Major Outer Membrane Lipoprotein Research Articles

Identification of Bacterial Drug-Resistant Cells by the Convolutional Neural Network in Transmission Electron Microscope Images.

The emergence of bacteria that are resistant to antibiotics is common in areas where antibiotics are used widely. The current standard procedure for detecting bacterial drug resistance is based on bacterial growth under antibiotic treatments. Here we describe the morphological changes in enoxacin-resistant Escherichia coli cells and the computational method used to identify these resistant cells in transmission electron microscopy (TEM) images without using antibiotics. Our approach was to create patches from TEM images of enoxacin-sensitive and enoxacin-resistant E. coli strains, use a convolutional neural network for patch classification, and identify the strains on the basis of the classification results. The proposed method was highly accurate in classifying cells, achieving an accuracy rate of 0.94. Using a gradient-weighted class activation mapping to visualize the region of interest, enoxacin-resistant and enoxacin-sensitive cells were characterized by comparing differences in the envelope. Moreover, Pearson’s correlation coefficients suggested that four genes, including lpp, the gene encoding the major outer membrane lipoprotein, were strongly associated with the image features of enoxacin-resistant cells.

Oral immunization of rat with chromosomal expression LipL32 in attenuated Salmonella vaccine induces immune respond against pathogenic Leptospira.

PurposeLeptospirosis caused by Leptospira spp. remains a global health problem. Available commercial leptospiral vaccines have shown an ineffective prevention for leptospiral infection. The aim of this study was to develop leptospirosis vaccine using recombinant attenuated Salmonella vaccine (RASV) as a platform. We expected that this vaccine has ability to continuous and strongly stimulate immune systems including protective mucosal, humoral, and cell mediated immunity in rat model.Materials and MethodsIn this study, we engineered RASV, NRSL32 strain containing chromosomal fusion between nucleotides encoding secretion signal of SPI-2 effector protein, SspH2 and gene encoding major pathogenic leptospiral outer membrane lipoprotein, LipL32. Subsequently, our modified RASV was oral vaccination to rat and blood samples were taken for assessment of immune responses.ResultsOur Salmonella NRSL32 strain showed expression and secretion of SspH21-215-LipL32 recombinant protein via SPI-2 T3SS. After oral administration of NRSL32 strain to rats, significant titers of total immunoglobulin G (IgG) and immunoglobulin A against rLipL32 were observed in long period up to 77 days after vaccination. The stimulated antibody showed ability to specific bind with LipL32 protein on surface of pathogenic Leptospira spp. Additionally, the balance level of IgG2a/IgG1 ratio and level of interferon-γ and interleukin-4 secretion were detected.ConclusionThe results showed that our RASV platform with chromosomal expression elicited effective immune responses to leptospiral antigen. Moreover, this platform was capable for simultaneous stimulation of Th1 and Th2-biased responses. Further investigation is necessary study of protective efficacy against leptospiral infection in animal models.

Lipid-tagged single domain antibodies for improved enzyme-linked immunosorbent assays

Anti-Staphylococcal Enterotoxin B single domain antibodies were engineered to include the N-terminal peptide sequence of the major outer membrane lipoprotein from Escherichia coli, which directs the N-terminal addition of lipid to the single domain antibody. We produced and purified two different single domain antibodies as well as a variant and dimer construct of one of the two, all with and without the added lipid. Their ability to function as the capture antibody in standard enzyme-linked immunosorbent assays were evaluated, finding that coating polystyrene microtiter plates with the lipid-tagged single domain antibodies gave a 3-fold improvement in the observed limit of detection. This increase was likely due to an increased amount of single domain antibody adsorbed to the microtiter plate, which translated to improved limits of detection of Staphylococcal Enterotoxin B over using the same single domain antibody sans lipid-tag. However, improved orientation may also play a role. Regardless of the mechanism, the biosynthetic lipid-tagging of single domain antibodies represent a facile modality that can enhance their ability to be utilized as immunoassay capture reagent as well as facilitate their incorporation into liposome targeting applications in the future.

