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

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37’s ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.     

致病钩体表层膜蛋白新基因（Lslp）的克隆表达及免疫原性研究

克隆表达钩端螺旋体表层膜蛋白新基因Lslp并分析表达产物的免疫原性。根据前期研究得到的致病钩体新基因Lslp(GenBankAF32 5 80 7)的序列设计引物 ,在 6株致病钩体中扩增Lslp基因并测序。以BamHⅠ酶切Lslp和pGEX 1 λT ,构建重组质粒并用酶切和PCR鉴定 ,进一步在大肠杆菌中诱导表达 ,并进行免疫印迹分析 ;纯化表达产物免疫家兔 ,ELISA检测血清抗体滴度。结果显示Lslp在 6株致病钩体中均能扩增出相应片段 ,且序列同源性达到99 6 % ;构建高效原核表达重组质粒pGST LslP ,经IPTG诱导在大肠杆菌中可表达出 6 6kDGST融合蛋白 ,并能与全钩抗血清发生免疫印迹反应 ;将上述融合蛋白免疫新西兰大白兔产生 1 :5 1 2 0高滴度的IgG抗体。研究结果提示致病钩体膜蛋白新基因Lslp可在大肠杆菌进行高效表达 ,表达产物能被全钩抗血清识别 ,为研究钩体的致病机制和筛选保护性抗原提供了基础     

Human Complement Regulators C4b-Binding Protein and C1 Esterase Inhibitor Interact with a Novel Outer Surface Protein of Borrelia recurrentis

The spirochete Borrelia recurrentis is the causal agent of louse-borne relapsing fever and is transmitted to humans by the infected body louse Pediculus humanus. We have recently demonstrated that the B. recurrentis surface receptor, HcpA, specifically binds factor H, the regulator of the alternative pathway of complement activation, thereby inhibiting complement mediated bacteriolysis. Here, we show that B. recurrentis spirochetes express another potential outer membrane lipoprotein, termed CihC, and acquire C4b-binding protein (C4bp) and human C1 esterase inhibitor (C1-Inh), the major inhibitors of the classical and lectin pathway of complement activation. A highly homologous receptor for C4bp was also found in the African tick-borne relapsing fever spirochete B. duttonii. Upon its binding to B. recurrentis or recombinant CihC, C4bp retains its functional potential, i.e. facilitating the factor I-mediated degradation of C4b. The additional finding that ectopic expression of CihC in serum sensitive B. burgdorferi significantly increased spirochetal resistance against human complement suggests this receptor to substantially contribute, together with other known strategies, to immune evasion of B. recurrentis.     

Identification of Seroreactive Proteins of Leptospira interrogans Serovar Copenhageni Using a High-Density Protein Microarray Approach

Background

Leptospirosis is a widespread zoonotic disease worldwide. The lack of an adequate laboratory test is a major barrier for diagnosis, especially during the early stages of illness, when antibiotic therapy is most effective. Therefore, there is a critical need for an efficient diagnostic test for this life threatening disease.

Methodology

In order to identify new targets that could be used as diagnostic makers for leptopirosis, we constructed a protein microarray chip comprising 61% of Leptospira interrogans proteome and investigated the IgG response from 274 individuals, including 80 acute-phase, 80 convalescent-phase patients and 114 healthy control subjects from regions with endemic, high endemic, and no endemic transmission of leptospirosis. A nitrocellulose line blot assay was performed to validate the accuracy of the protein microarray results.

Principal findings

We found 16 antigens that can discriminate between acute cases and healthy individuals from a region with high endemic transmission of leptospirosis, and 18 antigens that distinguish convalescent cases. Some of the antigens identified in this study, such as LipL32, the non-identical domains of the Lig proteins, GroEL, and Loa22 are already known to be recognized by sera from human patients, thus serving as proof-of-concept for the serodiagnostic antigen discovery approach. Several novel antigens were identified, including the hypothetical protein LIC10215 which showed good sensitivity and specificity rates for both acute- and convalescent-phase patients.

