in the cell surface and cell envelope fractions were routed through ESX-5. Additionally, saturated transposon-directed insertion-site sequencing (TraDIS) was applied to both wild-type M. marinum cells and cells expressing mspA to identify genes that are not essential anymore in the presence of MspA. This analysis confirmed the importance of esx-5, but we could not identify essential ESX-5 substrates, indicating that multiple of these substrates are together responsible for the essentiality. Finally, examination of phenotypes on defined carbon sources revealed that an esx-5 mutant is strongly impaired in the uptake and utilization of hydrophobic carbon sources. Based on these data, we propose a model in which the ESX-5 system is responsible for the transport of cell envelope proteins that are required for nutrient uptake. These proteins might in this way compensate for the lack of MspA-like porins in slow-growing mycobacteria.

The role of porins in copper acquisition by mycobacteria

Aims and objectivesCopper poisoning in macrophages plays an important role in immunity against invading pathogens. Many bacteria, including Mycobacterium tuberculosis (Mtb), have evolved mechanisms to combat copper-derived innate immune responses. Copper homeostasis in Mtb consists of several components, including a multi-copper oxidase, copper efflux pump, cytoplasmic metallothionein and copper-sensing transcriptional regulators. However, components involved in copper uptake are unknown, which prompted this study to investigate the possible role of porins in copper uptake in mycobacteria. MethodsMycobacterium smegmatis porin mutants were created and tested for their ability to grow under copper-reduced or copper-rich conditions. The M. smegmatis porin gene mspA was expressed in Mtb, and its copper susceptibility profile was investigated in the presence of different copper concentrations. The expression level of a copper detoxifying protein, mycobacterial multi-copper oxidase (MmcO), was monitored by western blot to assess intracellular copper content. ResultsDeletion of porin genes from M. smegmatis caused a severe growth defect on trace copper medium. Copper supplementation alleviated this phenotype. The inability to acquire copper in sufficient amounts due to lack of porins can explain this phenomenon. Moreover, porin mutants showed elevated tolerance to copper at concentrations that were toxic for wild-type strains, indicating that the lack of porins protects these strains from copper poisoning. On the other hand, heterologous expression of mspA in Mtb significantly impaired growth at 2.5μM copper and eliminated growth at 15μM, while wild-type Mtb eventually reached its normal cell density at this copper concentration. Consistent with a role of porins in copper uptake, expression levels of MmcO in Mtb expressing the M. smegmatis porin mspA was above wild-type levels, indicating that cytoplasmic copper-sensing transcriptional regulators respond by derepressing the expression of copper resistance genes. Moreover, the polyamine spermine, a known inhibitor of porin activity in gram-negative bacteria, increased the tolerance of wild-type Mtb for copper suggesting that endogenous outer membrane proteins with channel-forming activity exist and contribute to copper acquisition and toxicity in Mtb. ConclusionsIt was concluded from these results that porins are involved in copper uptake in mycobacteria. Moreover, the outer membrane of Mtb was found to be an important barrier against copper intoxication so that permeabilization of this barrier (e.g., by porins) renders Mtb extremely vulnerable to copper. Consequently, copper homeostasis of Mtb provides a promising drug target for the development of a new class of anti-tuberculosis compounds that can induce a copper hypersensitivity phenotype in Mtb.

Parallel Reconstitution of Bacterial Toxins, Porins and Ion Channels into Suspended Lipid Membrane Microarrays for High-Throughput Electrophysiology