The Miller Hypothesis.

The immune response is a cornerstone in the body's struggle against microbial pathogens. In ways that we do not yet completely understand, the mammalian immune response has evolved to identify proteins of pathogens that are either important virulence factors or key immunoprotective targets. Professor James N. Miller suggested that one way to discover such proteins is to harness the power of the immune system in the laboratory.This general concept, referred to here as the Miller Hypothesis, took several different manifestations in the discovery of some of the best known and widely studied leptospiral proteins: The porin OmpL1 was identified by surface immunoprecipitation, leptospiral immunoglobulin-like proteins were uncovered by screening a genomic library with sera from leptospirosis patients, and the major outer-membrane lipoprotein LipL32 was recognized through immunoblot studies. Such approaches will continue to bear fruit for both the leptospiral research field and research on other invasive pathogens.

In Silico Evaluation of Different Signal Peptides for the Secretory Production of Human Growth Hormone in E. coli

Various advantages of protein secretion have prompted scientists to search for secretory production of heterologous proteins. Signal peptides are one of the most important factors for prosperous secretion of the recombinant proteins. The aim of this study was to evaluate 23 different signal peptides and theoretically determine suitable ones for secretory production of the human growth hormone (hGH) in the E. coli host. The signal peptide sequences and the precise location of their cleavage sites were predicted using SignalP 4.0 server. Accordingly, six of the signal peptides, including hGH, major outer membrane lipoprotein, protease VII, protein TolB, periplasmic protein TorT and beta-lactamase TEM were excluded from the further study, due to their inappropriate cleavage scores. Different physico-chemical properties, which are essential for selecting a proper signal peptide, were evaluated using ProtParam and Solpro as the most accurate and reliable servers. Computational analysis of the above-mentioned factors, indicates that outer membrane protein C, fimbrial chaperone SfmC, outer membrane protein F and disulfide interchange protein DsbA can theoretically be suitable signal peptides for hGH secretion.

MicL, a new σE-dependent sRNA, combats envelope stress by repressing synthesis of Lpp, the major outer membrane lipoprotein.

In enteric bacteria, the transcription factor σ(E) maintains membrane homeostasis by inducing synthesis of proteins involved in membrane repair and two small regulatory RNAs (sRNAs) that down-regulate synthesis of abundant membrane porins. Here, we describe the discovery of a third σ(E)-dependent sRNA, MicL (mRNA-interfering complementary RNA regulator of Lpp), transcribed from a promoter located within the coding sequence of the cutC gene. MicL is synthesized as a 308-nucleotide (nt) primary transcript that is processed to an 80-nt form. Both forms possess features typical of Hfq-binding sRNAs but surprisingly target only a single mRNA, which encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. We show that the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. We also report that σ(E) activity is sensitive to Lpp abundance and that MicL and Lpp comprise a new σ(E) regulatory loop that opposes membrane stress. Together MicA, RybB, and MicL allow σ(E) to repress the synthesis of all abundant outer membrane proteins in response to stress.

Docking studies of doxycycline on pathogenic Leptospira Species with common pharmacopore

Every disease can be cured if its roots are traced; means there is cure for every disease. Leptospirosis is a hidden and emerging public health problem as it causes fever, acute renal failure and jaundice. The major outer membrane lipoprotein “LipL32” is expressed during infection which is highly conserved among pathogenic leptospira. It is absent in non-pathogenic avirulent leptospira. Insilico comparison of amino acid sequences of LipL32 showed 97.8% average sequence identity among the pathogenic species. In this study some 3D model were built for LipL32 protein using molecular modelling. Protein hydrophobicity plots were utilized to locate the antigenic sites. Docking studies of retrieved structures and modeled LipL32 proteins with doxycycline revealed that doxycycline can inhibit LipL32 proteins of Leptospira santarosai, Leptospira weilii and Leptospira kirschner as they have bound docking with doxycycline. They share a common pharmacopore within a genus and a common drug may be utilized to combat all the related species.

