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  • 1
    Online Resource
    Online Resource
    Intracellular Trafficking of AIP56, an NF-κB-Cleaving Toxin from Photobacterium damselae subsp. piscicida
    American Society for Microbiology ; 2014
    In:  Infection and Immunity Vol. 82, No. 12 ( 2014-12), p. 5270-5285
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 82, No. 12 ( 2014-12), p. 5270-5285
    Abstract: AIP56 (apoptosis-inducing protein of 56 kDa) is a metalloprotease AB toxin secreted by Photobacterium damselae subsp. piscicida that acts by cleaving NF-κB. During infection, AIP56 spreads systemically and depletes phagocytes by postapoptotic secondary necrosis, impairing the host phagocytic defense and contributing to the genesis of infection-associated necrotic lesions. Here we show that mouse bone marrow-derived macrophages (mBMDM) intoxicated by AIP56 undergo NF-κB p65 depletion and apoptosis. Similarly to what was reported for sea bass phagocytes, intoxication of mBMDM involves interaction of AIP56 C-terminal region with cell surface components, suggesting the existence of a conserved receptor. Biochemical approaches and confocal microscopy revealed that AIP56 undergoes clathrin-dependent endocytosis, reaches early endosomes, and follows the recycling pathway. Translocation of AIP56 into the cytosol requires endosome acidification, and an acidic pulse triggers translocation of cell surface-bound AIP56 into the cytosol. Accordingly, at acidic pH, AIP56 becomes more hydrophobic, interacting with artificial lipid bilayer membranes. Altogether, these data indicate that AIP56 is a short-trip toxin that reaches the cytosol using an acidic-pH-dependent mechanism, probably from early endosomes. Usually, for short-trip AB toxins, a minor pool reaches the cytosol by translocating from endosomes, whereas the rest is routed to lysosomes for degradation. Here we demonstrate that part of endocytosed AIP56 is recycled back and released extracellularly through a mechanism requiring phosphoinositide 3-kinase (PI3K) activity but independent of endosome acidification. So far, we have been unable to detect biological activity of recycled AIP56, thereby bringing into question its biological relevance as well as the importance of the recycling pathway.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.02623-14
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2014
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  • 2
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    Online Resource
    The C Terminus of Component C2II of Clostridium botulinum C2 Toxin Is Essential for Receptor Binding
    American Society for Microbiology ; 2000
    In:  Infection and Immunity Vol. 68, No. 8 ( 2000-08), p. 4566-4573
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 68, No. 8 ( 2000-08), p. 4566-4573
    Abstract: The binary Clostridium botulinum C2 toxin consists of two separate proteins, the binding component C2II (80.5 kDa) and the actin-ADP-ribosylating enzyme component C2I (49.4 kDa). For its cytotoxic action, C2II binds to a cell membrane receptor and induces cell entry of C2I via receptor-mediated endocytosis. Here we studied the structure-function relationship of C2II by constructing truncated C2II proteins and producing polyclonal antisera against selective regions of C2II. An antibody raised against the C terminus (amino acids 592 to 721) of C2II inhibited binding of C2II to cells. The antibody prevented pore formation by C2II oligomers in artificial membranes but did not influence the properties of existing channels. To further define the region responsible for receptor binding, we constructed proteins with deletions in C2II; specifically, they lacked amino acid residues 592 to 721 and the 7 C-terminal amino acid residues. The truncated proteins still formed sodium dodecyl sulfate-stable oligomers but were unable to bind to cells. Our data indicate that the C terminus of C2II mediates binding of the protein to cells and that the 7 C-terminal amino acids are structurally important for receptor binding.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.68.8.4566-4573.2000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2000
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  • 3
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    Online Resource
    Characterization of OpdH, a Pseudomonas aeruginosa Porin Involved in the Uptake of Tricarboxylates
    American Society for Microbiology ; 2007
    In:  Journal of Bacteriology Vol. 189, No. 3 ( 2007-02), p. 929-939
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 189, No. 3 ( 2007-02), p. 929-939
