scholarly journals Importance of Conserved Residues of the Serine Protease Autotransporter β-Domain in Passenger Domain Processing and β-Barrel Assembly

2010 ◽  
Vol 78 (8) ◽  
pp. 3516-3528 ◽  
Author(s):  
Yihfen T. Yen ◽  
Casey Tsang ◽  
Todd A. Cameron ◽  
Dennis O. Ankrah ◽  
Athina Rodou ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Serine Protease ◽  
Cell Fractionation ◽  
Passenger Domain ◽  
Double Mutation ◽  
Conserved Residues ◽  
Virulence Proteins ◽  
Secretion Pathway ◽  
Residue Substitution ◽  
Cellular Compartments

ABSTRACT Serine protease autotransporters of the family Enterobacteriaceae (SPATE) comprise a family of virulence proteins secreted by enteric Gram-negative bacteria via the autotransporter secretion pathway. A SPATE polypeptide contains a C-terminal translocator domain that inserts into the bacterial outer membrane as a β-barrel structure and mediates secretion of the passenger domain to the extracellular environment. In the present study, we examined the role of conserved residues located in the SPATE β-barrel-forming region in passenger domain secretion. Thirty-nine fully conserved residues in Tsh were mutated by single-residue substitution, and defects in their secretion phenotypes were assessed by cell fractionation and immunochemistry. A total of 22 single mutants exhibited abnormal phenotypes in different cellular compartments. Most mutants affecting secretion are charged residues with side chains pointing into the β-barrel interior. Seven mutants showed notable abnormalities in processing (constructs with the E1231A, E1249A, and R1374A mutations) and β-barrel assembly or insertion into the outer membrane (constructs with the G1158Y, F1360A, Y1375A, and F1377A mutations). The phenotypes of the β-barrel assembly/insertion mutants and the presence of a processed Tsh passenger domain in the periplasm support the possibility that the translocator domain must undergo extensive folding prior to insertion into the outer membrane. Results from double-mutation experiments further demonstrate that F1360 and F1377 affect β-barrel insertion/assembly at different times. In light of these new data, a more refined model for the mechanism of SPATE secretion is presented.

scholarly journals Intramolecular Interactions between the Protease and Structural Domains Are Important for the Functions of Serine Protease Autotransporters

2010 ◽  
Vol 78 (8) ◽  
pp. 3335-3345 ◽  
Author(s):  
Casey Tsang ◽  
Huma Malik ◽  
Deana Nassman ◽  
Antony Huang ◽  
Fayha Tariq ◽  
...  
Keyword(s):  
Serine Protease ◽  
Signal Sequence ◽  
Catalytic Triad ◽  
Intramolecular Interactions ◽  
Site Directed Mutagenesis ◽  
Temperature Sensitive ◽  
Molecular Graphics ◽  
Passenger Domain ◽  
Conserved Residues ◽  
Functional Roles

ABSTRACT Autotransporter (AT) is a protein secretion pathway found in Gram-negative bacteria featuring a multidomain polypeptide with a signal sequence, a passenger domain, and a translocator domain. An AT subfamily named serine protease ATs of the family Enterobacteriaceae (SPATEs) is characterized by the presence of a conserved serine protease motif in the passenger domain which contributes to bacterial pathogenesis. The goal of the current study is to determine the importance of the passenger domain conserved residues in the SPATE proteolytic and adhesive functions using the temperature-sensitive hemagglutinin (Tsh) protein as our model. To begin, mutations of 21 fully conserved residues in the four passenger domain conserved motifs were constructed by PCR-based site-directed mutagenesis. Seventeen mutants exhibited a wild-type secretion level; among these mutants, eight displayed reduced proteolytic activities in Tsh-specific oligopeptide and mucin cleavage assays. These eight mutants also demonstrated lower affinities to extracellular matrix proteins, collagen IV, and fibronectin. These eight conserved residues were analyzed by molecular graphics modeling to demonstrate their intramolecular interactions with the catalytic triad and other key residues. Additional mutations were made to confirm the above interactions in order to demonstrate their significance to the SPATE functions. Altogether our data suggest that certain conserved residues in the SPATE passenger domain are important for both the proteolytic and adhesive activities of SPATE by maintaining the proper protein structure via intramolecular interactions between the protease and β-helical domains. Here, we provide new insight into the structure-function relationship of the SPATEs and the functional roles of their conserved residues.