Overexpression of LolCDE allows deletion of the Escherichia coli gene encoding apolipoprotein N-acyltransferase.

Bacterial lipoproteins represent a subset of membrane-associated proteins that are covalently modified with lipids at the N-terminal cysteine. The final step of lipoprotein modification, N-acylation of apolipoproteins, is mediated by apolipoprotein N-acyltransferase (Lnt). Examinations with reconstituted proteoliposomes and a conditional mutant previously indicated that N-acylation of lipoproteins is required for their efficient release from the inner membrane catalyzed by LolA and LolCDE, the lipoprotein-specific chaperone and ABC transporter, respectively. Because Lnt is essential for Escherichia coli, a mutant lacking Lnt activity has not been isolated. However, we report here that lnt-null strains can be constructed when LolCDE is overproduced in strains lacking either the major outer membrane lipoprotein Lpp or transpeptidases that cross-link Lpp with peptidoglycan. Lipoproteins purified from the lnt-null strain exhibited increased mobility on SDS-PAGE compared to those from wild-type cells and could be sequenced by Edman degradation, indicating that lipoproteins in this mutant exist as apolipoproteins that lack N-acylation. Overexpression of Lpp in the lnt-null strain resulted in the accumulation of apoLpp in the inner membrane and caused growth arrest. In contrast to the release of mature Lpp in the presence of LolA and LolCDE, that of apoLpp from the inner membrane was significantly retarded. Furthermore, the amount of lipoproteins copurified with LolCDE was significantly reduced in the lnt-null strain. These results indicate that the affinity of LolCDE for apolipoprotein is very low, and therefore, overexpression of LolCDE is required for its release and sorting to the outer membrane.

Phosphatidic Acid and N-Acylphosphatidylethanolamine Form Membrane Domains in Escherichia coli Mutant Lacking Cardiolipin and Phosphatidylglycerol

The pgsA null Escherichia coli strain, UE54, lacks the major anionic phospholipids phosphatidylglycerol and cardiolipin. Despite these alterations the strain exhibits relatively normal cell division. Analysis of the UE54 phospholipids using negativeion electrospray ionization mass spectrometry resulted in identification of a new anionic phospholipid, N-acylphosphatidylethanolamine. Staining with the fluorescent dye 10-N-nonyl acridine orange revealed anionic phospholipid membrane domains at the septal and polar regions. Making UE54 null in minCDE resulted in budding off of minicells from polar domains. Analysis of lipid composition by mass spectrometry revealed that minicells relative to parent cells were significantly enriched in phosphatidic acid and N-acylphosphatidylethanolamine. Thus despite the absence of cardiolipin, which forms membrane domains at the cell pole and division sites in wild-type cells, the mutant cells still maintain polar/septal localization of anionic phospholipids. These three anionic phospholipids share common physical properties that favor polar/septal domain formation. The findings support the proposed role for anionic phospholipids in organizing amphitropic cell division proteins at specific sites on the membrane surface.

In LipL32, the Major Leptospiral Lipoprotein, the C Terminus Is the Primary Immunogenic Domain and Mediates Interaction with Collagen IV and Plasma Fibronectin

