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.

scholarly journals Characterization of BcaA, a Putative Classical Autotransporter Protein in Burkholderia pseudomallei

2013 ◽  
Vol 81 (4) ◽  
pp. 1121-1128 ◽  
Author(s):  
Cristine G. Campos ◽  
Luke Borst ◽  
Peggy A. Cotter
Keyword(s):  
Burkholderia Pseudomallei ◽  
Efflux Pump ◽  
Outer Membrane Proteins ◽  
Lung Epithelial Cells ◽  
Wild Type ◽  
Passenger Domain ◽  
Content Type ◽  
Reading Frame ◽  
Type V

ABSTRACTBurkholderia pseudomalleiis a tier 1 select agent, and the causative agent of melioidosis, a disease with effects ranging from chronic abscesses to fulminant pneumonia and septic shock, which can be rapidly fatal. Autotransporters (ATs) are outer membrane proteins belonging to the type V secretion system family, and many have been shown to play crucial roles in pathogenesis. The open reading frame Bp1026b_II1054 (bcaA) inB. pseudomalleistrain 1026b is predicted to encode a classical autotransporter protein with an approximately 80-kDa passenger domain that contains a subtilisin-related domain. Immediately 3′ tobcaAis Bp11026_II1055 (bcaB), which encodes a putative prolyl 4-hydroxylase. To investigate the role of these genes in pathogenesis, large in-frame deletion mutations ofbcaAandbcaBwere constructed in strain Bp340, an efflux pump mutant derivative of the melioidosis clinical isolate 1026b. Comparison of Bp340ΔbcaAand Bp340ΔbcaBmutants to wild-typeB. pseudomalleiin vitrodemonstrated similar levels of adherence to A549 lung epithelial cells, but the mutant strains were defective in their ability to invade these cells and to form plaques. In a BALB/c mouse model of intranasal infection, similar bacterial burdens were observed after 48 h in the lungs and liver of mice infected with Bp340ΔbcaA, Bp340ΔbcaB, and wild-type bacteria. However, significantly fewer bacteria were recovered from the spleen of Bp340ΔbcaA-infected mice, supporting the idea of a role for this AT in dissemination or in survival in the passage from the site of infection to the spleen.

scholarly journals Sequential Translocation of Polypeptides across the Bacterial Outer Membrane through the Trimeric Autotransporter Pathway

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Rakesh Sikdar ◽  
Harris D. Bernstein
Keyword(s):  
Outer Membrane ◽  
Alternative Energy ◽  
Protein Translocation ◽  
Outer Membrane Proteins ◽  
Passenger Domain ◽  
Experimental Conditions ◽  
Content Type ◽  
Intrinsically Disordered ◽  
Extracellular Domains ◽  
Bacterial Outer Membrane

ABSTRACT Trimeric autotransporter adhesins (TAAs) are a family of bacterial outer membrane (OM) proteins that are comprised of three identical subunits. Each subunit contains an N-terminal extracellular (“passenger”) domain and a short C-terminal segment that contributes four β strands to a single 12-stranded β barrel. The mechanism by which the passenger domains are translocated across the OM and the energetics of the translocation reaction are poorly understood. To address these issues, we examined the secretion of modified versions of the passenger domain of UpaG, a TAA produced by Escherichia coli CFT073. Using the SpyTag-SpyCatcher system to probe passenger domain localization, we found that both intrinsically disordered polypeptides fused to the UpaG passenger domain and artificially disulfide-bonded polypeptides were secreted effectively but relatively slowly. Surprisingly, we also found that in some cases, the three nonnative passenger domain segments associated with a single trimer were secreted sequentially. Photo-cross-linking experiments indicated that incompletely assembled UpaG derivatives remained bound to the barrel assembly machinery (Bam) complex until all three passenger domains were fully secreted. Taken together, our results strongly suggest that the secretion of polypeptides through the TAA pathway is coordinated with the assembly of the β barrel domain and that the folding of passenger domains in the extracellular space maximizes the rate of secretion. Furthermore, our work provides evidence for an unprecedented sequential mode of protein translocation, at least under specific experimental conditions. IMPORTANCE Trimeric autotransporter adhesins (TAAs) are specialized bacterial outer membrane proteins consisting of three identical subunits. TAAs contain large extracellular domains that trimerize and promote virulence, but the mechanism by which they are secreted is poorly understood. We found that the extracellular domains of a native TAA were secreted rapidly but that disordered and artificially folded polypeptides fused to native passenger domains were secreted in a slow, sequential fashion. Our results strongly suggest that the efficient secretion of native extracellular domains is driven by their trimerization following export but that alternative energy sources can be harnessed to secrete nonnative polypeptides. Furthermore, we obtained evidence that TAA extracellular domains are secreted before the assembly of the linked membrane spanning domain is completed.