scholarly journals Autotransporter protein secretion

2011 ◽  
Vol 2 (6) ◽  
pp. 525-536 ◽  
Author(s):  
Jeremy R.H. Tame
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Large Family ◽  
Helical Structure ◽  
Passenger Domain ◽  
True Nature ◽  
Structural Characterisation ◽  
Secretion Pathway ◽  
Hard Information

AbstractAutotransporter proteins are a large family of virulence factors secreted from Gram-negative bacteria by a unique mechanism. First described in the 1980s, these proteins have a C-terminal region that folds into a β-barrel in the bacterial outer membrane. The so-called passenger domain attached to this barrel projects away from the cell surface and may be liberated from the cell by self-cleavage or surface proteases. Although the majority of passenger domains have a similar β-helical structure, they carry a variety of sub­domains, allowing them to carry out widely differing functions related to pathogenesis. Considerable biochemical and structural characterisation of the barrel domain has shown that ‘autotransporters’ in fact require a conserved and essential protein complex in the outer membrane for correct folding. Although the globular domains of this complex projecting into the periplasmic space have also been structurally characterised, the overall secretion pathway of the autotransporters remains highly puzzling. It was presumed for many years that the passenger domain passed through the centre of the barrel domain to reach the cell surface, driven at least in part by folding. This picture is complicated by conflicting data, and there is currently little hard information on the true nature of the secretion intermediates. As well as their medical importance therefore, autotransporters are proving to be an excellent system to study the folding and membrane insertion of outer membrane proteins in general. This review focuses on structural aspects of autotransporters; their many functions in pathogenesis are beyond its scope.

scholarly journals Role of the α-Helical Linker of the C-Terminal Translocator in the Biogenesis of the Serine Protease Subfamily of Autotransporters

2006 ◽  
Vol 74 (9) ◽  
pp. 4961-4969 ◽  
Author(s):  
Maria Kostakioti ◽  
Christos Stathopoulos
Keyword(s):  
Outer Membrane ◽  
Serine Protease ◽  
Vaccine Development ◽  
Passenger Transport ◽  
Amino Acid Motif ◽  
Passenger Domain ◽  
Conserved Motif ◽  
Potential Applications ◽  
Hydrophobic Amino Acids

ABSTRACT Autotransporters are secreted virulence factors that comprise three domains: an N-terminal signal peptide, an internal passenger domain, and a C-terminal β-domain. The mechanism of passenger translocation across the outer membrane remains undefined, with four models having been proposed: the “hairpin,” the “threading,” the “multimeric,” and the “Omp85 (YaeT)” models. In an attempt to understand autotransporter biogenesis, we screened the sequences of the serine protease subfamily of autotransporters (SPATEs) for conserved features indicative of a common secretion mechanism. Our analyses revealed a strictly conserved 14-amino-acid motif within the predicted α-helical linker region, upstream of the β-domain of SPATEs. We investigated the function of this motif through a mutagenesis approach using Tsh as a model. Our studies demonstrate that mutations throughout the conserved motif do not block insertion of the β-domain into the outer membrane. However, nonconservative mutations of four hydrophobic (V1099, L1102, G1107, and L1109) and three polar (N1100, K1104, and R1105) residues of the motif severely decrease or even abolish Tsh biogenesis. Further studies showed that these mutations interfere with passenger transport across the outer membrane. Bioinformatical analyses suggest that the critical polar and hydrophobic amino acids localize on opposite sides of the helix that runs through the β-barrel pore. Our data indicate that the conserved motif is important for passenger secretion across the outer membrane and that mutations in certain residues severely affect the secretion process. We discuss how these results fit with the four proposed models for autotransporter secretion and potential applications in antimicrobial and vaccine development.

scholarly journals Soluble versions of outer membrane cytochromes function as exporters for heterologously produced cargo proteins