LipL32 is the major leptospiral outer membrane lipoprotein expressed during infection and is the immunodominant antigen recognized during the humoral immune response to leptospirosis in humans. In this study, we investigated novel aspects of LipL32. In order to define the immunodominant domains(s) of the molecule, subfragments corresponding to the N-terminal, intermediate, and C-terminal portions of the LipL32 gene were cloned and the proteins were expressed and purified by metal affinity chromatography. Our immunoblot results indicate that the C-terminal and intermediate domains of LipL32 are recognized by sera of patients with laboratory-confirmed leptospirosis. An immunoglobulin M response was detected exclusively against the LipL32 C-terminal fragment in both the acute and convalescent phases of illness. We also evaluated the capacity of LipL32 to interact with extracellular matrix (ECM) components. Dose-dependent, specific binding of LipL32 to collagen type IV and plasma fibronectin was observed, and the binding capacity could be attributed to the C-terminal portion of this molecule. Both heparin and gelatin could inhibit LipL32 binding to fibronectin in a concentration-dependent manner, indicating that the 30-kDa heparin-binding and 45-kDa gelatin-binding domains of fibronectin are involved in this interaction. Taken together, our results provide evidence that the LipL32 C terminus is recognized early in the course of infection and is the domain responsible for mediating interaction with ECM proteins.

In vivo production of oligodeoxyribonucleotides of specific sequences: application to antisense DNA.

Retrons, bacterial retroelements found in Gram-negative bacteria, are integrated into the bacterial genome expressing a reverse transcriptase related to eukaryotic reverse transcriptase. The bacterial reverse transcriptases are responsible for the production of multicopy, single-stranded (ms) DNA consisting of a short single-stranded DNA that is attached to an internal guanosine residue of an RNA molecule by a 2',5'-phosphodiester linkage. Reverse transcriptases use an RNA transcript from the retrons, not only as primer, but also as template for msDNA synthesis. By studying the structural requirement, it was found that for msDNA synthesis an internal region of msDNA can be replaced with other sequences. msDNA can thus be used as a vector for in vivo production of an oligodeoxyribonucleotide of a specific sequence. Artificial msDNAs containing a sequence complementary to part of the mRNA for the major outer membrane lipoprotein of Escherichia coli effectively inhibited lipoprotein biosynthesis upon induction of msDNA synthesis. This is the first demonstration of in vivo synthesis of oligodeoxyribonucleotides having antisense function. Since we have previously demonstrated that bacterial retrons are functional in eukaryotes producing msDNA in yeast and in mouse NIH/3T3 fibroblasts, the present system may also be used to produce a specific oligodeoxyribonucleotide inside the cells to regulate eukaryotic gene expression artificially. We also describe a method to produce cDNA to a specific cellular mRNA using the retron system.

Interaction of a Neurotropic Strain ofBorrelia turicataewith the Cerebral Microcirculation System

Relapsing fever (RF) is a spirochetal infection characterized by relapses of a febrile illness and spirochetemia due to the sequential appearance and disappearance of isogenic serotypes in the blood. The only difference between isogenic serotypes is the variable major outer membrane lipoprotein. In the absence of specific antibody, established serotypes cause persistent infection. Studies in our laboratory indicate that another consequence of serotype switching in RF is a change in neuroinvasiveness. As the next step to elucidate this phenomenon, we studied the interaction of the neurotropic Oz1 strain of the RF agent Borrelia turicatae with the cerebral microcirculation. During persistent infection of antibody-deficient mice, we found that serotype 1 entered the brain in larger numbers and caused more severe cerebral microgliosis than isogenic serotype 2. Microscopic examination revealed binding of B. turicatae to brain microvascular endothelial cells in vivo. In vitro we found that B. turicatae associated with brain microvascular endothelial cells (BMEC) significantly more than with fibroblasts or arachnoidal cells. The binding was completely eliminated by pretreatment of BMEC with proteinase K. Using transwell chambers with BMEC barriers, we found that serotype 1 crossed into the lower compartment significantly better than serotype 2. Heat killing significantly reduced BMEC crossing but not binding. We concluded that the interaction of B. turicatae with the cerebral microcirculation involves both binding and crossing brain microvascular endothelial cells, with significant differences among isogenic serotypes.

