The Experts below are selected from a list of 5253 Experts worldwide ranked by ideXlab platform
Aleksandr Noy - One of the best experts on this subject based on the ideXlab platform.
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synthesis lipid membrane incorporation and ion permeability testing of carbon nanotube Porins
Nature Protocols, 2016Co-Authors: Ramya Tunuguntla, Aleksandr Noy, Vadim A Frolov, Artur EscaladaAbstract:Incorporation of carbon nanotube Porins (CNTPs) into phospholipid membranes allows for the nanofluidic replication of biological ion channels. This protocol describes the fabrication and testing of CNTPs at both bulk and single-pore levels.
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Ultrafast proton transport in sub-1-nm diameter carbon nanotube Porins
Nature nanotechnology, 2016Co-Authors: Ramya H. Tunuguntla, Frances I. Allen, Kyunghoon Kim, Allison Belliveau, Aleksandr NoyAbstract:Proton transport plays an important role in many biological processes due to the ability of protons to rapidly translocate along chains of hydrogen-bonded water molecules. Molecular dynamics simulations have predicted that confinement in hydrophobic nanochannels should enhance the rate of proton transport. Here, we show that 0.8-nm-diameter carbon nanotube Porins, which promote the formation of one-dimensional water wires, can support proton transport rates exceeding those of bulk water by an order of magnitude. The transport rates in these narrow nanotube pores also exceed those of biological channels and Nafion. With larger 1.5-nm-diameter nanotube Porins, proton transport rates comparable to bulk water are observed. We also show that the proton conductance of these channels can be modulated by the presence of Ca(2+) ions. Our results illustrate the potential of small-diameter carbon nanotube Porins as a proton conductor material and suggest that strong spatial confinement is a key factor in enabling efficient proton transport.
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live cell interactions with biocompatible ultra short carbon nanotube Porins
Biophysical Journal, 2015Co-Authors: Kyunghoon Kim, Jia Geng, Whitney Stannard, Arthur Escalada, Michael P Thelen, Vadim A Frolov, Aleksandr NoyAbstract:Various techniques, such as peptide chemistry, DNA origami, or nanotubes cutting, have been tried to develop synthetic analogues of biological membrane channels for transmembrane transport of ions and molecules, but challenges still remain to achieve the desired affinity, transport properties and biocompatibility for delivery applications. Here we report the development of CNT Porins, an ultra-short biocompatible carbon nanotube with the dimension of 5-15 nm in length and 1.5 nm in diameters and with structure and function resembling membrane channel. We explored concentration-dependent interaction of the CNT Porins with CHO cells using microscopy and OmniLog phenotype microarray system. Interestingly, the CNT Porins didn’t inhibit the cell viability at low concentration over a 72 hr co-incubation period. Single channel recording experiments also showed well-defined channels formation on the cell membrane. Those results indicated those artificial transmembrane channels could be established as a promising biomimetic platform for developing cell interfaces, studying transport in biological channels, and creating stochastic sensors. Their inherent robustness towards biological and chemical challenges and exceptional biocompatibility should prove valuable for bionanofluidic and cellular interface applications.
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osmotically driven transport in carbon nanotube Porins
Nano Letters, 2014Co-Authors: Ramya H. Tunuguntla, Kyunghoon Kim, Aleksandr Noy, Jia Geng, Luis R Comolli, Costas P Grigoropoulos, Caroline M AjofranklinAbstract:We report the measurements of transport of ions and uncharged species through carbon nanotube (CNT) Porins—short segments of CNTs inserted into a lipid bilayer membrane. Rejection characteristics of the CNT Porins are governed by size exclusion for the uncharged species. In contrast, rejection of ionic species is governed by the electrostatic repulsion and Donnan membrane equilibrium. Permeability of monovalent cations follows the general trend in the hydrated ion size, except in the case of Cs+ ions.
Jeanmarie Pages - One of the best experts on this subject based on the ideXlab platform.
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Outer Membrane Porins.