scholarly journals Simultaneous detection of LipL32 and LipL21 genes of pathogenic leptospira from serum samples of bovines by multiplex PCR

2011 ◽  
Vol 1 (1) ◽  
pp. 15
Author(s):  
Timiri V. Meenambigai ◽  
Gopalakrishnan Ravikumar ◽  
Andy Srithar ◽  
Govindan Balakrishnan ◽  
Chidambaram Saranya ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Multiplex Pcr ◽  
Outer Membrane ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Simultaneous Detection ◽  
Serum Samples ◽  
Pathogenic Leptospira ◽  
Annealing Conditions ◽  
Reference Strains

<p>Leptospirosis is a worldwide zoonotic disease of cattle associated with pathogenic leptospiral infection. This study focuses in the use of a molecular tool to detect pathogenic leptospiral infection in bovines by targeting the outer membrane proteins LipL32 and LipL21 simultaneously in a multiplex PCR. Sixteen pathogenic reference strains and 10 bovine serum samples were analyzed for simultaneous detection of both genes at appropriate annealing conditions. These findings are suggestive of the fact that multiplex PCR can be used to detect major outer membrane proteins of pathogenic leptospira from serum samples. Further it aided in the differentiation of pathogenic and non-pathogenic species of leptospires too. This study will definitely serve as a valuable tool, as it suggests the importance of <em>LipL32</em> genes as potential candidates for vaccine development to control animal Leptospirosis.</p>

scholarly journals Expression of Members of the 28-Kilodalton Major Outer Membrane Protein Family of Ehrlichia chaffeensis during Persistent Infection

2004 ◽  
Vol 72 (8) ◽  
pp. 4336-4343 ◽  
Author(s):  
Jian-zhi Zhang ◽  
Hong Guo ◽  
Gary M. Winslow ◽  
Xue-jie Yu
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Multigene Family ◽  
Outer Membrane Proteins ◽  
Enzyme Linked Immunosorbent Assay ◽  
Ehrlichia Chaffeensis ◽  
Antigenic Peptides ◽  
Indirect Measure

ABSTRACT The 28-kDa immunodominant outer membrane proteins (P28 OMPs) of Ehrlichia chaffeensis are encoded by a multigene family. As an indirect measure of the in vivo expression of the members of the p28 multigene family of E. chaffeensis, sera from two beagle dogs experimentally infected with E. chaffeensis were evaluated for the presence of specific antibodies to P28 OMPs by enzyme-linked immunosorbent assay. Antigenic peptides unique to each of the P28s were identified within the first hypervariable region of each P28 OMP. Serological responses to peptides derived from all P28 OMPs were detected from day 30 postinoculation to day 468 and from day 46 until day 159 in the two beagles. Although antibody titers to the peptides fluctuated, the peak response to all of the peptides appeared simultaneously in each dog. The antibody responses to another outer membrane protein of E. chaffeensis (GP120) showed similar temporal and quantitative changes. These data suggest that the P28 OMPs are expressed concurrently during persistent Ehrlichia infection.