Conclusions

Our study is the first large-scale evaluation of immunodominant antigens associated with naturally acquired leptospiral infection, and novel as well as known serodiagnostic leptospiral antigens that are recognized by antibodies in the sera of leptospirosis cases were identified. The novel antigens identified here may have potential use in both the development of new tests and the improvement of currently available assays for diagnosing this neglected tropical disease. Further research is needed to assess the utility of these antigens in more deployable diagnostic platforms.     

Multiple Activities of LigB Potentiate Virulence of Leptospira interrogans: Inhibition of Alternative and Classical Pathways of Complement

Microbial pathogens acquire the immediate imperative to avoid or counteract the formidable defense of innate immunity as soon as they overcome the initial physical barriers of the host. Many have adopted the strategy of directly disrupting the complement system through the capture of its components, using proteins on the pathogen's surface. In leptospirosis, pathogenic Leptospira spp. are resistant to complement-mediated killing, in contrast to the highly vulnerable non-pathogenic strains. Pathogenic L. interrogans uses LenA/LfhA and LcpA to respectively sequester and commandeer the function of two regulators, factor H and C4BP, which in turn bind C3b or C4b to interrupt the alternative or classical pathways of complement activation. LigB, another surface-proximal protein originally characterized as an adhesin binding multiple host proteins, has other activities suggesting its importance early in infection, including binding extracellular matrix, plasma, and cutaneous repair proteins and inhibiting hemostasis. In this study, we used a recent model of ectopic expression of LigB in the saprophyte, L. biflexa, to test the hypothesis that LigB also interacts with complement proteins C3b and C4b to promote the virulence of L. interrogans. The surface expression of LigB partially rescued the non-pathogen from killing by 5% normal human serum, showing 1.3- to 48-fold greater survival 4 to 6 d following exposure to complement than cultures of the non-expressing parental strain. Recombinant LigB7'-12 comprising the LigB-specific immunoglobulin repeats binds directly to human complement proteins, C3b and C4b, with respective K(d)s of 43±26 nM and 69±18 nM. Repeats 9 to 11, previously shown to contain the binding domain for fibronectin and fibrinogen, are also important in LigB-complement interactions, which interfere with the alternative and classical pathways measured by complement-mediated hemolysis of erythrocytes. Thus, LigB is an adaptable interface for L. interrogans to efficiently counteract the multiple homeostatic processes of the host.     

The Terminal Immunoglobulin-Like Repeats of LigA and LigB of Leptospira Enhance Their Binding to Gelatin Binding Domain of Fibronectin and Host Cells

Leptospira spp. are pathogenic spirochetes that cause the zoonotic disease leptospirosis. Leptospiral immunoglobulin (Ig)-like protein B (LigB) contributes to the binding of Leptospira to extracellular matrix proteins such as fibronectin, fibrinogen, laminin, elastin, tropoelastin and collagen. A high-affinity Fn-binding region of LigB has been localized to LigBCen2, which contains the partial 11th and full 12th Ig-like repeats (LigBCen2R) and 47 amino acids of the non-repeat region (LigBCen2NR) of LigB. In this study, the gelatin binding domain of fibronectin was shown to interact with LigBCen2R (K_D = 1.91±0.40 µM). Not only LigBCen2R but also other Ig-like domains of Lig proteins including LigAVar7''-8, LigAVar10, LigAVar11, LigAVar12, LigAVar13, LigBCen7''-8, and LigBCen9 bind to GBD. Interestingly, a large gain in affinity was achieved through an avidity effect, with the terminal domains, 13th (LigA) or 12th (LigB) Ig-like repeat of Lig protein (LigAVar7''-13 and LigBCen7''-12) enhancing binding affinity approximately 51 and 28 fold, respectively, compared to recombinant proteins without this terminal repeat. In addition, the inhibited effect on MDCKs cells can also be promoted by Lig proteins with terminal domains, but these two domains are not required for gelatin binding domain binding and cell adhesion. Interestingly, Lig proteins with the terminal domains could form compact structures with a round shape mediated by multidomain interaction. This is the first report about the interaction of gelatin binding domain of Fn and Lig proteins and provides an example of Lig-gelatin binding domain binding mediating bacterial-host interaction.     

