scholarly journals Major Surface Protein LipL32 Is Not Required for Either Acute or Chronic Infection with Leptospira interrogans

2008 ◽  
Vol 77 (3) ◽  
pp. 952-958 ◽  
Author(s):  
Gerald L. Murray ◽  
Amporn Srikram ◽  
David E. Hoke ◽  
Elsio A. Wunder ◽  
Rebekah Henry ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Chronic Infection ◽  
Matrix Protein ◽  
Severe Disease ◽  
Extracellular Matrix Protein ◽  
Leptospira Interrogans ◽  
Renal Tubules ◽  
Wild Type

ABSTRACT Leptospira interrogans is responsible for leptospirosis, a zoonosis of worldwide distribution. LipL32 is the major outer membrane protein of pathogenic leptospires, accounting for up to 75% of total outer membrane protein. In recent times LipL32 has become the focus of intense study because of its surface location, dominance in the host immune response, and conservation among pathogenic species. In this study, an lipL32 mutant was constructed in L. interrogans using transposon mutagenesis. The lipL32 mutant had normal morphology and growth rate compared to the wild type and was equally adherent to extracellular matrix. Protein composition of the cell membranes was found to be largely unaffected by the loss of LipL32, with no obvious compensatory increase in other proteins. Microarray studies found no obvious stress response or upregulation of genes that may compensate for the loss of LipL32 but did suggest an association between LipL32 and the synthesis of heme and vitamin B12. When hamsters were inoculated by systemic and mucosal routes, the mutant caused acute severe disease manifestations that were indistinguishable from wild-type L. interrogans infection. In the rat model of chronic infection, the LipL32 mutant colonized the renal tubules as efficiently as the wild-type strain. In conclusion, this study showed that LipL32 does not play a role in either the acute or chronic models of infection. Considering the abundance and conservation of LipL32 among all pathogenic Leptospira spp. and its absence in saprophytic Leptospira, this finding is remarkable. The role of this protein in leptospiral biology and pathogenesis thus remains elusive.

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 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.

Inactivation of a fibronectin-binding TonB-dependent protein increases adhesion properties of Bacteroides fragilis

2013 ◽  
Vol 62 (10) ◽  
pp. 1524-1530 ◽  
Author(s):  
Heidi Pauer ◽  
Soraia N. V. Cavalcanti ◽  
Felipe L. Teixeira ◽  
Joaquim Santos-Filho ◽  
Rossiane C. Vommaro ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Bacteroides Fragilis ◽  
Strictly Anaerobic ◽  
Wild Type ◽  
Extracellular Matrix Components ◽  
Mutant Strains ◽  
Fibronectin Binding

Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.

scholarly journals Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate

PLoS ONE ◽  
2015 ◽  
Vol 10 (11) ◽  
pp. e0142821 ◽  
Author(s):  
Thaís Larré Oliveira ◽  
André Alex Grassmann ◽  
Rodrigo Andrade Schuch ◽  
Amilton Clair Pinto Seixas Neto ◽  
Marcelo Mendonça ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Vaccine Candidate ◽  
Leptospira Interrogans

scholarly journals Putative Riemerella anatipestifer Outer Membrane Protein H Affects Virulence

2021 ◽  
Vol 12 ◽  
Author(s):  
Qun Gao ◽  
Shuwei Lu ◽  
Mingshu Wang ◽  
Renyong Jia ◽  
Shun Chen ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Pathogenic Bacteria ◽  
Lethal Dose ◽  
Protein A ◽  
Economic Losses ◽  
Wild Type ◽  
Riemerella Anatipestifer ◽  
Similar Growth

