@article{BenzJonesYounasetal.2015, author = {Benz, Roland and Jones, Michael D. and Younas, Farhan and Maier, Elke and Modi, Niraj and Mentele, Reinhard and Lottspeich, Friedrich and Kleinekath{\"o}fer, Ulrich and Smit, John}, title = {OmpW of Caulobacter crescentus functions as an outer membrane channel for cations}, series = {PLoS ONE}, volume = {10}, journal = {PLoS ONE}, number = {11}, doi = {10.1371/journal.pone.0143557}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-145114}, pages = {e0143557}, year = {2015}, abstract = {Caulobacter crescentus is an oligotrophic bacterium that lives in dilute organic environments such as soil and freshwater. This bacterium represents an interesting model for cellular differentiation and regulation because daughter cells after division have different forms: one is motile while the other is non-motile and can adhere to surfaces. Interestingly, the known genome of C. crescentus does not contain genes predicted to code for outer membrane porins of the OmpF/C general diffusion type present in enteric bacteria or those coding for specific porins selective for classes of substrates. Instead, genes coding for 67 TonB-dependent outer membrane receptors have been identified, suggesting that active transport of specific nutrients may be the norm. Here, we report that high channel-forming activity was observed with crude outer membrane extracts of C. crescentus in lipid bilayer experiments, indicating that the outer membrane of C. crescentus contained an ion-permeable channel with a single-channel conductance of about 120 pS in 1M KCl. The channel-forming protein with an apparent molecular mass of about 20 kDa was purified to homogeneity. Partial protein sequencing of the protein indicated it was a member of the OmpW family of outer membrane proteins from Gram-negative bacteria. This channel was not observed in reconstitution experiments with crude outer membrane extracts of an OmpW deficient C. crescentus mutant. Biophysical analysis of the C. crescentus OmpW suggested that it has features that are special for general diffusion porins of Gram-negative outer membranes because it was not a wide aqueous channel. Furthermore, OmpW of C. crescentus seems to be different to known OmpW porins and has a preference for ions, in particular cations. A putative model for OmpW of C. crescentus was built on the basis of the known 3D-structures of OmpW of Escherichia coli and OprG of Pseudomonas aeruginosa using homology modeling. A comparison of the two known structures with the model of OmpW of C. crescentus suggested that it has a more hydrophilic interior and possibly a larger diameter.}, language = {en} } @article{KronhardtBeitzingerBarthetal.2016, author = {Kronhardt, Angelika and Beitzinger, Christoph and Barth, Holger and Benz, Roland}, title = {Chloroquine Analog Interaction with C2-and Iota-Toxin in Vitro and in Living Cells}, series = {Toxins}, volume = {8}, journal = {Toxins}, number = {8}, doi = {10.3390/toxins8080237}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-168286}, pages = {237}, year = {2016}, abstract = {C2-toxin from Clostridium botulinum and Iota-toxin from Clostridium perfringens belong both to the binary A-B-type of toxins consisting of two separately secreted components, an enzymatic subunit A and a binding component B that facilitates the entry of the corresponding enzymatic subunit into the target cells. The enzymatic subunits are in both cases actin ADP-ribosyltransferases that modify R177 of globular actin finally leading to cell death. Following their binding to host cells' receptors and internalization, the two binding components form heptameric channels in endosomal membranes which mediate the translocation of the enzymatic components Iota a and C2I from endosomes into the cytosol of the target cells. The binding components form ion-permeable channels in artificial and biological membranes. Chloroquine and related 4-aminoquinolines were able to block channel formation in vitro and intoxication of living cells. In this study, we extended our previous work to the use of different chloroquine analogs and demonstrate that positively charged aminoquinolinium salts are able to block channels formed in lipid bilayer membranes by the binding components of C2- and Iota-toxin. Similarly, these molecules protect cultured mammalian cells from intoxication with C2- and Iota-toxin. The aminoquinolinium salts did presumably not interfere with actin ADP-ribosylation or receptor binding but blocked the pores formed by C2IIa and Iota b in living cells and in vitro. The blocking efficiency of pores formed by Iota b and C2IIa by the chloroquine analogs showed interesting differences indicating structural variations between the types of protein-conducting nanochannels formed by Iota b and C2IIa.}, language = {en} } @article{AbdaliYounasMafakherietal.2018, author = {Abdali, Narges and Younas, Farhan and Mafakheri, Samaneh and Pothula, Karunakar R. and Kleinekath{\"o}fer, Ulrich and Tauch, Andreas and Benz, Roland}, title = {Identification and characterization of smallest pore-forming protein in the cell wall of pathogenic Corynebacterium urealyticum DSM 7109}, series = {BMC Biochemistry}, volume = {19}, journal = {BMC Biochemistry}, doi = {10.1186/s12858-018-0093-9}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226959}, year = {2018}, abstract = {Background: Corynebacterium urealyticum, a pathogenic, multidrug resistant member of the mycolata, is known as causative agent of urinary tract infections although it is a bacterium of the skin flora. This pathogenic bacterium shares with the mycolata the property of having an unusual cell envelope composition and architecture, typical for the genus Corynebacterium. The cell wall of members of the mycolata contains channel-forming proteins for the uptake of solutes. Results: In this study, we provide novel information on the identification and characterization of a pore-forming protein in the cell