scholarly journals LipL21 Is a Novel Surface-Exposed Lipoprotein of Pathogenic Leptospira Species

2003 ◽  
Vol 71 (5) ◽  
pp. 2414-2421 ◽  
Author(s):  
Paul A. Cullen ◽  
David A. Haake ◽  
Dieter M. Bulach ◽  
Richard L. Zuerner ◽  
Ben Adler
Keyword(s):  
Outer Membrane ◽  
Global Analysis ◽  
Outer Membrane Proteins ◽  
Sucrose Density Gradient ◽  
Etiologic Agent ◽  
Signal Peptidase ◽  
Gene Encoding ◽  
Pathogenic Leptospira ◽  
Outer Membrane Lipoprotein ◽  
Triton X

ABSTRACT Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.

scholarly journals Temperature-Regulated Protein Synthesis by Leptospira interrogans

2001 ◽  
Vol 69 (1) ◽  
pp. 400-404 ◽  
Author(s):  
Jarlath E. Nally ◽  
John F. Timoney ◽  
Brian Stevenson
Keyword(s):  
Protein Synthesis ◽  
Outer Membrane ◽  
Pathogenic Bacteria ◽  
Outer Membrane Proteins ◽  
Pathogen Transmission ◽  
Leptospira Interrogans ◽  
Outer Membrane Lipoprotein ◽  
Higher Temperature ◽  
Response To Temperature ◽  
Triton X

ABSTRACT Leptospira interrogans is an important mammalian pathogen. Transmission from an environmental source requires adaptations to a range of new environmental conditions in the organs and tissues of the infected host. Since many pathogenic bacteria utilize temperature to discern their environment and regulate the synthesis of appropriate proteins, we investigated the effects of temperature on protein synthesis in L. interrogans. Bacteria were grown for several days after culture temperatures were shifted from 30 to 37°C. Triton X-114 cellular fractionation identified several proteins of the cytoplasm, periplasm, and outer membrane for which synthesis was dependent on the culture temperature. Synthesis of a cytoplasmic protein of 20 kDa was switched off at 37°C, whereas synthesis of a 66-kDa periplasmic protein was increased at the higher temperature. Increased synthesis of a 25-kDa outer membrane protein was observed when the organisms were shifted from 30 to 37°C. A 36-kDa protein synthesized at 30 but not at 37°C was identified as LipL36, an outer membrane lipoprotein. In contrast, expression of another lipoprotein, LipL41, was the same at either temperature. Immunoblotting with convalescent equine sera revealed that some proteins exhibiting thermoregulation of synthesis elicited antibody responses during infection. Our results show that sera from horses which aborted as a result of naturally acquired infection withL. interrogans serovar pomona type kennewicki recognize periplasmic and outer membrane proteins which are differentially synthesized in response to temperature and which therefore may be important in the host-pathogen interaction during infection.

scholarly journals Outer Membrane Proteins Omp10, Omp16, and Omp19 of Brucella spp. Are Lipoproteins

1999 ◽  
Vol 67 (9) ◽  
pp. 4960-4962 ◽  
Author(s):  
Anne Tibor ◽  
Béatrice Decelle ◽  
Jean-Jacques Letesson
Keyword(s):  
Monoclonal Antibodies ◽  
Membrane Proteins ◽  
Palmitic Acid ◽  
Outer Membrane ◽  
Brucella Abortus ◽  
Outer Membrane Proteins ◽  
Specific Inhibitor ◽  
Signal Peptidase ◽  
Processing Sequence ◽  
Triton X

ABSTRACT The deduced sequences of the Omp10, Omp16, and Omp19 outer membrane proteins of Brucella spp. contain a potential bacterial lipoprotein processing sequence. After extraction with Triton X-114, these three proteins partitioned into the detergent phase. Processing of the three proteins is inhibited by globomycin, a specific inhibitor of lipoprotein signal peptidase. The three proteins were radioimmunoprecipitated from [3H]palmitic acid-labeledBrucella abortus lysates with monoclonal antibodies. These results demonstrate that Omp10, Omp16, and Omp19 are lipoproteins.

scholarly journals LipL46 is a novel surface-exposed lipoprotein expressed during leptospiral dissemination in the mammalian host

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3777-3786 ◽  
Author(s):  
James Matsunaga ◽  
Kristian Werneid ◽  
Richard L. Zuerner ◽  
Ami Frank ◽  
David A. Haake
Keyword(s):  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Signal Peptidase ◽  
Mammalian Host ◽  
Renal Tubules ◽  
Phase Partitioning ◽  
Peripheral Membrane Protein ◽  
Major Antigen ◽  
Triton X ◽  
Reservoir Hosts