Recombinant Mannheimia haemolytica serotype 1 outer membrane protein PlpE enhances commercial M. haemolytica vaccine-induced resistance against serotype 6 challenge

Mannheimia haemolytica outer membrane protein PlpE, a major immunogenic outer membrane lipoprotein has identical sequences in serotypes 1 (S1) and S6. Recombinant outer membrane lipoprotein PlpE (rPLpE) from M. haemolytica S1 was added to commercial M. haemolytica S1 vaccines to determine if it would enhance vaccine-induced immunity against heterotypic M. haemolytica S6 challenge. Serum antibody responses to M. haemolytica whole cells, leukotoxin and rPlpE were measured. Experiment 1 consisted of four vaccine groups: controls, 100 μg rPlpE, M. haemolytica Bacterin-Toxoid (One Shot™) and M. haemolytica Bacterin-Toxoid + 100 μg rPlpE. Vaccines were given on day 0. On day 21, calves were challenged transthoracically with M. haemolytica S6. Lung lesion scores and percentage lesion reduction were 6.3 ± 2.0 for controls, 3.6 ± 2.4 for rPlpE vaccinates (42.9% reduction), 3.4 ± 1.5 for One Shot-vaccinates (46.0% reduction), and 2.4 ± 1.4 for One Shot/rPlpE vaccinates (61.9% reduction). Experiment 2 consisted of four vaccine groups: controls, 100 μg rPlpE, M. haemolytica toxoid (Presponse ®), and M. haemolytica toxoid + 100 μg rPlpE. On day 28, calves were challenged transthoracically with M. haemolytica S6. Lung lesion scores and percentage lesion reduction were 8.1 ± 2.2 for controls, 4.4 ± 4.7 for the rPlpE vaccinates (45.7% reduction), 4.8 ± 2.2 for Presponse-vaccinates (40.7% reduction), and 2.0 ± 1.2 for Presponse/rPlpE vaccinates (75.3% reduction). These results indicate that addition of rPlpE from M. haemolytica S1 can enhance commercial M. haemolytica vaccine-induced resistance against experimental challenge with M. haemolytica S6.

Activation of the Rcs signal transduction system is responsible for the thermosensitive growth defect of an Escherichia coli mutant lacking phosphatidylglycerol and cardiolipin.

The lethal effect of an Escherichia coli pgsA null mutation, which causes a complete lack of the major acidic phospholipids, phosphatidylglycerol and cardiolipin, is alleviated by a lack of the major outer membrane lipoprotein encoded by the lpp gene, but an lpp pgsA strain shows a thermosensitive growth defect. Using transposon mutagenesis, we found that this thermosensitivity was suppressed by disruption of the rcsC, rcsF, and yojN genes, which code for a sensor kinase, accessory positive factor, and phosphotransmitter, respectively, of the Rcs phosphorelay signal transduction system initially identified as regulating the capsular polysaccharide synthesis (cps) genes. Disruption of the rcsB gene coding for the response regulator of the system also suppressed the thermosensitivity, whereas disruption of cpsE did not. By monitoring the expression of a cpsB'-lac fusion, we showed that the Rcs system is activated in the pgsA mutant and is reverted to a wild-type level by the rcs mutations. These results indicate that envelope stress due to an acidic phospholipid deficiency activates the Rcs phosphorelay system and thereby causes the thermosensitive growth defect independent of the activation of capsule synthesis.

Construction and evaluation of a plasmid vector for the expression of recombinant lipoproteins in Escherichia coli