Mapping of the Coronavirus Membrane Protein Domains Involved in Interaction with the Spike Protein

The coronavirus membrane (M) protein is the key player in virion assembly. One of its functions is to mediate the incorporation of the spikes into the viral envelope. Heterotypic interactions between M and the spike (S) protein can be demonstrated by coimmunoprecipitation and by immunofluorescence colocalization, after coexpression of their genes in eukaryotic cells. Using these assays in a mutagenetic approach, we have mapped the domains in the M protein that are involved in complex formation between M and S. It appeared that the 25-residue luminally exposed amino-terminal domain of the M protein is not important for M-S interaction. A 15-residue deletion, the insertion of a His tag, and replacement of the ectodomain by that of another coronavirus M protein did not affect the ability of the M protein to associate with the S protein. However, complex formation was sensitive to changes in the transmembrane domains of this triple-spanning protein. Deletion of either the first two or the last two transmembrane domains, known not to affect the topology of the protein, led to a considerable decrease in complex formation, but association was not completely abrogated. Various effects of changes in the part of the M protein that is located at the cytoplasmic face of the membrane were observed. Deletions of the extreme carboxy-terminal tail appeared not to interfere with M-S complex formation. However, deletions in the amphipathic domain severely affected M-S interaction. Interestingly, changes in the amino-terminal and extreme carboxy-terminal domains of M, which did not disrupt the interaction with S, are known to be fatal to the ability of the protein to engage in virus particle formation (C. A. M. de Haan, L. Kuo, P. S. Masters, H. Vennema, and P. J. M. Rottier, J. Virol. 72:6838-6850, 1998). Apparently, the structural requirements of the M protein for virus particle assembly differ from the requirements for the formation of M-S complexes.     

Evolutionary Implication of Outer Membrane Lipoprotein-Encoding Genes ompL1,lipL32 and lipL41 of Pathogenic Leptospira Species

Leptospirosis is recognized as the most widespread zoonosis with a global distribution. In this study, the antigenic variation in Leptospira interrogans and Leptospira borgpetersenii isolated from human urine and field rat kidney was preliminarily confirmed by microscopic agglutination test using monoclonal antibodies, and was further subjected to amplification and identification of outer membrane lipoproteins with structural gene variation. Sequence similarity analysis revealed that these protein sequences...     

Evolutionary Implication of Outer Genes ompL1, lipL32 and lipL41 Membrane Lipoprotein-Encoding of Pathogenic Leptospira Species

Leptospirosis is recognized as the most widespread zoonosis with a global distribution. In this study, the antigenic variation in Leptospira interrogans and Leptospira borgpetersenii isolated from human urine and field rat kidney was preliminarily confirmed by microscopic agglutination test using monoclonal antibodies, and was further subjected to amplification and identification of outer membrane lipoproteins with structural gene variation. Sequence similarity analysis revealed that these protein sequences, namely OmpL1, LipL32 and LipL41, showed no more homologies to outer membrane lipoproteins of non-pathogenic Leptospira and other closely related Spirochetes, but showed a strong identity within L. interrogans, suggesting intra-specific phylogenetic lineages that might be originated from a common pathogenic leptospiral origin. Moreover, the ompL1 gene showed more antigenic variation than lipL32 and lipL41 due to less conservation in secondary structural evolution within closely related species. Phylogenetically, ompL1 and lipL41 of these strains gave a considerable proximity to L. weilii and L. santarosai. The ompL1 gene of L. interrogans clustered distinctly from other pathogenic and non-pathogenic leptospiral species. The diversity of ompL genes has been analyzed and it envisaged that sequence-specific variations at antigenic determinant sites would result in slow evolutionary changes along with new serovar origination within closely related species. Thus, a crucial work on effective recombinant vaccine development and engineered antibodies will hopefully meet to solve the therapeutic challenges.Key words: Leptospira, ompL1, lipL32, lipL41, phylogeny, antigenic variation     

Outer Membrane Proteins of Escherichia coli IV. Differences in Outer Membrane Proteins Due To Strain and Cultural Differences