The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease

Microbiology ◽  
2009 ◽  
Vol 155 (12) ◽  
pp. 3982-3991 ◽  
Author(s):  
Ana Sauri ◽  
Zora Soprova ◽  
David Wickström ◽  
Jan-Willem de Gier ◽  
Roel C. Van der Schors ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Critical Role ◽  
Structural Data ◽  
Intermediate Stage ◽  
Gram Negative Bacteria ◽  
Passenger Domain ◽  
Gram Negative ◽  
Periplasmic Chaperone ◽  
Folded Structures

Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms a β-barrel pore in the outer membrane implicated in translocation of the upstream ‘passenger’ domain across the outer membrane. However, recent structural data suggest that the diameter of the β-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage the β-domain was not inserted and folded as an integral β-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Bam (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane.

Comparison of the class 1 outer membrane proteins of eight serological reference strains of Neisseria meningitidis

1991 ◽  
Vol 5 (3) ◽  
pp. 727-736 ◽  
Author(s):  
M. C. J. Maiden ◽  
J. Suker ◽  
A. J. McKenna ◽  
J. A. Bygraves ◽  
I. M. Feavers
Keyword(s):  
Membrane Proteins ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Class 1 ◽  
Reference Strains

scholarly journals Within-host evolution of a Klebsiella pneumoniae clone: selected mutations associated with the alteration of outer membrane protein expression conferred multidrug resistance

2020 ◽  
Author(s):  
Masamune Aihara ◽  
Ruriko Nishida ◽  
Masaru Akimoto ◽  
Yasuhiro Gotoh ◽  
Makiko Kiyosuke ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Genetic Differences ◽  
Single Amino Acid ◽  
Upstream Region ◽  
Genome Comparison ◽  
Single Amino Acid Residue ◽  
Multiple Drugs ◽  
Host Evolution

Abstract Background A patient repeatedly developed bacteraemia despite the continuous use of antibiotics. We obtained two Klebsiella pneumoniae isolates from the patient’s blood on Days 72 and 105 after hospitalization. Each of the two isolates belonged to ST45, but while the first isolate was susceptible to most antibiotics, the second one was resistant to multiple drugs including carbapenems. Objectives To identify the genetic differences between the two isolates and uncover alterations formed by the within-host bacterial evolution leading to the antimicrobial resistance. Methods Whole-genome comparison of the two isolates was carried out to identify their genetic differences. We then profiled their outer membrane proteins related to membrane permeability to drugs. To characterize a ramR gene mutation found in the MDR isolate, its WT and mutant genes were cloned and expressed in the MDR isolate. Results The two isolates showed only three genomic differences, located in mdoH, ramR and upstream of ompK36. In the MDR isolate, a single nucleotide substitution in the ompK36 upstream region attenuated OmpK36 expression. A single amino acid residue insertion in RamR in the MDR isolate impaired its function, leading to the down-regulation of OmpK35 and the subsequent up-regulation of the AcrAB-TolC transporter, which may contribute to the MDR. Conclusions We identified very limited genomic changes in the second K. pneumoniae clone during within-host evolution, but two of the three identified mutations conferred the MDR phenotype on the clone by modulating drug permeability.

scholarly journals Sequence variation of rare outer membrane protein β-barrel domains in clinical strains provides insights into the evolution ofTreponema pallidumsubsp.pallidum, the syphilis spirochete

2018 ◽  
Author(s):  
Sanjiv Kumar ◽  
Melissa J. Caimano ◽  
Arvind Anand ◽  
Abhishek Dey ◽  
Kelly L. Hawley ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Evolutionary Biology ◽  
Sequence Variation ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Clinical Strains ◽  
Extracellular Loops ◽  
Reference Strains ◽  
Reference Genomes

ABSTRACTIn recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) ofTreponema pallidumsubspeciespallidum(TPA), the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions oftprC,tprD, andbamA, in 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we usedtp0548genotyping andtp0558sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants inTPAreference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. Based upon structural models, non-conservative amino acid substitutions in predicted transmembrane β-strands of TprC and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures driveTPAOMP diversity and that genetic exchange contributes to the evolutionary biology ofTPA. They also set the stage for topology-based analysis of antibody responses against OMPs and help frame strategies for syphilis vaccine development.IMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) ofTreponema pallidum(TPA), little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci,tprC,tprD, andbamA,inTPAfrom a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurred predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function also were noted. Our findings suggest that selection pressures exerted by human populations driveTPAOMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance ofTPAand frame strategies for vaccine development based upon conserved OMP extracellular loops.