Outer membrane lipoproteins are emerging as key targets for protective immunity to many bacterial pathogens. Heterologous expression of lipoproteins in Escherichia coli does not always result in high level expression of acylated recombinant protein. Thus, these proteins do not take up their correct membrane topology and are lacking the immunostimulatory properties endowed by the lipid. To this end, we have designed a lipoprotein expression vector (pDUMP) that results in the production of fusion proteins containing the E. coli major outer membrane lipoprotein (Lpp) signal sequence, lipoprotein signal peptidase recognition site, and the +2 outer membrane sorting signal at their N termini. To test the ability of pDUMP to express lipoproteins from heterologous hosts, the surface lipoprotein PsaA from the Gram-positive organism Streptococcus pneumoniae and the outer membrane lipoproteins MlpA from the Gram-negative Pasteurella multocida and BlpA from the spirochete Brachyspira hyodysenteriae were cloned into both hexahistidine fusion vectors and pDUMP. High level expression of antigenically active protein from both the hexahistidine fusion vectors and pDUMP resulted in abundant bands of the predicted molecular masses when analyzed by SDS–PAGE. When grown in the presence of 3[H]palmitic acid, proteins encoded by pDUMP were observed to incorporate palmitic acid whilst the hexahistidine fusion proteins did not. Using mass spectrometry and image analysis we determined the efficiency of lipidation between the three clones to vary from 31.7 to 100%. In addition, lipidated, but not hexahistidine, forms of the proteins were presented on the E. coli surface.

Envelope Disorder of Escherichia coli Cells Lacking Phosphatidylglycerol

Phosphatidylglycerol, the most abundant acidic phospholipid in Escherichia coli, is considered to play specific roles in various cellular processes that are essential for cell viability. A null mutation of pgsA, which encodes phosphatidylglycerophosphate synthase, does indeed confer lethality. However, pgsA null mutants are viable if they lack the major outer membrane lipoprotein (Lpp) (lpp mutant) (S. Kikuchi, I. Shibuya, and K. Matsumoto, J. Bacteriol. 182:371-376, 2000). Here we show that Lpp expressed from a plasmid causes cell lysis in a pgsA lpp double mutant. The envelopes of cells harvested just before lysis could not be separated into outer and inner membrane fractions by sucrose density gradient centrifugation. In contrast, expression of a mutant Lpp (LppdeltaK) lacking the COOH-terminal lysine residue (required for covalent linking to peptidoglycan) did not cause lysis and allowed for the clear separation of the outer and inner membranes. We propose that in pgsA mutants LppdeltaK could not be modified by the addition of a diacylglyceryl moiety normally provided by phosphatidylglycerol and that this defect caused unmodified LppdeltaK to accumulate in the inner membrane. Although LppdeltaK accumulation did not lead to lysis, the accumulation of unmodified wild-type Lpp apparently led to the covalent linking to peptidoglycan, causing the inner membrane to be anomalously anchored to peptidoglycan and eventually leading to lysis. We suggest that this anomalous anchoring largely explains a major portion of the nonviable phenotypes of pgsA null mutants.

Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in leukotoxin

Actinobacillus actinomycetemcomitans is associated with early onset periodontal diseases and secretes membranous vesicles that appear to contain several virulence-associated proteins. However, the composition of these vesicles and the process leading to their secretion are not well defined. Electron micrographs of thin sectioned bacterial cells and purified vesicle preparations showed that vesicles are spherical lipid bilayers, 50–100 nm in diameter, that appear to form by budding from the outer membrane of the bacterium. Thin layer chromatography identified the predominant lipid components of vesicles as lipopolysaccharide, phosphatidylethanolamine and cardiolipin, similar to the main lipid constituents of the outer membrane. However, vesicles also contained minor lipids that were not detected in outer membrane samples. The major protein constituents of vesicles co-migrated with proteins in outer membrane extracts of A. actinomycetemcomitans, but the outer membrane preparations possessed polypeptides that were not detected in vesicles. Three vesicle proteins were identified; the heat-modifiable OmpA homologue of A. actinomycetemcomitans, a 28 kDa lipoprotein related to the major outer membrane lipoprotein of Mannheimia haemolytica and leukotoxin. Incubation of leukotoxin-sensitive human HL60 cells with vesicles from A. actinomycetemcomitans strains JP2 and 652 resulted in cell lysis, indicating that vesicle-associated leukotoxin is biologically active. Vesicles from the highly leukotoxic strain JP2 were five- to 10-fold more toxic than vesicles from the minimally leukotoxic 652 strain. Furthermore, the specific leukotoxic activity of JP2 vesicles was approximately four- to five-fold higher than isolated outer membrane preparations from JP2, suggesting that vesicles are enriched in leukotoxin. Together, these results suggest that the formation of A. actinomycetemcomitans vesicles occurs by a process that results in the enrichment of leukotoxin.