When the 42,000-dalton major outer membrane protein of Escherichia coli O111 is examined on alkaline polyacrylamide gels containing sodium dodecyl sulfate, it is resolved into three distinct bands designated as proteins 1, 2, and 3. Band 3 consists of two distinct polypeptides, proteins 3a and 3b. E. coli K-12 does not make any protein 2, but makes proteins similar to 1, 3a, and 3b as indicated by comparison of cyanogen bromide peptide patterns. Several Shigella species and most other strains of E. coli resemble E. coli K-12 in that they lack protein 2, whereas Salmonella typhimurium is more similar to E. coli O111. In addition to these species and strain differences, cultural differences resulted in differences in the outer membrane protein profiles. Under conditions of catabolite repression, the level of protein 2 in E. coli O111 decreased while the level of protein 1 increased. An enterotoxin-producing strain similar to E. coli O111 produced no protein 1 and an elevated level of protein 2 under conditions of low catabolite repression. The levels of proteins 1 and 3 are also different in different phases of the growth curve, with protein 1 being the major species in the exponential-phase cells and protein 3 being the major species in stationary-phase cells. A multiply phage-resistant mutant of E. coli K-12 with no obvious cell wall defects produced no protein 1 or 2, but made increased amounts of protein 3. Thus, the major outer membrane proteins of E. coli and related species may vary considerably without affecting outer membrane integrity.     

A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli

Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM.     

Outer Membrane Proteins and Serosubtyping with Outer Membrane Vesicles from Clinical Isolates of Neisseria meningitidis

The currently practiced protocol for routine serosubtyping of Neisseria meningitidis relies on reactivity of whole cells to monoclonal antibodies against the class 1 outer membrane protein (OMP) in ELISAs or dot-blots. This procedure, however, failed to yield serosubtyping information in 28% (48/174) of clinical isolates (1993–1994) in the province of Québec, Canada. These 48 strains were characterized by OMP profiles and ELISAs with outer membrane vesicles (OMVs). Forty out of the 48 strains expressed class 1 OMP, indicating that the inability to assign a serosubtype was not owing to the absence of the class 1 OMP. Of these, 15 (38%) were serosubtypable in ELISAs with outer membrane vesicles. Thus, 81% (141/174) of all meningococcal strains were serosubtypable with ELISAs using whole-cells or OMVs. Because the routinely used procedure for serosubtyping of meningococci is limited in providing serosubtype information, alternate procedures are proposed to obtain comprehensive information for epidemiological identification of this bacterium. Received: 11 June 1996 / Accepted: 5 July 1996     

Rapid distinction between Leptospira interrogans and Leptospira biflexa by PCR amplification of 23S ribosomal DNA

Bacterial specific primers were used to amplify 23S rRNA genes from a representative strain from each of the 23 serogroups of the pathogenic Leptospira interrogans and 8 strains from 6 serogroups of the non-pathogenic Leptospira biflexa. Only regions of extreme variability, which had been identified on the basis of homology-based search of all the 23S rRNA sequences available in GenBank database, were sequenced from the amplified products. PCR primers that had the potential to distinguish L. interrogans from L. biflexa species were designed from the derived sequences and a sensitive PCR protocol developed. The PCR method enabled the differentiation of the 59 strains of the 23 serogroups of L. interrogans from the 8 strains of 6 serogroups of L. biflexa. Further investigation by 16S rDNA sequencing of two strains of L. interrogans, which gave unexpected PCR results, provided evidence that they had been misclassified and hence we propose to reassign them to L. biflexa.     

Outer Membrane Proteins of Escherichia coli III. Evidence that the Major Protein of Escherichia coli O111 Outer Membrane Consists of Four Distinct Polypeptide Species

Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.     