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 Sequence Variation of Rare Outer Membrane Protein β-Barrel Domains in Clinical Strains Provides Insights into the Evolution ofTreponema pallidumsubsp.pallidum, the Syphilis Spirochete

mBio ◽  
2018 ◽  
Vol 9 (3) ◽  
Author(s):  
Sanjiv Kumar ◽  
Melissa J. Caimano ◽  
Arvind Anand ◽  
Abhishek Dey ◽  
Kelly L. Hawley ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Evolutionary Biology ◽  
Vaccine Development ◽  
Outer Membrane Proteins ◽  
Treponema Pallidum ◽  
Content Type ◽  
Clinical Strains ◽  
Extracellular Loops ◽  
Reference Strains ◽  
Reference Genomes

ABSTRACTIn recent years, considerable progress has been made in topologically and functionally characterizing integral outer membrane proteins (OMPs) ofTreponema pallidumsubspeciespallidum, the syphilis spirochete, and identifying its surface-exposed β-barrel domains. Extracellular loops in OMPs of Gram-negative bacteria are known to be highly variable. We examined the sequence diversity of β-barrel-encoding regions oftprC,tprD, andbamAin 31 specimens from Cali, Colombia; San Francisco, California; and the Czech Republic and compared them to allelic variants in the 41 reference genomes in the NCBI database. To establish a phylogenetic framework, we usedT. pallidum0548 (tp0548) genotyping andtp0558sequences to assign strains to the Nichols or SS14 clades. We found that (i) β-barrels in clinical strains could be grouped according to allelic variants inT. pallidumsubsp.pallidumreference genomes; (ii) for all three OMP loci, clinical strains within the Nichols or SS14 clades often harbored β-barrel variants that differed from the Nichols and SS14 reference strains; and (iii) OMP variable regions often reside in predicted extracellular loops containing B-cell epitopes. On the basis of structural models, nonconservative amino acid substitutions in predicted transmembrane β-strands ofT. pallidumrepeat C (TprC) and TprD2 could give rise to functional differences in their porin channels. OMP profiles of some clinical strains were mosaics of different reference strains and did not correlate with results from enhanced molecular typing. Our observations suggest that human host selection pressures driveT. pallidumsubsp.pallidumOMP diversity and that genetic exchange contributes to the evolutionary biology ofT. pallidumsubsp.pallidum. They also set the stage for topology-based analysis of antibody responses to OMPs and help frame strategies for syphilis vaccine development.IMPORTANCEDespite recent progress characterizing outer membrane proteins (OMPs) ofTreponema pallidum, little is known about how their surface-exposed, β-barrel-forming domains vary among strains circulating within high-risk populations. In this study, sequences for the β-barrel-encoding regions of three OMP loci,tprC,tprD, andbamA, inT. pallidumsubsp.pallidumisolates from a large number of patient specimens from geographically disparate sites were examined. Structural models predict that sequence variation within β-barrel domains occurs predominantly within predicted extracellular loops. Amino acid substitutions in predicted transmembrane strands that could potentially affect porin channel function were also noted. Our findings suggest that selection pressures exerted within human populations driveT. pallidumsubsp.pallidumOMP diversity and that recombination at OMP loci contributes to the evolutionary biology of syphilis spirochetes. These results also set the stage for topology-based analysis of antibody responses that promote clearance ofT. pallidumsubsp.pallidumand frame strategies for vaccine development based upon conserved OMP extracellular loops.

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