Leptospirosis is a widespread zoonosis caused by invasive spirochaetes belonging to the genus Leptospira. Pathogenic leptospires disseminate via the bloodstream to colonize the renal tubules of reservoir hosts. Little is known about leptospiral outer-membrane proteins expressed during the dissemination stage of infection. In this study, a novel surface-exposed lipoprotein is described; it has been designated LipL46 to distinguish it from a previously described 31 kDa peripheral membrane protein, P31LipL45, which is exported as a 45 kDa probable lipoprotein. The lipL46 gene encodes a 412 aa polypeptide with a 21 aa signal peptide. Lipid modification of cysteine at the lipoprotein signal peptidase cleavage site FSISC is supported by the finding that Leptospira interrogans intrinsically labels LipL46 during incubation in medium containing [14C]palmitate. LipL46 appears to be exported to the leptospiral outer membrane as a 46 kDa lipoprotein, based on Triton X-114 solubilization and phase partitioning studies, which included the outer and inner membrane controls LipL32 and LipL31, respectively. Surface immunoprecipitation and whole-cell ELISA experiments indicate that LipL46 is exposed on the leptospiral surface. Immunohistochemistry studies demonstrated expression of LipL46 by leptospires found in the bloodstream of acutely infected hamsters. Leptospires expressing LipL46 were also found in the intercellular spaces of the liver, within splenic phagocytes, and invading the glomerular hilum of the kidney. Infection-associated expression is supported by the finding that LipL46 is a major antigen recognized by sera from infected hamsters. These findings indicate that LipL46 may be important in leptospiral dissemination, and that it may serve as a useful serodiagnostic antigen.

Identification and cloning of the gene encoding BmpC: an outer-membrane lipoprotein associated with Brachyspira pilosicoli membrane vesicles

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 1041-1053 ◽  
Author(s):  
Darren J. Trott ◽  
David P. Alt ◽  
Richard L. Zuerner ◽  
Dieter M. Bulach ◽  
Michael J. Wannemuehler ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Host Species ◽  
Membrane Vesicles ◽  
Immunogold Labelling ◽  
Gene Encoding ◽  
Brachyspira Pilosicoli ◽  
Density Fraction ◽  
Outer Membrane Lipoprotein ◽  
Protein Contamination ◽  
Triton X

The intestinal spirochaete Brachyspira pilosicoli causes colitis in a wide variety of host species. Little is known about the structure or protein constituents of the B. pilosicoli outer membrane (OM). To identify surface-exposed proteins in this species, membrane vesicles were isolated from B. pilosicoli strain 95-1000 cells by osmotic lysis in dH2O followed by isopycnic centrifugation in sucrose density gradients. The membrane vesicles were separated into a high-density fraction (HDMV; ρ=1·18 g cm−3) and a low-density fraction (LDMV; ρ=1·12 g cm−3). Both fractions were free of flagella and soluble protein contamination. LDMV contained predominantly OM markers (lipo-oligosaccharide and a 29 kDa B. pilosicoli OM protein) and was used as a source of antigens to produce mAbs. Five B. pilosicoli-specific mAbs reacting with proteins with molecular masses of 23, 24, 35, 61 and 79 kDa were characterized. The 23 kDa protein was only partially soluble in Triton X-114, whereas the 24 and 35 kDa proteins were enriched in the detergent phase, implying that they were integral membrane proteins or lipoproteins. All three proteins were localized to the B. pilosicoli OM by immunogold labelling using specific mAbs. The gene encoding the abundant, surface-exposed 23 kDa protein was identified by screening a B. pilosicoli 95-1000 genome library with the mAb and was expressed in Escherichia coli. Sequence analysis showed that it encoded a unique lipoprotein, designated BmpC. Recombinant BmpC partitioned predominantly in the OM fraction of E. coli strain SOLR. The mAb to BmpC was used to screen a collection of 13 genetically heterogeneous strains of B. pilosicoli isolated from five different host species. Interestingly, only strain 95-1000 was reactive with the mAb, indicating that either the surface-exposed epitope on BmpC is variable between strains or that the protein is restricted in its distribution within B. pilosicoli.

scholarly journals Correction: Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans

2015 ◽  
Vol 9 (12) ◽  
pp. e0004286 ◽  
Author(s):  
Leandro C. D. Breda ◽  
Ching-Lin Hsieh ◽  
Mónica M. Castiblanco Valencia ◽  
Ludmila B. da Silva ◽  
Angela S. Barbosa ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Fine Mapping ◽  
Binding Protein ◽  
Outer Membrane Proteins ◽  
Leptospira Interrogans ◽  
Pathogenic Leptospira

scholarly journals Fine Mapping of the Interaction between C4b-Binding Protein and Outer Membrane Proteins LigA and LigB of Pathogenic Leptospira interrogans

2015 ◽  
Vol 9 (10) ◽  
pp. e0004192 ◽  
Author(s):  
Leandro C. D. Breda ◽  
Ching-Lin Hsieh ◽  
Mónica M. Castiblanco Valencia ◽  
Ludmila B. da Silva ◽  
Angela S. Barbosa ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Fine Mapping ◽  
Binding Protein ◽  
Outer Membrane Proteins ◽  
Leptospira Interrogans ◽  
Pathogenic Leptospira

scholarly journals Putative β-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity

2020 ◽  
Vol 88 (5) ◽  
Author(s):  
G. J. Staton ◽  
S. D. Carter ◽  
S. Ainsworth ◽  
J. Mullin ◽  
R. F. Smith ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Functional Characterization ◽  
Signal Peptidase ◽  
Effective Vaccine ◽  
Digital Dermatitis ◽  
Diverse Range ◽  
Content Type ◽  
Signal Peptidase I

ABSTRACT Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

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