Riemerella anatipestifer causes serious contagious disease in ducks, geese, and other fowl. However, as a harmful pathogen causing significant economic losses in the poultry industry, R. anatipestifer is still poorly understood for its pathogenesis mechanisms. In a previous study, we developed an indirect ELISA method for detecting R. anatipestifer infection using B739_0832 protein, a putative outer membrane protein H (OmpH) that is conserved among different serotypes of R. anatipestifer. Although OmpH in some pathogenic bacteria, such as Pasteurella, has been reported as a virulence factor, it is still not clear whether B739_0832 protein contributes to the virulence of R. anatipestifer. In this study, we confirmed that B739_0832 protein in R. anatipestifer localizes to the outer membrane. We constructed a B739_0832 deletion mutant strain (ΔB739_0832) and assayed various effects from the deletion of B739_0832. ΔB739_0832 strain had a similar growth rate to wild-type R. anatipestifer CH-1. However, the survival rate of ducklings in 10 days after infection from ΔB739_0832 strain was 50%, whereas no ducklings survived from wild-type R. anatipestifer infection. Furthermore, the median lethal dose (LD50) of the ΔB739_0832 strain was approximately 150 times higher than that of the wild-type strain. Pathology examinations on infected ducklings found that, at 36 h after infection, bacterial loads in blood, liver, and brain tissues from ΔB739_0832-infected ducklings were considerably lower than those from wild-type infected ducklings. These results demonstrate that the B739_0832 protein contributes to the virulence of R. anatipestifer CH-1.

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 Identification of an Iron-Regulated Hemin-Binding Outer Membrane Protein, HupO, in Vibrio fluvialis: Effects on Hemolytic Activity and the Oxidative Stress Response

2005 ◽  
Vol 73 (2) ◽  
pp. 722-729 ◽  
Author(s):  
Sun-Hee Ahn ◽  
Jeong-Hyun Han ◽  
Jong-Hee Lee ◽  
Kee-Jai Park ◽  
In-Soo Kong
Keyword(s):  
Oxidative Stress ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Hemolytic Activity ◽  
Lethal Dose ◽  
Sodium Dodecyl ◽  
Watery Diarrhea ◽  
Wild Type ◽  
Vibrio Fluvialis

ABSTRACT In pathogenic bacteria, iron acquisition is important for colonization and proliferation in the host under iron-limited conditions. The ability of Vibrio spp. to acquire iron is often critical to their virulence, causing gastroenteritis or excessive watery diarrhea in humans. In the study described here, we cloned the 2,100-bp heme utilization protein gene hupO in Vibrio fluvialis. HupO had high homology to iron-regulated outer membrane receptor proteins in Vibrio sp. and contained motifs that are common to bacterial heme receptors, including a consensus TonB box, a FRAP domain, and an NPNL domain. To characterize the hemin-binding activity of HupO, we purified the recombinant HupO protein (rHupO) from Escherichia coli by using an overexpression system. HupO was found to bind to hemin but not to hemoglobin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting demonstrated that the 77-kDa outer membrane protein HupO of V. fluvialis was induced under iron-restricted conditions. We constructed a hupO mutant, HP1, to investigate the biochemical function of HupO in V. fluvialis. The hemolytic activity of HP1 was reduced compared to that of wild-type cells and, when exposed to hydrogen peroxide, significantly lower numbers of HP1 survived than was the case in the wild type. These results suggest that HupO is associated with virulence expression in V. fluvialis through stimulation of hemolysin production and resistance to oxidative stress. In experimentally infected mice, the 50% lethal dose value of the wild-type was lower than that of the mutant, HP1.

scholarly journals Crystal structure of the outer membrane protein OmpU fromVibrio choleraeat 2.2 Å resolution

2018 ◽  
Vol 74 (1) ◽  
pp. 21-29 ◽  
Author(s):  
Huanyu Li ◽  
Weijiao Zhang ◽  
Changjiang Dong
Keyword(s):  
Crystal Structure ◽  
Membrane Protein ◽  
Host Cell ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Severe Disease ◽  
Cell Interaction ◽  
Cell Binding ◽  
Important Virulence Factor ◽  
Host Cell Interaction

