scholarly journals Metalloadsorption by Escherichia coliCells Displaying Yeast and Mammalian Metallothioneins Anchored to the Outer Membrane Protein LamB

1998 ◽  
Vol 180 (9) ◽  
pp. 2280-2284 ◽  
Author(s):  
Carolina Sousa ◽  
Pavel Kotrba ◽  
Tomas Ruml ◽  
Angel Cebolla ◽  
Víctor De Lorenzo
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Metal Binding ◽  
Wild Type ◽  
Lambda Phage ◽  
E Coli ◽  
Hybrid Proteins ◽  
Mammalian Metallothioneins

ABSTRACT Yeast (CUP1) and mammalian (HMT-1A) metallothioneins (MTs) have been efficiently expressed in Escherichia coli as fusions to the outer membrane protein LamB. A 65-amino-acid sequence from the CUP1 protein of Saccharomyces cerevisiae (yeast [Y] MT) was genetically inserted in permissive site 153 of the LamB sequence, which faces the outer medium. A second LamB fusion at position 153 was created with 66 amino acids recruited from the form of human (H) MT that is predominant in the adipose tissue, HMT-1A. Both LamB153-YMT and LamB153-HMT hybrids were produced in vivo as full-length proteins, without any indication of instability or proteolytic degradation. Each of the two fusion proteins was functional as the port of entry of lambda phage variants, suggesting maintenance of the overall topology of the wild-type LamB. Expression of the hybrid proteins in vivo multiplied the natural ability of E. colicells to bind Cd2+ 15- to 20-fold, in good correlation with the number of metal-binding centers contributed by the MT moiety of the fusions.

scholarly journals ExbBD-Dependent Transport of Maltodextrins through the Novel MalA Protein across the Outer Membrane of Caulobacter crescentus

2005 ◽  
Vol 187 (24) ◽  
pp. 8300-8311 ◽  
Author(s):  
Heidi Neugebauer ◽  
Christina Herrmann ◽  
Winfried Kammer ◽  
Gerold Schwarz ◽  
Alfred Nordheim ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Caulobacter Crescentus ◽  
Vitamin B ◽  
Wild Type ◽  
E Coli ◽  
Iron Source ◽  
Maltose Transport ◽  
Dependent Transport

ABSTRACT Analysis of the genome sequence of Caulobacter crescentus predicts 67 TonB-dependent outer membrane proteins. To demonstrate that among them are proteins that transport nutrients other than chelated Fe3+ and vitamin B12—the substrates hitherto known to be transported by TonB-dependent transporters—the outer membrane protein profile of cells grown on different substrates was determined by two-dimensional electrophoresis. Maltose induced the synthesis of a hitherto unknown 99.5-kDa protein, designated here as MalA, encoded by the cc2287 genomic locus. MalA mediated growth on maltodextrins and transported [14C]maltodextrins from [14C]maltose to [14C]maltopentaose. [14C]maltose transport showed biphasic kinetics, with a fast initial rate and a slower second rate. The initial transport had a Kd of 0.2 μM, while the second transport had a Kd of 5 μM. It is proposed that the fast rate reflects binding to MalA and the second rate reflects transport into the cells. Energy depletion of cells by 100 μM carbonyl cyanide 3-chlorophenylhydrazone abolished maltose binding and transport. Deletion of the malA gene diminished maltose transport to 1% of the wild-type malA strain and impaired transport of the larger maltodextrins. The malA mutant was unable to grow on maltodextrins larger than maltotetraose. Deletion of two C. crescentus genes homologous to the exbB exbD genes of Escherichia coli abolished [14C]maltodextrin binding and transport and growth on maltodextrins larger than maltotetraose. These mutants also showed impaired growth on Fe3+-rhodotorulate as the sole iron source, which provided evidence of energy-coupled transport. Unexpectedly, a deletion mutant of a tonB homolog transported maltose at the wild-type rate and grew on all maltodextrins tested. Since Fe3+-rhodotorulate served as an iron source for the tonB mutant, an additional gene encoding a protein with a TonB function is postulated. Permeation of maltose and maltotriose through the outer membrane of the C. crescentus malA mutant was slower than permeation through the outer membrane of an E. coli lamB mutant, which suggests a low porin activity in C. crescentus. The pores of the C. crescentus porins are slightly larger than those of E. coli K-12, since maltotetraose supported growth of the C. crescentus malA mutant but failed to support growth of the E. coli lamB mutant. The data are consistent with the proposal that binding of maltodextrins to MalA requires energy and MalA actively transports maltodextrins with Kd values 1,000-fold smaller than those for the LamB porin and 100-fold larger than those for the vitamin B12 and ferric siderophore outer membrane transporters. MalA is the first example of an outer membrane protein for which an ExbB/ExbD-dependent transport of a nutrient other than iron and vitamin B12 has been demonstrated.