Sub-cellular biochemistry, 2019Co-Authors: Muriel Masi, Mathias Winterhalter, Jeanmarie PagesAbstract:The transport of small molecules across membranes is essential for the import of nutrients and other energy sources into the cell and, for the export of waste and other potentially harmful byproducts out of the cell. While hydrophobic molecules are permeable to membranes, ions and other small polar molecules require transport via specialized membrane transport proteins . The two major classes of membrane transport proteins are transporters and channels. With our focus here on Porins-major class of non-specific diffusion channel proteins , we will highlight some recent structural biology reports and functional assays that have substantially contributed to our understanding of the mechanism that mediates uptake of small molecules, including antibiotics, across the outer membrane of Enterobacteriaceae . We will also review advances in the regulation of porin expression and porin biogenesis and discuss these pathways as new therapeutic targets.
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getting drugs into gram negative bacteria rational rules for permeation through general Porins
ACS Infectious Diseases, 2018Co-Authors: Silvia Acostagutierrez, Mathias Winterhalter, M. Pathania, Igor Bodrenko, Luana Ferrara, Muriel Masi, Jiajun Wang, Michael Zahn, Robert A Stavenger, Jeanmarie PagesAbstract:Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by Porins. We have determined the X-ray crystal structures of the principal general Porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the Porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through Porins and help to guide the optimization of antibiotics with poor permeation.
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structure function and regulation of outer membrane proteins involved in drug transport in enterobactericeae the ompf c tolc case
The Open Microbiology Journal, 2013Co-Authors: Muriel Masi, Jeanmarie PagesAbstract:Antibiotic translocation across membranes of Gram-negative bacteria is a key step for the activity on their spe- cific intracellular targets. Resistant bacteria control their membrane permeability as a first line of defense to protect them- selves against external toxic compounds such as antibiotics and biocides. On one hand, resistance to small hydrophilic an- tibiotics such as s-lactams and fluoroquinolones frequently results from the « closing » of their way in: the general outer membrane Porins. On the other hand, an effective way out for a wide range of antibiotics is provided by TolC-like pro- teins, which are outer membrane components of multidrug efflux pumps. Accordingly, altered membrane permeability, including porin modifications and/or efflux pumps' overexpression, is always associated to multidrug resistance (MDR) in a number of clinical isolates. Several recent studies have highlighted our current understanding of Porins/TolC structures and functions in Enterobacte- riaceae. Here, we review the transport of antibiotics through the OmpF/C general Porins and the TolC-like channels with regards to recent data on their structure, function, assembly, regulation and contribution to bacterial resistance. Because MDR strains have evolved global strategies to identify and fight our antibiotic arsenal, it is important to con- stantly update our global knowledge on antibiotic transport.
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the porin and the permeating antibiotic a selective diffusion barrier in gram negative bacteria
Nature Reviews Microbiology, 2008Co-Authors: Jeanmarie Pages, Chloe E James, Mathias WinterhalterAbstract:Gram-negative bacteria are responsible for a large proportion of antibiotic-resistant bacterial diseases. These bacteria have a complex cell envelope that comprises an outer membrane and an inner membrane that delimit the periplasm. The outer membrane contains various protein channels, called Porins, which are involved in the influx of various compounds, including several classes of antibiotics. Bacterial adaptation to reduce influx through Porins is an increasing problem worldwide that contributes, together with efflux systems, to the emergence and dissemination of antibiotic resistance. An exciting challenge is to decipher the genetic and molecular basis of membrane impermeability as a bacterial resistance mechanism. This Review outlines the bacterial response towards antibiotic stress on altered membrane permeability and discusses recent advances in molecular approaches that are improving our knowledge of the physico-chemical parameters that govern the translocation of antibiotics through porin channels.
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Multiple facets of bacterial Porins
FEMS microbiology letters, 2001Co-Authors: Wafa Achouak, Thierry Heulin, Jeanmarie PagesAbstract:Porins form channels allowing the transport of molecules across lipid bilayer membranes. Their structure, location and large number on the bacterial surface lend them multiple functions. Porin loops are potential targets for adhesion to other cells and binding of bactericidal compounds to the surface of Gram-negative bacteria. Variation of the loop structure as a mechanism to escape immune pressure, or modulation of the porin expression in response to the presence of antibiotics, are survival strategies developed by some pathogenic bacteria. Porins may play a significant role as pathogenesis effectors.
Lee M Wetzler - One of the best experts on this subject based on the ideXlab platform.