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Helge M. Dietrich ◽  
Miriam Edel ◽  
Thea Bursac ◽  
Manfred Meier ◽  
Katrin Sturm-Richter ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Culture Supernatant ◽  
Cellulase Activity ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Recognition Sequence ◽  
Secretion Pathway ◽  
N Terminus ◽  
Cargo Proteins ◽  
Lipid Anchor

AbstractThis study reveals that it is possible to secrete truncated versions of outer membrane cytochromes into the culture supernatant and that these proteins can provide a basis for the export of heterologously produced proteins. Different soluble and truncated versions of the outer membrane cytochrome MtrF were analyzed for their suitability to be secreted. A protein version with a very short truncation of the N-terminus to remove the recognition sequence for the addition of a lipid anchor is secreted efficiently to the culture supernatant, and moreover this protein could be further truncated by a deletion of 160 amino acid and still is detectable in the supernatant. By coupling a cellulase to this soluble outer membrane cytochrome, the export efficiency was measured by means of relative cellulase activity. We conclude that outer membrane cytochromes of S. oneidensis can be applied as transporters for the export of target proteins into the medium using the type II secretion pathway.

scholarly journals The Role of Virulence Proteins in Protection Conferred by Bordetella pertussis Outer Membrane Vesicle Vaccines

Vaccines ◽  
2020 ◽  
Vol 8 (3) ◽  
pp. 429
Author(s):  
René H. M. Raeven ◽  
Naomi van Vlies ◽  
Merijn L. M. Salverda ◽  
Larissa van der Maas ◽  
Joost P. Uittenbogaard ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Bordetella Pertussis ◽  
Protective Immunity ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Outer Membrane Vesicle ◽  
Protective Capacity ◽  
T Helper 1 ◽  
Virulence Proteins ◽  
Humoral Responses

The limited protective immunity induced by acellular pertussis vaccines demands development of novel vaccines that induce broader and longer-lived immunity. In this study, we investigated the protective capacity of outer membrane vesicle pertussis vaccines (omvPV) with different antigenic composition in mice to gain insight into which antigens contribute to protection. We showed that total depletion of virulence factors (bvg(-) mode) in omvPV led to diminished protection despite the presence of high antibody levels. Antibody profiling revealed overlap in humoral responses induced by vaccines in bvg(-) and bvg(+) mode, but the potentially protective responses in the bvg(+) vaccine were mainly directed against virulence-associated outer membrane proteins (virOMPs) such as BrkA and Vag8. However, deletion of either BrkA or Vag8 in our outer membrane vesicle vaccines did not affect the level of protection. In addition, the vaccine-induced immunity profile, which encompasses broad antibody and mixed T-helper 1, 2 and 17 responses, was not changed. We conclude that the presence of multiple virOMPs in omvPV is crucial for protection against Bordetella pertussis. This protective immunity does not depend on individual proteins, as their absence or low abundance can be compensated for by other virOMPs.

Cyclin I and p35 determine the subcellular distribution of Cdk5

2015 ◽  
Vol 308 (4) ◽  
pp. C339-C347 ◽  
Author(s):  
Henning Hagmann ◽  
Yoshinori Taniguchi ◽  
Jeffrey W. Pippin ◽  
Hans-Michael Kauerz ◽  
Thomas Benzing ◽  
...  
Keyword(s):  
Subcellular Distribution ◽  
Cell Biology ◽  
Kinase Activity ◽  
Brain Slices ◽  
Axonal Guidance ◽  
Cell Fractionation ◽  
Cyclin Dependent Kinase ◽  
Immunofluorescence Labeling ◽  
Cellular Compartments ◽  
Cyclin I

The atypical cyclin-dependent kinase 5 (Cdk5) serves an array of different functions in cell biology. Among these are axonal guidance, regulation of intercellular contacts, cell differentiation, and prosurvival signaling. The variance of these functions suggests that Cdk5 activation comes to pass in different cellular compartments. The kinase activity, half-life, and substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39, and cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in murine kidney and brain slices of wild-type and cyclin I- and/or p35-null mice by immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling, as well as cell fractionation experiments. The predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These findings were further substantiated by overexpression of cyclin I and p35 with altered targeting characteristics in human embryonic kidney 293T cells. These studies reveal that the subcellular localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 kinase activity in specific cellular compartments dependent on the activator present and allows Cdk5 to serve multiple independent roles.