The porin OmpC ofSalmonella typhimuriummediates adherence to macrophages

1999 ◽  
Vol 45 (8) ◽  
pp. 658-669 ◽  
Author(s):  
Robert S Negm ◽  
Thomas G Pistole
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Virulent Strain ◽  
Salmonella Typhi ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
E Coli ◽  
Host Cell Recognition

Macrophages recognize, adhere to, and phagocytose Salmonella typhimurium. The major outer membrane protein OmpC is a candidate ligand for macrophage recognition. To confirm this we used transposon mutagenesis to develop an ompC-deficient mutant in a known virulent strain of S. typhimurium; mutant and wild type were compared in macrophage adherence and association assays. Radiolabeled wild type S. typhimurium bound to macrophages at five-fold higher levels than did the ompC mutant. In association assays, macrophages in monolayers bound and internalized three-fold more wild type than mutant, while macrophages in suspension bound and internalized 40-fold more wild type than mutant. The ompC gene of our test strain of S. typhimurium contains several discrete differences compared with the ompC genes of Salmonella typhi and Escherichia coli. The deduced OmpC amino acid sequence of S. typhimurium shares 77 and 98% identity with OmpC amino acid sequences of E. coli and S. typhi, respectively. Evidence from this study supports a role for the OmpC protein in initial recognition by macrophages and distinguishes regions of this protein that potentially participate in host-cell recognition of bacteria by phagocytic cells.Key words: Salmonella, porin, macrophage, outer membrane protein, DNA sequencing.

scholarly journals The Helicobacter pylori Autotransporter ImaA (HP0289) Modulates the Immune Response and Contributes to Host Colonization

2012 ◽  
Vol 80 (7) ◽  
pp. 2286-2296 ◽  
Author(s):  
William E. Sause ◽  
Andrea R. Castillo ◽  
Karen M. Ottemann
Keyword(s):  
Immune Response ◽  
Helicobacter Pylori ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Content Type ◽  
H Pylori ◽  
Murine Infections

ABSTRACTThe human pathogenHelicobacter pyloriemploys a diverse collection of outer membrane proteins to colonize, persist, and drive disease within the acidic gastric environment. In this study, we sought to elucidate the function of the host-induced geneHP0289, which encodes an uncharacterized outer membrane protein. We first generated an isogenicH. pylorimutant that lacksHP0289and found that the mutant has a colonization defect in single-strain infections and is greatly outcompeted in mouse coinfection experiments with wild-typeH. pylori. Furthermore, we used protease assays and biochemical fractionation coupled with an HP0289-targeted peptide antibody to verify that the HP0289 protein resides in the outer membrane. Our previous findings showed that theHP0289promoter is upregulated in the mouse stomach, and here we demonstrate thatHP0289expression is induced under acidic conditions in an ArsRS-dependent manner. Finally, we have shown that theHP0289mutant induces greater expression of the chemokine interleukin-8 (IL-8) and the cytokine tumor necrosis factor alpha (TNF-α) in gastric carcinoma cells (AGS). Similarly, transcription of the IL-8 homolog keratinocyte-derived chemokine (KC) is elevated in murine infections with the HP0289 mutant than in murine infections with wild-typeH. pylori. On the basis of this phenotype, we renamed HP0289 ImaA forimmunomodulatoryautotransporter protein. Our work has revealed that genes inducedin vivoplay an important role inH. pyloripathogenesis. Specifically, the outer membrane protein ImaA modulates a component of the host inflammatory response, and thus may allowH. pylorito fine tune the host immune response based on ImaA expression.

scholarly journals Localization of Haemophilus ducreyi at the Pustular Stage of Disease in the Human Model of Infection

2000 ◽  
Vol 68 (4) ◽  
pp. 2309-2314 ◽  
Author(s):  
Margaret E. Bauer ◽  
Stanley M. Spinola
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Human Subjects ◽  
Haemophilus Ducreyi ◽  
Human Model ◽  
Stage Of Disease ◽  
The One ◽  
Secondary Antibodies

ABSTRACT To localize Haemophilus ducreyi in vivo, human subjects were experimentally infected with H. ducreyi until they developed a painful pustule or for 14 days. Lesions were biopsied, and biopsy samples were fixed in 4% paraformaldehyde, and cryosectioned. Sections were stained with polyclonal anti-H. ducreyi antiserum or H. ducreyi-specific monoclonal antibodies (MAbs) and fluorescently tagged secondary antibodies and examined by confocal microscopy. We identified H. ducreyi in 16 of 18 pustules but did not detect bacteria in the one papule examined. H. ducreyi was observed as individual cells and in clumps or chains. Staining with MAbs 2D8, 5C9, 3B9, 2C7, and 9D12 demonstrated that H. ducreyi expresses the major pilus subunit, FtpA, the 28-kDa outer membrane protein Hlp, the 18-kDa outer membrane protein PAL, and the major outer membrane protein (MOMP) or OmpA2 in vivo. By dual staining with polyclonal anti-H. ducreyi antiserum and MAbs that recognize human skin components, we observed bacteria within the neutrophilic infiltrates of all positively staining pustules and in the dermis of 10 of 16 pustules. We were unable to detect bacteria associated with keratinocytes in the samples examined. The data suggest that H. ducreyi is found primarily in association with neutrophils and in the dermis at the pustular stage of disease in the human model of infection.