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innate immune function of the neisserial Porins and the relationship to vaccine adjuvant activity
Future Microbiology, 2010Co-Authors: Lee M WetzlerAbstract:Neisseria meningitidis is a Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial Porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells. Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of antigen-presenting cells, an effect that is dependent on Toll-like receptor (TLR)2 and MyD88, through a cascade of signal transduction events mediated by direct binding of the porin to the TLR2-TLR1 heterodimer. This article summarizes work carried out investigating the mechanisms of the immune stimulating capacity of the neisserial Porins (specifically meningococcal PorB), emphasizing cellular events involved in antigen-presenting cell activation and induction of expression of cell surface molecules involved in the immune response.
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the porb porin from commensal neisseria lactamica induces th1 and th2 immune responses to ovalbumin in mice and is a potential immune adjuvant
Vaccine, 2008Co-Authors: Xiuping Liu, Lee M Wetzler, Paola MassariAbstract:Porins from pathogenic Neisseriae are among several bacterial products with immune adjuvant activity. Neisseria meningitidis (Nme) PorB, has been shown to induce immune cells activation in a TLR2-dependent manner and acts as a vaccine immune adjuvant. The PorB porin from Neisseria lactamica (Nlac), a common nasopharyngeal commensal, shares significant structural and functional similarities with Nme PorB. In this work we ask whether the immune adjuvant ability of Porins from pathogenic Neisserial strains is a characteristic shared with Porins from non-pathogenic Neisserial species or whether it is unique for bacterial products derived from microorganisms capable of inducing inflammation and disease. We evaluated the potential immune adjuvant effect of Nlac PorB in mice using ovalbumin (OVA) as a prototype antigen. Immunization with Nlac PorB/OVA induced high OVA-specific IgG and IgM titers compared to OVA alone, similar to other adjuvants such as Nme PorB and alum. High titers of IgG1 and IgG2b were detected as well as production of IL-4, IL-10, IL-12 and INF-γ in response to Nlac PorB, consistent with induction of both a Th1-type and a Th2-type immune response. OVA-specific proliferation was also determined in splenocytes from Nlac PorB/OVA-immunized mice. In addition, B cell activation in vitro and cytokine production in response to Nlac PorB was found to be mediated by TLR2, in a similar manner to Nme PorB.
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The role of Porins in neisserial pathogenesis and immunity.
Trends in microbiology, 2003Co-Authors: Paola Massari, Sanjay Ram, Heather Macleod, Lee M WetzlerAbstract:Neisseria meningitidis and Neisseria gonorrhoeae are Gram-negative pathogenic bacteria responsible for bacterial meningitis and septicemia, and the sexually transmitted disease gonorrhea, respectively. Porins are the most represented outer membrane proteins in the pathogenic Neisseria species, functioning as pores for the exchange of ions, and are characterized by a trimeric beta-barrel structure. Neisserial Porins have been shown to act as adjuvants in the immune response via activation of B cells and other antigen-presenting cells (APCs). Their effect on the immune response is mediated by upregulation of the costimulatory molecule B7-2 (CD86) on the surface of APCs, an effect that is Toll-like receptor 2- and MyD88-dependent. The effect of neisserial Porins on the immune system also involves interaction with components of the complement cascade. Furthermore, neisserial Porins co-localize with mitochondria of target cells, where they appear to modulate apoptosis.
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cutting edge immune stimulation by neisserial Porins is toll like receptor 2 and myd88 dependent
Journal of Immunology, 2002Co-Authors: Paola Massari, Philipp Henneke, Eicke Latz, Douglas T Golenbock, Lee M WetzlerAbstract:The immunopotentiating activity of neisserial Porins, the major outer membrane protein of the pathogenic Neisseria, is mediated by its ability to stimulate B cells and up-regulate the surface expression of B7-2. This ability is dependent on MyD88 and Toll-like receptor (TLR)2 expression, as demonstrated by a lack of a response by B cells from MyD88 or TLR2 knockout mice to the Porins. Using previously described TLR2-dependent reporter constructs, these results were confirmed and were shown to be due to induction of NF-κB nuclear translocation. This is the first demonstration of known vaccine adjuvant to stimulate immune cells via TLR2.