Deletion of lolB, encoding an outer membrane lipoprotein, is lethal for Escherichia coli and causes accumulation of lipoprotein localization intermediates in the periplasm.

Outer membrane lipoproteins of Escherichia coli are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal lolB gene under the control of the lac promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal lolB null mutant harboring a temperature-sensitive helper plasmid carrying the lolB gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the lolB gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for E. coli and that its depletion inhibits the upstream reactions of lipoprotein trafficking.

Escherichia coli Braun lipoprotein induces a lipopolysaccharide-like endotoxic response from primary human endothelial cells.

All bacteria contain proteins in which their amino-terminal cysteine residue is modified with N-acyl S-diacylglycerol functions, and peptides and proteins bearing this modification are immunomodulatory. The major outer membrane lipoprotein of Escherichia coli, the Braun lipoprotein (BLP), is the prototypical triacylated cysteinyl-modified protein. We find it is as active as LPS in stimulating human endothelial cells to an inflammatory phenotype, and a BLP-negative mutant of E. coli was less inflammatory than its parental strain. While the lipid modification was essential, the lipidated protein was more potent than a lipid-modified peptide. BLP associates with CD14, but this interaction, unlike that with LPS, was not required to elicit endothelial cell activation. BLP stimulated endothelial cell E-selectin surface expression, IL-6 secretion, and up-regulation of the same battery of cytokine mRNAs induced by LPS. Quantitative microarray analysis of 4400 genes showed the same 30 genes were induced by BLP and LPS, and that there was near complete concordance in the level of gene induction. We conclude that the lipid modification of at least one abundant Gram-negative protein is essential for endotoxic activity, but that the protein component also influences activity. The equivalent potency of BLP and LPS, and their complete concordance in the nature and extent of endothelial cell activation show that E. coli endotoxic activity is not due to just LPS. The major outer membrane protein of E. coli is a fully active endotoxic agonist for endothelial cells.

Leptospiral proteins recognized during the humoral immune response to leptospirosis in humans.

Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus Leptospira. An understanding of leptospiral protein expression regulation is needed to develop new immunoprotective and serodiagnostic strategies. We used the humoral immune response during human leptospirosis as a reporter of protein antigens expressed during infection. Qualitative and quantitative immunoblot analysis was performed using sera from 105 patients from Brazil and Barbados. Sera from patients with other diseases and healthy individuals were evaluated as controls. Seven proteins, p76, p62, p48, p45, p41, p37, and p32, were identified as targets of the humoral response during natural infection. In both acute and convalescent phases of illness, antibodies to lipopolysaccharide were predominantly immunoglobulin M (IgM) while antibodies to proteins were exclusively IgG. Anti-p32 reactivity had the greatest sensitivity and specificity: positive reactions were observed in 37 and 84% of acute- and convalescent-phase sera, respectively, while only 5% of community control individuals demonstrated positive reactions. Six immunodominant antigens were expressed by all pathogenic leptospiral strains tested; only p37 was inconsistently expressed. Two-dimensional immunoblots identified four of the seven infection-associated antigens as being previously characterized proteins: LipL32 (the major outer membrane lipoprotein), LipL41 (a surface-exposed outer membrane lipoprotein), and heat shock proteins GroEL and DnaK. Fractionation studies demonstrated LipL32 and LipL41 reactivity in the outer membrane fraction and GroEL and DnaK in the cytoplasmic fraction, while p37 appeared to be a soluble periplasmic protein. Most of the other immunodominant proteins, including p48 and p45, were localized to the inner membrane. These findings indicate that leptospiral proteins recognized during natural infection are potentially useful for serodiagnosis and may serve as targets for vaccine design.