    Abstract: The Pseudomonas aeruginosa outer membrane is intrinsically impermeable to many classes of antibiotics, due in part to its relative lack of general uptake pathways. Instead, this organism relies on a large number of substrate-specific uptake porins. Included in this group are the 19 members of the OprD family, which are involved in the uptake of a diverse array of metabolites. One of these porins, OpdH, has been implicated in the uptake of cis -aconitate. Here we demonstrate that this porin may also enable P. aeruginosa to take up other tricarboxylates. Isocitrate and citrate strongly and specifically induced the opdH gene via a mechanism involving derepression by the putative two-component regulatory system PA0756-PA0757. Planar bilayer analysis of purified OpdH demonstrated that it was a channel-forming protein with a large single-channel conductance (230 pS in 1 M KCl; 10-fold higher than that of OprD); however, we were unable to demonstrate the presence of a tricarboxylate binding site within the channel. Thus, these data suggest that the requirement for OpdH for efficient growth on tricarboxylates was likely due to the specific expression of this large-channel porin under particular growth conditions.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.01296-06
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2007
    detail.hit.zdb_id: 1481988-0
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  • 4
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    Online Resource
    Oms38 Is the First Identified Pore-Forming Protein in the Outer Membrane of Relapsing Fever Spirochetes
    American Society for Microbiology ; 2008
    In:  Journal of Bacteriology Vol. 190, No. 21 ( 2008-11), p. 7035-7042
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 190, No. 21 ( 2008-11), p. 7035-7042
    Abstract: Relapsing fever is a worldwide, endemic disease caused by several spirochetal species belonging to the genus Borrelia . During the recurring fever peaks, borreliae proliferate remarkably quickly compared to the slow dissemination of Lyme disease Borrelia and therefore require efficient nutrient uptake from the blood of their hosts. This study describes the identification and characterization of the first relapsing fever porin, which is present in the outer membranes of B. duttonii , B. hermsii , B. recurrentis , and B. turicatae . The pore-forming protein was purified by hydroxyapatite chromatography and designated Oms38, for outer membrane-spanning protein of 38 kDa. Biophysical characterization of Oms38 was done by using the black lipid bilayer method, demonstrating that Oms38 forms small, water-filled channels of 80 pS in 1 M KCl that did not exhibit voltage-dependent closure. The Oms38 channel is slightly selective for anions and shows a ratio of permeability for cations over anions of 0.41 in KCl. Analysis of the deduced amino acid sequences demonstrated that Oms38 contains an N-terminal signal sequence which is processed under in vivo conditions. Oms38 is highly conserved within the four studied relapsing fever species, sharing an overall amino acid identity of 58% and with a strong indication for the presence of amphipathic β-sheets.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00818-08
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2008
    detail.hit.zdb_id: 1481988-0
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  • 5
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    Online Resource
    Mutations Affecting Export and Activity of Cytolysin A from Escherichia coli
    American Society for Microbiology ; 2010
    In:  Journal of Bacteriology Vol. 192, No. 15 ( 2010-08), p. 4001-4011
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 192, No. 15 ( 2010-08), p. 4001-4011
    Abstract: Cytolysin A (known as ClyA, HlyE, and SheA) is a cytolytic pore-forming protein toxin found in several Escherichia coli and Salmonella enterica strains. The structure of its water-soluble monomeric form and that of dodecameric ClyA pores is known, but the mechanisms of ClyA export from bacterial cells and of pore assembly are only partially understood. Here we used site-directed mutagenesis to study the importance of different regions of the E. coli ClyA protein for export and activity. The data indicate that ClyA translocation to the periplasm requires several protein segments located closely adjacent to each other in the “tail” domain of the ClyA monomer, namely, the N- and C-terminal regions and the hydrophobic sequence ranging from residues 89 to 101. Deletion of most of the “head” domain of the monomer (residues 181 to 203), on the other hand, did not strongly affect ClyA secretion, suggesting that the tail domain plays a particular role in export. Furthermore, we found that the N-terminal amphipathic helix αA1 of ClyA is crucial for the formation and the properties of the transmembrane channel, and hence for hemolytic activity. Several mutations affecting the C-terminal helix αG, the “β-tongue” region in the head domain, or the hydrophobic region in the tail domain of the ClyA monomer strongly impaired the hemolytic activity and reduced the activity toward planar lipid bilayer membranes but did not totally prevent formation of wild-type-like channels in these artificial membranes. The latter regions thus apparently promote membrane interaction without being directly required for pore formation in a lipid bilayer.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.01283-09