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neisseria meningitidis porin porb interacts with mitochondria and protects cells from apoptosis
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Paola Massari, Lee M WetzlerAbstract:Abstract Neisserial Porins are strong immune adjuvants and B cell activators. The effect of neisserial porin PorB on activation-induced cell death was investigated, as a potential additional mechanism of the porin's immunopotentiating ability. Neisserial Porins interact with target cells to localize intracellularly in the mitochondrial compartment without negatively affecting cellular survival. Pretreatment with Neisseria meningitidis PorB porin decreased or abrogated the mitochondrial damage induced by apoptotic stimuli. In addition, end stage determinants of apoptosis, including DNA breakdown, were diminished by PorB. Immunoprecipitation experiments revealed that PorB interacts with the mitochondrial porin VDAC (voltage-dependent anion channel). The mechanism of the antiapoptotic effect of neisserial Porins could be explained by the protein–protein interaction of PorB with VDAC, similar to the interaction of VDAC with antiapoptotic Bcl-2 proteins, resulting in an enhancement of cell survival and continued activation of B cells.
Mathias Winterhalter - One of the best experts on this subject based on the ideXlab platform.
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kanamycin uptake into escherichia coli is facilitated by ompf and ompc porin channels located in the outer membrane
ACS Infectious Diseases, 2020Co-Authors: Jayesh Arun Bafna, Mathias Winterhalter, Eulalia Sansserramitjana, Silvia Acostagutierrez, Igor Bodrenko, Daniel Horompoli, Anne Berscheid, Heike Brotzoesterhelt, Matteo CeccarelliAbstract:Despite decades of therapeutic application of aminoglycosides, it is still a matter of debate if Porins contribute to the translocation of the antibiotics across the bacterial outer membrane. Here,...
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structural insights into the main s layer unit of deinococcus radiodurans reveal a massive protein complex with porin like features
Journal of Biological Chemistry, 2020Co-Authors: Domenica Farci, Mathias Winterhalter, Jayesh Arun Bafna, Igor Bodrenko, Matteo Ceccarelli, Mehmet Alphan Aksoyoglu, Stefano Francesco Farci, Joanna Kirkpatrick, Sami KereicheAbstract:In the extremophile bacterium Deinococcus radiodurans, the outermost surface layer is tightly connected with the rest of the cell wall. This integrated organization provides a compact structure that shields the bacterium against environmental stresses. The fundamental unit of this surface layer (S-layer) is the S-layer deinoxanthin-binding complex (SDBC), which binds the carotenoid deinoxanthin and provides both, thermostability and UV radiation resistance. However, the structural organization of the SDBC awaits elucidation. Here, we report the isolation of the SDBC with a gentle procedure consisting of lysozyme treatment and solubilization with the nonionic detergent n-dodecyl-β-d-maltoside, which preserved both hydrophilic and hydrophobic components of the SDBC and allows the retention of several minor subunits. As observed by low-resolution single-particle analysis, we show that the complex possesses a porin-like structural organization, but is larger than other known Porins. We also noted that the main SDBC component, the protein DR_2577, shares regions of similarity with known Porins. Moreover, results from electrophysiological assays with membrane-reconstituted SDBC disclosed that it is a nonselective channel that has some peculiar gating properties, but also exhibits behavior typically observed in pore-forming proteins, such as Porins and ionic transporters. The functional properties of this system and its porin-like organization provide information critical for understanding ion permeability through the outer cell surface of S-layer-carrying bacterial species.
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Outer Membrane Porins.
Sub-cellular biochemistry, 2019Co-Authors: Muriel Masi, Mathias Winterhalter, Jeanmarie PagesAbstract:The transport of small molecules across membranes is essential for the import of nutrients and other energy sources into the cell and, for the export of waste and other potentially harmful byproducts out of the cell. While hydrophobic molecules are permeable to membranes, ions and other small polar molecules require transport via specialized membrane transport proteins . The two major classes of membrane transport proteins are transporters and channels. With our focus here on Porins-major class of non-specific diffusion channel proteins , we will highlight some recent structural biology reports and functional assays that have substantially contributed to our understanding of the mechanism that mediates uptake of small molecules, including antibiotics, across the outer membrane of Enterobacteriaceae . We will also review advances in the regulation of porin expression and porin biogenesis and discuss these pathways as new therapeutic targets.