wall of C. urealyticum DSM 7109. Detergent extracts of whole C. urealyticum cultures formed in lipid bilayer membranes slightly cation-selective pores with a single-channel conductance of 1.75 nS in 1 M KCl. Experiments with different salts and non-electrolytes suggested that the cell wall pore of C. urealyticum is wide and water-filled and has a diameter of about 1.8 nm. Molecular modelling and dynamics has been performed to obtain a model of the pore. For the search of the gene coding for the cell wall pore of C. urealyticum we looked in the known genome of C. urealyticum for a similar chromosomal localization of the porin gene to known porH and porA genes of other Corynebacterium strains. Three genes are located between the genes coding for GroEL2 and polyphosphate kinase (PKK2). Two of the genes (cur_1714 and cur_1715) were expressed in different constructs in C. glutamicum Delta porA Delta porH and in porin-deficient BL21 DE3 Omp8 E. coli strains. The results suggested that the gene cur_1714 codes alone for the cell wall channel. The cell wall porin of C. urealyticum termed PorACur was purified to homogeneity using different biochemical methods and had an apparent molecular mass of about 4 kDa on tricine-containing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Conclusions: Biophysical characterization of the purified protein (PorACur) suggested indeed that cur_1714 is the gene coding for the pore-forming protein in C. urealyticum because the protein formed in lipid bilayer experiments the same pores as the detergent extract of whole cells. The study is the first report of a cell wall channel in the pathogenic C. urealyticum.}, language = {en} } @article{KnappBenz2020, author = {Knapp, Oliver and Benz, Roland}, title = {Membrane activity and channel formation of the adenylate cyclase toxin (CyaA) of Bordetella pertussis in lipid bilayer membranes}, series = {Toxins}, volume = {12}, journal = {Toxins}, number = {3}, issn = {2072-6651}, doi = {10.3390/toxins12030169}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-203362}, year = {2020}, abstract = {The Gram-negative bacterium Bordetella pertussis is the cause of whooping cough. One of its pathogenicity factors is the adenylate cyclase toxin (CyaA) secreted by a Type I export system. The 1706 amino acid long CyaA (177 kDa) belongs to the continuously increasing family of repeat in toxin (RTX) toxins because it contains in its C-terminal half a high number of nine-residue tandem repeats. The protein exhibits cytotoxic and hemolytic activities that target primarily myeloid phagocytic cells expressing the αMβ2 integrin receptor (CD11b/CD18). CyaA represents an exception among RTX cytolysins because the first 400 amino acids from its N-terminal end possess a calmodulin-activated adenylate cyclase (AC) activity. The entry of the AC into target cells is not dependent on the receptor-mediated endocytosis pathway and penetrates directly across the cytoplasmic membrane of a variety of epithelial and immune effector cells. The hemolytic activity of CyaA is rather low, which may have to do with its rather low induced permeability change of target cells and its low conductance in lipid bilayer membranes. CyaA forms highly cation-selective channels in lipid bilayers that show a strong dependence on aqueous pH. The pore-forming activity of CyaA but not its single channel conductance is highly dependent on Ca\(^{2+}\) concentration with a half saturation constant of about 2 to 4 mM.}, language = {en} } @article{Benz2020, author = {Benz, Roland}, title = {RTX-Toxins}, series = {Toxins}, volume = {12}, journal = {Toxins}, number = {6}, issn = {2072-6651}, doi = {10.3390/toxins12060359}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-205860}, year = {2020}, abstract = {No abstract available.}, language = {en} } @article{PiselliBenz2021, author = {Piselli, Claudio and Benz, Roland}, title = {Fosmidomycin transport through the phosphate-specific porins OprO and OprP of Pseudomonas aeruginosa}, series = {Molecular Microbiology}, volume = {116}, journal = {Molecular Microbiology}, number = {1}, doi = {10.1111/mmi.14693}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-238905}, pages = {97 -- 108}, year = {2021}, abstract = {The Gram-negative bacterium Pseudomonas aeruginosa is an opportunistic pathogen, responsible for many hospital-acquired infections. The bacterium is quite resistant toward many antibiotics, in particular because of the fine-tuned permeability of its outer membrane (OM). General diffusion outer membrane pores are quite rare in this organism. Instead, its OM contains many substrate-specific porins. Their expression is varying according to growth conditions and virulence. Phosphate limitations, as well as pathogenicity factors, result in the induction of the two mono- and polyphosphate-specific porins, OprP and OprO, respectively, together with an inner membrane uptake mechanism and a periplasmic binding protein. These outer membrane channels could serve as outer membrane pathways for the uptake of phosphonates. Among them are not only herbicides, but also potent antibiotics, such as fosfomycin and fosmidomycin. In this study, we investigated the interaction between OprP and OprO and fosmidomycin in detail. We could demonstrate that fosmidomycin is able to bind to the phosphate-specific binding site inside the two porins. The inhibition of chloride conductance of OprP and OprO by fosmidomycin is considerably less than that of phosphate or diphosphate, but it can be measured in titration experiments of chloride conductance and also in single-channel experiments. The results suggest that fosmidomycin transport across the OM of P. aeruginosa occurs through OprP and OprO. Our data with the ones already known in the literature show that phosphonic acid-containing antibiotics are in general good candidates to treat the infections of P. aeruginosa at the very beginning through a favorable OM transport system.}, language = {en} }