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2010
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  • 6
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    Online Resource
    Characterization of Dominantly Negative Mutant ClyA Cytotoxin Proteins in Escherichia coli
    American Society for Microbiology ; 2003
    In:  Journal of Bacteriology Vol. 185, No. 18 ( 2003-09-15), p. 5491-5499
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 185, No. 18 ( 2003-09-15), p. 5491-5499
    Abstract: We report studies of the subcellular localization of the ClyA cytotoxic protein and of mutations causing defective translocation to the periplasm in Escherichia coli . The ability of ClyA to translocate to the periplasm was abolished in deletion mutants lacking the last 23 or 11 amino acid residues of the C-terminal region. A naturally occurring ClyA variant lacking four residues (183 to 186) in a hydrophobic subdomain was retained mainly in the cytosolic fraction. These mutant proteins displayed an inhibiting effect on the expression of the hemolytic phenotype of wild-type ClyA. Studies in vitro with purified mutant ClyA proteins revealed that they were defective in formation of pore assemblies and that their activity in hemolysis assays and in single-channel conductance tests was at least 10-fold lower than that of the wild-type ClyA. Tests with combinations of the purified proteins indicated that mutant and wild-type ClyA interacted and that formation of heteromeric assemblies affected the pore-forming activity of the wild-type protein. The observed protein-protein interactions were consistent with, and provided a molecular explanation for, the dominant negative feature of the mutant ClyA variants.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.185.18.5491-5499.2003
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
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  • 7
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    Online Resource
    Differences in Purinergic Amplification of Osmotic Cell Lysis by the Pore-Forming RTX Toxins Bordetella pertussis CyaA and Actinobacillus pleuropneumoniae ApxIA: the Role of Pore Size
    American Society for Microbiology ; 2013
    In:  Infection and Immunity Vol. 81, No. 12 ( 2013-12), p. 4571-4582
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 81, No. 12 ( 2013-12), p. 4571-4582
    Abstract: A large subgroup of the r epeat in t o x in (RTX) family of leukotoxins of Gram-negative pathogens consists of pore-forming hemolysins. These can permeabilize mammalian erythrocytes (RBCs) and provoke their colloid osmotic lysis (hemolytic activity). Recently, ATP leakage through pannexin channels and P2X receptor-mediated opening of cellular calcium and potassium channels were implicated in cell permeabilization by pore-forming toxins. In the study described here, we examined the role played by purinergic signaling in the cytolytic action of two RTX toxins that form pores of different sizes. The cytolytic potency of ApxIA hemolysin of Actinobacillus pleuropneumoniae , which forms pores about 2.4 nm wide, was clearly reduced in the presence of P2X 7 receptor antagonists or an ATP scavenger, such as pyridoxalphosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS), Brilliant Blue G, ATP oxidized sodium salt, or hexokinase. In contrast, antagonists of purinergic signaling had no impact on the hemolytic potency of the adenylate cyclase toxin-hemolysin (CyaA) of Bordetella pertussis , which forms pores of 0.6 to 0.8 nm in diameter. Moreover, the conductance of pores formed by ApxIA increased with the toxin concentration, while the conductance of the CyaA single pore units was constant at various toxin concentrations. However, the P2X 7 receptor antagonist PPADS inhibited in a concentration-dependent manner the exacerbated hemolytic activity of a CyaA-ΔN489 construct (lacking 489 N-terminal residues of CyaA), which exhibited a strongly enhanced pore-forming propensity ( 〉 20-fold) and also formed severalfold larger conductance units in planar lipid bilayers than intact CyaA. These results point to a pore size threshold of purinergic amplification involvement in cell permeabilization by pore-forming RTX toxins.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00711-13
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2013