High-throughput electrophysiological recordings in artificial membranes have a great potential for pharmaceutical screening and nanopore analytics. In addition to parallelization and automation of lipid bilayers formation, different protein reconstitution protocols are required depending on the concrete application.We report on functional reconstitutions of three classes of pore-forming proteins into suspended lipid bilayers on Micro Electrode Cavity Array (MECA). Three reconstitution strategies have been employed and optimized depending on the protein structure: self-insertion for pore-forming toxins, transfer from detergent micelles for porins and vesicle fusion for ion channels.By optimizing protein concentration and facilitating spontaneous insertion with short voltage pulses just below the electroporation threshold, a controlled insertion of alpha-hemolysin was achieved. Exactly one pore per bilayer was inserted in over 50% of bilayers in the array, which is substantially higher than the 37% predicted by the Poisson distribution.We investigated and optimized insertion of outer membrane porins OmpF, MspA and OccK1 into bilayer arrays via detergent dilution. Best strategy was to use mild nonionic detergents and sequentially dilute protein stock solution to minimize influence of the detergent on the membrane. Protein injection in the immediate vicinity of a bilayer array and thorough mixing of the solution were decisive factors for homogeneous distribution of functional porins.Reconstitution via detergent micelles was found to be problematic for ion channels, but proteovesicle fusion was successful. The tetrameric potassium channel KcsA was expressed in vitro with cotranslational insertion into lipid vesicles or nanodiscs. Transfer from nanodiscs into suspended bilayers was shown to be inefficient. Additionally, the presence of nanodiscs substantially destabilized the membranes. For proteovesicles best fusion rates were achieved upon addition of negatively charged phospholipids followed by extrusion of the recovered proteovesicles through a polycarbonate filter.

DNA Capture and Translocation through Nanoscale Pores—a Fine Balance of Electrophoresis and Electroosmosis

DNA Capture and Translocation through Nanoscale Pores—a Fine Balance of Electrophoresis and Electroosmosis

A Hybrid Soft Solar Cell Based on the Mycobacterial Porin MspA Linked to a Sensitizer–Viologen Diad

A prototype of a nano solar cell containing the mycobacterial channel protein MspA has been successfully designed. MspA, an octameric transmembrane channel protein from Mycobacterium smegmatis, is one of the most stable proteins known to date. Eight Ruthenium(II) aminophenanthroline-viologen maleimide Diads (Ru-Diads) have been successfully bound to the MspA mutant MspAA96C via cysteine-maleimide bonds. MspA is known to form double layers in which it acts as nanoscopic surfactant. The nanostructured layer that is formed by (Ru-Diad)8MspA at the TiO2 electrode is photochemically active. The resulting "protein nano solar cell" features an incident photon conversion efficiency of 1% at 400 nm. This can be regarded as a proof-of-principle that stable proteins can be successfully integrated into the design of solar cells.

Nanoscopic surfactant behavior of the porin MspA in aqueous media

The mycobacterial porin MspA is one of the most stable channel proteins known to date. MspA forms vesicles at low concentrations in aqueous buffers. Evidence from dynamic light scattering, transmission electron microscopy and zeta-potential measurements by electrophoretic light scattering indicate that MspA behaves like a nanoscale surfactant. The extreme thermostability of MspA allows these investigations to be carried out at temperatures as high as 343 K, at which most other proteins would quickly denature. The principles of vesicle formation of MspA as a function of temperature and the underlying thermodynamic factors are discussed here. The results obtained provide crucial evidence in support of the hypothesis that, during vesicle formation, nanoscopic surfactant molecules, such as MspA, deviate from the principles underlined in classical surface chemistry.

Detection and Mapping of 5-Methylcytosine and 5-Hydroxymethylcytosine in Short Strands of ssDNA using Nanopore Sequencing with MspA

Epigenetic modifications of cytosines, such as 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in CpG sites of DNA, are both, inheritable and influenced by the environment. These nucleotide modifications have been shown to be important in gene regulation, cell programming, and carcinogenesis. It is therefore imperative that next-generation sequencing techniques are able to detect epigenetic modifications. Conventional methods for detection of 5-mCpG and 5-hmCpG, require chemical conversion of 5-mC to uracil or are limited to bulk analysis of relative ratios of C to 5-mC to 5-hmC. Here we demonstrate a technique for mapping individual 5-mCpG and 5-hmCpG sites within single molecules of ssDNA. We used nanopore sequencing whereby the phi29 DNA polymerase draws ssDNA through the porin MspA. An ion current passing through the pore directly detects and maps the location of such modifications in single molecules. We will present data on specific detection of both 5-mCpG and 5-hmCpG sites based on comparisons with unmodified sequences.