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getting drugs into gram negative bacteria rational rules for permeation through general Porins
ACS Infectious Diseases, 2018Co-Authors: Silvia Acostagutierrez, Mathias Winterhalter, M. Pathania, Igor Bodrenko, Luana Ferrara, Muriel Masi, Jiajun Wang, Michael Zahn, Robert A Stavenger, Jeanmarie PagesAbstract:Small, hydrophilic molecules, including most important antibiotics in clinical use, cross the Gram-negative outer membrane through the water-filled channels provided by Porins. We have determined the X-ray crystal structures of the principal general Porins from three species of Enterobacteriaceae, namely Enterobacter aerogenes, Enterobacter cloacae, and Klebsiella pneumoniae, and determined their antibiotic permeabilities as well as those of the orthologues from Escherichia coli. Starting from the structure of the Porins and molecules, we propose a physical mechanism underlying transport and condense it in a computationally efficient scoring function. The scoring function shows good agreement with in vitro penetration data and will enable the screening of virtual databases to identify molecules with optimal permeability through Porins and help to guide the optimization of antibiotics with poor permeation.
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molecular basis of filtering carbapenems by Porins from β lactam resistant clinical strains of escherichia coli
Journal of Biological Chemistry, 2016Co-Authors: Harsha Bajaj, Matteo Ceccarelli, Mariano Andrea Scorciapino, Lucile Moynie, Malcolm G P Page, James H Naismith, Mathias WinterhalterAbstract:Integral membrane proteins known as Porins are the major pathway by which hydrophilic antibiotics cross the outer membrane of Gram-negative bacteria. Single point mutations in Porins can decrease the permeability of an antibiotic, either by reduction of channel size or modification of electrostatics in the channel, and thereby confer clinical resistance. Here, we investigate four mutant OmpC proteins from four different clinical isolates of Escherichia coli obtained sequentially from a single patient during a course of antimicrobial chemotherapy. OmpC porin from the first isolate (OmpC20) undergoes three consecutive and additive substitutions giving rise to OmpC26, OmpC28, and finally OmpC33. The permeability of two zwitterionic carbapenems, imipenem and meropenem, measured using liposome permeation assays and single channel electrophysiology differs significantly between OmpC20 and OmpC33. Molecular dynamic simulations show that the antibiotics must pass through the constriction zone of Porins with a specific orientation, where the antibiotic dipole is aligned along the electric field inside the porin. We identify that changes in the vector of the electric field in the mutated porin, OmpC33, create an additional barrier by "trapping" the antibiotic in an unfavorable orientation in the constriction zone that suffers steric hindrance for the reorientation needed for its onward translocation. Identification and understanding the underlying molecular details of such a barrier to translocation will aid in the design of new antibiotics with improved permeation properties in Gram-negative bacteria.
Michael Niederweis - One of the best experts on this subject based on the ideXlab platform.
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importance of Porins for biocide efficacy against mycobacterium smegmatis
Applied and Environmental Microbiology, 2011Co-Authors: Elrike Frenzel, Michael Niederweis, Stefan Schmidt, Katrin SteinhauerAbstract:Mycobacteria are among the microorganisms least susceptible to biocides but cause devastating diseases, such as tuberculosis, and increasingly opportunistic infections. The exceptional resistance of mycobacteria to toxic solutes is due to an unusual outer membrane, which acts as an efficient permeability barrier, in synergy with other resistance mechanisms. Porins are channel-forming proteins in the outer membrane of mycobacteria. In this study we used the alamarBlue assay to show that the deletion of Msp Porins in isogenic mutants increased the resistance of Mycobacterium smegmatis to isothiazolinones (methylchloroisothiazolinone [MCI]/methylisothiazolinone [MI] and octylisothiazolinone [2-n-octyl-4-isothiazolin-3-one; OIT]), formaldehyde-releasing biocides {hexahydrotriazine [1,3,5-tris (2-hydroxyethyl)-hexahydrotriazine; HHT] and methylenbisoxazolidine [N,N'-methylene-bis-5-(methyloxazolidine); MBO]}, and the lipophilic biocides polyhexamethylene biguanide and octenidine dihydrochloride 2- to 16-fold. Furthermore, the susceptibility of the porin triple mutant against a complex disinfectant was decreased 8-fold compared to wild-type (wt) M. smegmatis. Efficacy testing in the quantitative suspension test EN 14348 revealed 100-fold improved survival of the porin mutant in the presence of this biocide. These findings underline the importance of Porins for the susceptibility of M. smegmatis to biocides.