scholarly journals Meningococcal Outer Membrane Protein NhhA Is Essential for Colonization and Disease by Preventing Phagocytosis and Complement Attack

2008 ◽  
Vol 76 (11) ◽  
pp. 5412-5420 ◽  
Author(s):  
Hong Sjölinder ◽  
Jens Eriksson ◽  
Lisa Maudsdotter ◽  
Helena Aro ◽  
Ann-Beth Jonsson
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Bacterial Colonization ◽  
Membrane Attack Complex ◽  
Rapid Onset ◽  
Bacterial Attachment ◽  
High Morbidity ◽  
Bacterial Survival

ABSTRACT Neisseria meningitidis is a leading cause of meningitis and septicemia worldwide, with a rapid onset of disease and a high morbidity and mortality. NhhA is a meningococcal outer membrane protein included in the family of trimeric autotransporter adhesins. The protein binds to the extracellular matrix proteins heparan sulfate and laminin and facilitates attachment to host epithelial cells. In this study, we show that NhhA is essential for bacterial colonization of the nasopharyngeal mucosa in a murine model of meningococcal disease. Successful colonization depends on bacterial attachment but also to the capacity to overcome innate host immune responses. We found that NhhA protected bacteria from phagocytosis, which is important for the mucosal survival of bacteria. In addition, NhhA mediated extensive serum resistance that increased bacterial survival in blood and promoted lethal sepsis. The presence of NhhA protected bacteria from complement-mediated killing by preventing the deposition of the membrane attack complex. Taken together, the results of this work reveal that NhhA inhibits phagocytosis and protects bacteria against complement-mediated killing, which enhances both nasal colonization and the development of sepsis in vivo.

scholarly journals Structure of BamA, an essential factor in outer membrane protein biogenesis

2014 ◽  
Vol 70 (6) ◽  
pp. 1779-1789 ◽  
Author(s):  
Reinhard Albrecht ◽  
Monika Schütz ◽  
Philipp Oberhettinger ◽  
Michaela Faulstich ◽  
Ivan Bermejo ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Cell Envelope ◽  
Significant Degree ◽  
State Function ◽  
E Coli ◽  
Functional Studies ◽  
C Terminus ◽  
Protein Biogenesis

Outer membrane protein (OMP) biogenesis is an essential process for maintaining the bacterial cell envelope and involves the β-barrel assembly machinery (BAM) for OMP recognition, folding and assembly. InEscherichia colithis function is orchestrated by five proteins: the integral outer membrane protein BamA of the Omp85 superfamily and four associated lipoproteins. To unravel the mechanism underlying OMP folding and insertion, the structure of theE. coliBamA β-barrel and P5 domain was determined at 3 Å resolution. These data add information beyond that provided in the recently published crystal structures of BamA fromHaemophilus ducreyiandNeisseria gonorrhoeaeand are a valuable basis for the interpretation of pertinent functional studies. In an `open' conformation,E. coliBamA displays a significant degree of flexibility between P5 and the barrel domain, which is indicative of a multi-state function in substrate transfer.E. coliBamA is characterized by a discontinuous β-barrel with impaired β1–β16 strand interactions denoted by only two connecting hydrogen bonds and a disordered C-terminus. The 16-stranded barrel surrounds a large cavity which implies a function in OMP substrate binding and partial folding. These findings strongly support a mechanism of OMP biogenesis in which substrates are partially folded inside the barrel cavity and are subsequently released laterally into the lipid bilayer.

scholarly journals In Vivo Transfer of Plasmid-Encoded ACC-1 AmpC from Klebsiella pneumoniae to Escherichia coli in an Infant and Selection of Impermeability to Imipenem in K. pneumoniae

2005 ◽  
Vol 49 (8) ◽  
pp. 3562-3565 ◽  
Author(s):  
Philippe Bidet ◽  
Béatrice Burghoffer ◽  
Valérie Gautier ◽  
Naïma Brahimi ◽  
Patricia Mariani-Kurkdjian ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Major Outer Membrane Protein ◽  
In Vivo Selection ◽  
Selection Of

ABSTRACT We describe in vivo selection of a Klebsiella pneumoniae strain with diminished imipenem susceptibility attributable to plasmid-encoded ACC-1 β-lactamase production and loss of a 36-kDa major outer membrane protein, together with transfer of this plasmid from K. pneumoniae to Escherichia coli in a Tunisian infant.

Sign in / Sign up

Export Citation Format

Share Document