Biological Nanopore MspA for DNA Sequencing

Nanopore sequencing is a single molecule technique that has the potential to provide fast and low-cost DNA sequencing. In nanopore sequencing, an ionic current passing through a small pore would directly map the sequence as single stranded DNA is driven through the constriction. The mutant pore protein, MspA, derived from Mycobacterium smegmatis forms a short and narrow channel ideal for nanopore sequencing. Here we present recent advances in nanopore sequencing using the biological porin MspA. Homopolymers of adenine, cytosine, thymine, and guanine each generate unique current signatures when held in MspA. Additionally, we show that individual nucleotides passing through MspA modulate the ionic current in a predictable manner providing sequence information. MspA has a high signal-to-noise ratio and the single nucleotide sensitivity desired for nanopore sequencing. These results indicate that MspA is an ideal candidate for nanopore DNA sequencing.

Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments

Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface-accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low-pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice.

Ethidium bromide transport across Mycobacterium smegmatiscell-wall: correlation with antibiotic resistance

BackgroundActive efflux systems and reduced cell-wall permeability are considered to be the main causes of mycobacterial intrinsic resistance to many antimicrobials. In this study, we have compared the Mycobacterium smegmatis wild-type strain mc2155 with knockout mutants for porins MspA (the main porin of M. smegmatis) and MspC, the efflux pump LfrA (the main efflux pump system of M. smegmatis) and its repressor LfrR for their ability to transport ethidium bromide (EtBr) on a real-time basis. This information was then correlated with minimum inhibitory concentrations (MICs) of several antibiotics in the presence or absence of the efflux inhibitors chlorpromazine, thioridazine and verapamil.ResultsIn the absence of porins MspA and MspC, accumulation of ethidium bromide decreased and the cells became more resistant to several antibiotics, whereas the knockout mutant for the LfrA pump showed increased accumulation of EtBr and increased susceptibility to EtBr, rifampicin, ethambutol and ciprofloxacin. Moreover, the efflux inhibitors caused a reduction of the MICs of streptomycin, rifampicin, amikacin, ciprofloxacin, clarithromycin and erythromycin in most of the strains tested.ConclusionsThe methodology used in this study demonstrated that porin MspA plays an important role in the influx of quaternary ammonium compounds and antibiotics and that efflux via the LfrA pump is involved in low-level resistance to several antimicrobial drugs in M. smegmatis. The results obtained with this non-pathogenic mycobacterium will be used in future studies as a model for the evaluation of the activity of the same efflux inhibitors on the susceptibility of multidrug resistant strains of Mycobacterium tuberculosis to isoniazid and rifampicin.

Determining the Resolution of Biological Porin Mspa for Nanopore Sequencing

Nanopore sequencing has the potential to become a fast and low-cost DNA sequencing platform. The ion current passing through a small pore identifies the sequence of single stranded DNA (ssDNA) electrophorically driven through the constriction. A mutant pore protein, MspA, derived from Mycobacterium smegmatis forms a short and narrow channel ideal for resolving single nucleotides. To study the resolution, we immobilize ssDNA within the pore using a biotin-NeutrAvidin complex. Each base, adenine, cytosine, thymine, and guanine, produces a distinct current signature and methylated cytosine is distinguishable from unmethylated cytosine. Single heteronucleotide substitutions within homopolymer ssDNA are used to characterize the resolution of the pore. Using single nucleotide polymorphisms, we show that single nucleotides within heteromeric ssDNA can be identified when the substitution is held in MspA's constriction. Our results indicate that MspA has high signal-to-noise and single nucleotide sensitivity for nanopore sequencing.

Single Nucleotide Discrimination in Single Stranded DNA Immobilized within Biological Nanopore MspA

Biological nanopores are currently being investigated as a fast, low cost DNA sequencing platform. Single stranded DNA (ssDNA) is electrophoretically driven through a protein pore as the ionic current through the constriction is measured. The porin MspA of Mycobacterium smegmatis was mutated to produce a channel highly suitable for nanopore DNA sequencing. To study the resolution of the mutated porin MspA, we immobilize ssDNA within the pore using a streptavidin ‘anchor’. Each base, adenine, cytosine, thymine, and guanine, produces a distinct current signature when it is held within the nanopore.