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Role of Porins in iron uptake by Mycobacterium smegmatis.
Journal of bacteriology, 2010Co-Authors: Christopher M. Jones, Michael NiederweisAbstract: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.
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role of Porins for uptake of antibiotics by mycobacterium smegmatis
Antimicrobial Agents and Chemotherapy, 2008Co-Authors: Olga Danilchanka, Mikhail Pavlenok, Michael NiederweisAbstract:The outer membrane of mycobacteria presents an effective permeability barrier for many antibiotics. Transport pathways across this membrane are unknown for most drugs. Here, we examined which antibiotics utilize the porin pathway across the outer membrane of the model organism Mycobacterium smegmatis. Deletion of the Porins MspA and MspC drastically increased the resistance of M. smegmatis ML10 to β-lactam antibiotics, while its β-lactamase activity remained unchanged. These results are consistent with the ninefold-reduced outer membrane permeability of the M. smegmatis porin mutants for cephaloridine and strongly indicate that β-lactam antibiotics rely on the porin pathway. The porin mutant ML10 accumulated less chloramphenicol and norfloxacin and was less susceptible to these antibiotics than wild-type M. smegmatis. These results demonstrated that small and hydrophilic antibiotics use the Msp Porins for entering the cell. In contrast to norfloxacin, the hydrophobic moxifloxacin was 32-fold more effective in inhibiting the growth of M. smegmatis, presumably because it was able to diffuse through the lipid membrane. Structural models indicated that erythromycin, kanamycin, and vancomycin are too large to move through the MspA channel. This study presents the first experimental evidence that hydrophilic fluoroquinolones and chloramphenicol diffuse through Porins in mycobacteria. Thus, mutations resulting in less efficient Porins or lower porin expression levels are likely to represent a mechanism for the opportunistic pathogens M. avium, M. chelonae, and M. fortuitum, which have Msp-like Porins, to acquire resistance to fluoroquinolones.
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Porins are required for uptake of phosphates by mycobacterium smegmatis
Journal of Bacteriology, 2007Co-Authors: Frank Wolschendorf, Michael Niederweis, Maysa MahfoudAbstract:Phosphorus is an essential nutrient, but how phosphates cross the mycobacterial cell wall is unknown. Phosphatase activity in whole cells of Mycobacterium smegmatis was significantly lower than that in lysed cells, indicating that access to the substrate was restricted. The loss of the outer membrane (OM) porin MspA also reduced the phosphatase activity in whole cells compared to that in lysed cells. A similar result was obtained for M. smegmatis that overexpressed endogenous alkaline phosphatase, indicating that PhoA is not a surface protein, contrary to a previous report. The uptake of phosphate by a mutant lacking the Porins MspA and MspC was twofold lower than that by wild-type M. smegmatis. Strikingly, the loss of these Porins resulted in a severe growth defect of M. smegmatis on low-phosphate plates. We concluded that the OM of M. smegmatis represents a permeability barrier for phosphates and that Msp Porins are the only OM channels for the diffusion of phosphate in M. smegmatis. However, phosphate diffusion through Msp pores is rather inefficient as shown by the 10-fold lower permeability of M. smegmatis for phosphate compared to that for glucose. This is likely due to the negative charges in the constriction zone of Msp Porins. The phosphatase activity in whole cells of Mycobacterium bovis BCG was significantly less than that in lysed cells, indicating a similar uptake pathway for phosphates in slow-growing mycobacteria. However, Porins that could mediate the diffusion of phosphates across the OM of M. bovis BCG and Mycobacterium tuberculosis are unknown.
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the mspa porin promotes growth and increases antibiotic susceptibility of both mycobacterium bovis bcg and mycobacterium tuberculosis
Microbiology, 2004Co-Authors: Claudia Mailaender, Harald Engelhardt, Norbert Reiling, Stefan H Bossmann, Stefan Ehlers, Michael NiederweisAbstract: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 microm(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 beta-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.