scholarly journals Antigenic Organization of the N-Terminal Part of the Polymorphic Outer Membrane Proteins 90, 91A, and 91B of Chlamydophilaabortus

2003 ◽  
Vol 71 (6) ◽  
pp. 3240-3250 ◽  
Author(s):  
Evangelia Vretou ◽  
Panagiota Giannikopoulou ◽  
David Longbottom ◽  
Evgenia Psarrou
Keyword(s):  
Outer Membrane ◽  
Binding Sites ◽  
Outer Membrane Proteins ◽  
Single Amino Acid ◽  
Antigenic Peptides ◽  
Terminal Part ◽  
Passenger Domain ◽  
Recombinant Peptides ◽  
Type V ◽  
Reference Strains

ABSTRACT A series of overlapping recombinant antigens, 61 to 74 residues in length, representing polymorphic outer membrane protein 90 (POMP90) of Chlamydophila abortus and two recombinant peptides spanning gene fragment p91Bf99 of POMP91B were assessed by immunoblotting to determine the antigen-binding sites of 20 monoclonal antibodies to POMP90, -91A, and -91B. The epitopes were further restricted by scanning 52 overlapping synthetic 12-mer peptides representing the N-terminal part of POMP90, and the 12-mer epitopes were then analyzed by using hexapeptides to the resolution of a single amino acid. Ten epitopes were defined: 1, TSEEFQVKETSSGT; 2, SGAIYTCEGNVCISYAGKDSPL; 3, SLVFHKNCSTAE; 4, AIYADKLTIVSGGPTLFS; 5, SPKGGAISIKDS; 6, ITFDGNKIIKTS; 7, LRAKDGFGIFFY; 7a, DGFGIF; 7b, GIFFYD; 8, IFFYDPITGGGS; 8a, FFYDPIT; 9, GKIVFSGE; and 10, DLGTTL. The 20-mer peptide LRAKDGFGIFFYDPITGGGS was a major epitope that was recognized by seven antibodies. Epitopes 7 to 10 were conserved in reference strains of the former species C. psittaci, whereas the strong antigenic peptides FYDPIT and IVFSGE were conserved among members of the genus Chlamydophila. Epitopes 3 to 8 were located within the best-scoring beta-helical wrap (residues 148 to 293) predicted for POMP91B by the program BETAWRAP. Other studies have suggested an association of the POMPs with type V secretory autotransporter proteins. The results presented in this study provide some evidence for a passenger domain that is folded as a beta-helix pyramid with compact antigenic organization.

Structural basis for the interaction of BamB with the POTRA3–4 domains of BamA

2016 ◽  
Vol 72 (2) ◽  
pp. 236-244 ◽  
Author(s):  
Zhen Chen ◽  
Li-Hong Zhan ◽  
Hai-Feng Hou ◽  
Zeng-Qiang Gao ◽  
Jian-Hua Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Escherichia Coli ◽  
Outer Membrane ◽  
Essential Role ◽  
Biochemical Analysis ◽  
Hydrophobic Interactions ◽  
Outer Membrane Proteins ◽  
Structural Basis ◽  
Conserved Residues ◽  
Bam Complex

InEscherichia coli, the Omp85 protein BamA and four lipoproteins (BamBCDE) constitute the BAM complex, which is essential for the assembly and insertion of outer membrane proteins into the outer membrane. Here, the crystal structure of BamB in complex with the POTRA3–4 domains of BamA is reported at 2.1 Å resolution. Based on this structure, the POTRA3 domain is associated with BamBviahydrogen-bonding and hydrophobic interactions. Structural and biochemical analysis revealed that the conserved residues Arg77, Glu127, Glu150, Ser167, Leu192, Leu194 and Arg195 of BamB play an essential role in interaction with the POTRA3 domain.

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