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  • 8
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    Online Resource
    PorA Represents the Major Cell Wall Channel of the Gram-Positive Bacterium Corynebacterium glutamicum
    American Society for Microbiology ; 2003
    In:  Journal of Bacteriology Vol. 185, No. 16 ( 2003-08-15), p. 4779-4786
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 185, No. 16 ( 2003-08-15), p. 4779-4786
    Abstract: The cell wall of the gram-positive bacterium Corynebacterium glutamicum contains a channel (porin) for the passage of hydrophilic solutes. The channel-forming polypeptide PorA is a 45-amino-acid acidic polypeptide with an excess of four negatively charged amino acids, which is encoded by the 138-bp gene porA . porA was deleted from the chromosome of C.glutamicum wild-type strain ATCC 13032 to obtain mutant ATCC 13032Δ porA . Southern blot analysis demonstrated that porA was deleted. Lipid bilayer experiments revealed that PorA was not present in the cell wall of the mutant strain. Searches within the known chromosome of C. glutamicum by using National Center for Biotechnology Information BLAST and reverse transcription-PCR showed that no other PorA-like protein is encoded on the chromosome or is expressed in the deletion strain. The porA deletion strain exhibited slower growth and longer growth times than the C. glutamicum wild-type strain. Experiments with different antibiotics revealed that the susceptibility of the mutant strain was much lower than that of the wild-type C. glutamicum strain. The results presented here suggest that PorA represents a major hydrophilic pathway through the cell wall and that C. glutamicum contains cell wall channels which are not related to PorA.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.185.16.4779-4786.2003
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2003
    detail.hit.zdb_id: 1481988-0
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  • 9
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    Online Resource
    The BBA01 Protein, a Member of Paralog Family 48 from Borrelia burgdorferi , Is Potentially Interchangeable with the Channel-Forming Protein P13
    American Society for Microbiology ; 2006
    In:  Journal of Bacteriology Vol. 188, No. 12 ( 2006-06-15), p. 4207-4217
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 188, No. 12 ( 2006-06-15), p. 4207-4217
    Abstract: The Borrelia burgdorferi genome exhibits redundancy, with many plasmid-carried genes belonging to paralogous gene families. It has been suggested that certain paralogs may be necessary in various environments and that they are differentially expressed in response to different conditions. The chromosomally located p13 gene which codes for a channel-forming protein belongs to paralog family 48, which consists of eight additional genes. Of the paralogous genes from family 48, the BBA01 gene has the highest homology to p13 . Herein, we have inactivated the BBA01 gene in B. burgdorferi strain B31-A. This mutant shows no apparent phenotypic difference compared to the wild type. However, analysis of BBA01 in a C-terminal protease A (CtpA)-deficient background revealed that like P13, BBA01 is posttranslationally processed at its C terminus. Elevated BBA01 expression was obtained in strains with the BBA01 gene introduced on the shuttle vector compared to the wild-type strain. We could further demonstrate that BBA01 is a channel-forming protein with properties surprisingly similar to those of P13. The single-channel conductance, of about 3.5 nS, formed by BBA01 is comparable to that of P13, which together with the high degree of sequence similarity suggests that the two proteins may have similar and interchangeable functions. This is further strengthened by the up-regulation of the BBA01 protein and its possible localization in the outer membrane in a p13 knockout strain, thus suggesting that P13 can be replaced by BBA01.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00302-06
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2006
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  • 10
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    Identification of the Outer Membrane Porin of Thermus thermophilus HB8: the Channel-Forming Complex Has an Unusually High Molecular Mass and an Extremely Large Single-Channel Conductance
    American Society for Microbiology ; 2001
    In:  Journal of Bacteriology Vol. 183, No. 2 ( 2001-01-15), p. 800-803
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 183, No. 2 ( 2001-01-15), p. 800-803
    Abstract: The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.183.2.800-803.2001
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2001
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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