问号钩端螺旋体属特异性OmpL1s抗原膜定位及其自然抗体应答

为了确定问号钩端螺旋体(简称钩体)属特异性OmpL1s抗原膜定位及其自然抗体应答情况和抗体类型,为OmpL1s用于研制通用性钩体基因工程疫苗及检测试剂盒抗原提供依据。采用显微镜凝集试验(MAT)检测四川地区156份钩体病人血清标本。用PCR和核苷酸序列分析,了解中国流行的钩体主要血清群ompL1基因型。采用常规基因工程技术构建ompL1/1和ompL1/2主要基因型原核表达系统,Ni-NTA亲和层析法提纯目的重组表达产物rOmpL1/1和rOmpL1/2。采用胶体金免疫电镜技术,对OmpL1s进行膜定位。建立了基于rOmpL1s的ELISAs,检测钩体病人血清中特异性抗体水平及其类型。试验结果表明,黄疸出血群钩体仍然是四川地区最主要的优势钩体血清群。中国流行的钩体主要血清群ompL1基因可有ompL1/1和ompL1/2两个基因型,两者核苷酸和氨基酸序列相似性之间有较明显的差异。OmpL1s是位于钩体外膜表面的蛋白分子。不同稀释度的156例钩体病人血清标本中,rOmpL1/1和rOmpL1/2特异性IgM阳性率分别为67.9%～79.5%和75.0%～75.6%,特异性IgG阳性率分别为71.8%～79.5%和75.0%～76.9%。上述试验结果提示,OmpL1s是位于钩体表面属特异性蛋白抗原。自然感染钩体时,rOmpL1/1和rOmpL1/2均可诱导机体体液免疫应答并产生IgM和IgG两类血清抗体,且两者之间有广泛的抗原交叉反应。rOmpL1/1和rOmpL1/2可作为研制通用性钩体基因工程疫苗和检测试剂盒的候选抗原。     

Quantification of Interaction Strengths between Chaperones and Tetratricopeptide Repeat Domain-containing Membrane Proteins

The three tetratricopeptide repeat domain-containing docking proteins Toc64, OM64, and AtTPR7 reside in the chloroplast, mitochondrion, and endoplasmic reticulum of Arabidopsis thaliana, respectively. They are suggested to act during post-translational protein import by association with chaperone-bound preprotein complexes. Here, we performed a detailed biochemical, biophysical, and computational analysis of the interaction between Toc64, OM64, and AtTPR7 and the five cytosolic chaperones HSP70.1, HSP90.1, HSP90.2, HSP90.3, and HSP90.4. We used surface plasmon resonance spectroscopy in combination with Interaction Map® analysis to distinguish between chaperone oligomerization and docking protein-chaperone interactions and to calculate binding affinities for all tested interactions. Complementary to this, we applied pulldown assays as well as microscale thermophoresis as surface immobilization independent techniques. The data revealed that OM64 prefers HSP70 over HSP90, whereas Toc64 binds all chaperones with comparable affinities. We could further show that AtTPR7 is able to bind HSP90 in addition to HSP70. Moreover, differences between the HSP90 isoforms were detected and revealed a weaker binding for HSP90.1 to AtTPR7 and OM64, showing that slight differences in the amino acid composition or structure of the chaperones influence binding to the tetratricopeptide repeat domain. The combinatory approach of several methods provided a powerful toolkit to determine binding affinities of similar interaction partners in a highly quantitative manner.     

Outer Membrane Proteins and Lipopolysaccharides in Pathovars of Xanthomonas campestris

Variations in the outer membrane proteins (OMPs) and lipopolysaccharides (LPSs) of 54 isolates belonging to 16 different pathovars of Xanthomonas campestris were characterized. OMP samples prepared by sarcosyl extraction of cell walls and LPS samples prepared by proteinase K treatment of sonicated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of 4 M urea. In general, the OMP and LPS profiles within each pathovar were very similar but different from the profiles of other pathovars. Heterogeneity in OMP and LPS profiles was observed within X. campestris pv. campestris, X. campestris pv. translucens, and X. campestris pv. vesicatoria. LPSs were isolated from six X. campestris pathovars, which fell into two major groups on the basis of O antigenicity. The O antigens of X. campestris pv. begoniae, X. campestris pv. graminis, and X. campestris pv. translucens cross-reacted with each other; the other group consisted of X. campestris pv. campestris, X. campestris pv. pelargonii, and X. campestris pv. vesicatoria. A chemical analysis revealed a significant difference between the compositions of the neutral sugars of the LPSs of those two groups; the LPSs of the first group contained xylose and a 6-deoxy-3-O-methyl hexose, whereas the LPSs of the other group lacked both sugars.