Vibrio choleraecauses a severe disease that kills thousands of people annually. The outer membrane protein OmpU is the most abundant outer membrane protein inV. cholerae, and has been identified as an important virulence factor that is involved in host-cell interaction and recognition, as well as being critical for the survival of the pathogenicV. choleraein the host body and in harsh environments. The mechanism of these processes is not well understood owing to a lack of the structure ofV. choleraeOmpU. Here, the crystal structure of theV. choleraeOmpU trimer is reported to a resolution of 2.2 Å. The protomer forms a 16-β-stranded barrel with a noncanonical N-terminal coil located in the lumen of the barrel that consists of residues Gly32–Ser42 and is observed to participate in forming the second gate in the pore. By mapping the published functional data onto the OmpU structure, the OmpU structure reinforces the notion that the long extracellular loop L4 with a β-hairpin-like motif may be critical for host-cell binding and invasion, while L3, L4 and L8 are crucially implicated in phage recognition byV. cholerae.

scholarly journals Heterogeneity among Helicobacter pylori Strains in Expression of the Outer Membrane Protein BabA

2004 ◽  
Vol 72 (6) ◽  
pp. 3429-3435 ◽  
Author(s):  
Ewa E. Hennig ◽  
Ray Mernaugh ◽  
Jennifer Edl ◽  
Ping Cao ◽  
Timothy L. Cover
Keyword(s):  
Helicobacter Pylori ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Recombinant Antibody ◽  
Wild Type ◽  
Single Chain ◽  
H Pylori ◽  
Different Strains

ABSTRACT The BabA adhesin of Helicobacter pylori is an outer membrane protein that binds to the fucosylated Lewis b histo-blood group antigen on the surface of gastric epithelial cells. We screened a phage-displayed ScFv (single-chain fragment variable) recombinant antibody library for antibodies reactive with a recombinant BabA fragment and identified two such antibodies. Each antibody recognized an ∼75-kDa protein present in wild-type H. pylori strain J99 but absent from an isogenic babA mutant strain. An immunoreactive BabA protein was detected by at least one of the antibodies in 18 (46%) of 39 different wild-type H. pylori strains and was detected more commonly in cagA-positive strains than in cagA-negative strains. Numerous amino acid polymorphisms were detected among BabA proteins expressed by different strains, with the greatest diversity occurring in the middle region of the proteins. Among the 18 strains that expressed a detectable BabA protein, there was considerable variation in the level of binding to Lewis b in vitro. Heterogeneity among H. pylori strains in expression of the BabA protein may be a factor that contributes to differing clinical outcomes among H. pylori-infected humans.

Effect of mutations in the general secretory pathway on outer membrane protein and surface layer assembly in Aeromonas spp.

1995 ◽  
Vol 41 (6) ◽  
pp. 525-532 ◽  
Author(s):  
S Peter Howard ◽  
Heather G. Meiklejhon
Keyword(s):  
Surface Layer ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Secretory Pathway ◽  
Immunoblot Analysis ◽  
Surface Proteins ◽  
Wild Type ◽  
Extracellular Secretion ◽  
In The Wild

Mutations in the exeC-N operon of Aeromonas hydrophila, which block extracellular protein secretion, also result in large decreases in the level of the major outer membrane porin, protein II. Immunoblot analysis demonstrated that the porin missing from the outer membrane of the mutant was not accumulating elsewhere in the cell. Pulse-chase and immunoprecipitation analyses showed that the porin was as stable in the mutant as in the wild type, but that far less porin was synthesized in the exe mutants. The relationship between extracellular secretion involving the exe genes and the assembly of other outer membrane and surface proteins was also examined. Both the wild type and exeE mutants of A. hydrophila were capable of assembling the protein I and protein III porins under inducing conditions. Aeromonas sohria As9071, which contains a surface array protein, could secrete A. hydrophila aerolysin and required homologues of the A. hydrophila exe genes to do so; however, an exe− derivative of this bacteria was unaffected in its ability to assemble its surface array. These results demonstrate that the exe genes are not required for general outer membrane protein assembly in these bacteria, but that the synthesis of protein II is specifically downregulated in the exe mutants.Key words: extracellular secretion, outer membrane assembly, surface layer, Aeromonas.

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