Characterization of proteins in the outer membrane preparation of a murine pathogen, Helicobacter bilis

Helicobacter bilis is a bacterial pathogen associated with multifocal hepatitis and inflammatory bowel disease in certain strains of mice. This bacterium colonizes the liver, bile, and lower intestine in mice and has also been isolated from a wide spectrum of laboratory animals. In this study, proteins present in the outer membrane preparation (OMP) of four H. bilis strains isolated from a mouse, a dog, a rat, and a gerbil were characterized and compared with that of Helicobacter pylori, a human gastric pathogen. All four H. bilis strains had similar OMP protein profiles that were distinct from those of H. pylori. Immunoblotting demonstrated that OMP proteins from H. bilis and H. pylori have little cross-reactivity, except for their flagellins. Nine major immunogenic polypeptides were present in the H. bilis OMPs. By using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, five heat-modifiable proteins with molecular masses of 82, 66, 52, 47 and 37 kDa were identified. The N-terminal sequences of the 46- and 47-kDa OMP proteins had no homology with protein sequences available in public databases. These results indicate that H. bilis has a conserved, unique OMP protein profile that is distinct from those of H. pylori.     

Expression of Helicobacter pylori virulence factors and associated expression profiles of inflammatory genes in the human gastric mucosa

Helicobacter pylori virulence factors have been suggested to be important in determining the outcome of infection. The H. pylori adhesion protein BabA2 is thought to play a crucial role in bacterial colonization and in induction of severe gastric inflammation, particularly in combination with expression of CagA and VacA. However, the influence of these virulence factors on the pathogenesis of H. pylori infection is still poorly understood. To address this question, the inflammatory gene expression profiles for two groups of patients infected with triple-negative strains (lacking expression of cagA, babA2, and vacAs1 but expressing vacAs2) and triple-positive strains (expressing cagA, vacAs1, and babA2 but lacking expression of vacAs2) were investigated. The gene expression patterns in the antrum gastric mucosa from patients infected with different H. pylori strains were very similar, and no differentially expressed genes could be identified by pairwise comparisons. Our data thus suggest that there is a lack of correlation between the host inflammatory responses in the gastric mucosa and expression of the babA2, cagA, and vacAs1 genes.     

B-cell and T-cell immune responses to experimental Helicobacter pylori infection in humans

The acute antibody and T-cell immune response to Helicobacter pylori infection in humans has not been studied systematically. Serum from H. pylori-naive volunteers challenged with H. pylori and cured after 4 or 12 weeks was tested by enzyme-linked immunosorbent assays for anti-H. pylori-specific immunoglobulin M (IgM) and IgA established using bacterial lysates from homologous (the infecting strain) and heterologous H. pylori. Proteins recognized by IgM antibody were identified by mass spectrometry of immunoreactive bands separated by two-dimensional gel electrophoresis. Mucosal T-cell subsets (CD4, CD8, CD3, and CD30 cells) were assessed by immunohistochemistry. All 18 infected volunteers developed H. pylori-specific IgM responses to both homologous or heterologous H. pylori antigens. H. pylori antigens reacted with IgM antibody at 4 weeks postinfection. IgM Western blotting showed immunoreactivity of postinfection serum samples to multiple H. pylori proteins with molecular weights ranging between 9,000 (9K) to 150K with homologous strains but only a 70K band using heterologous antigens. Two-dimensional electrophoresis demonstrated that production of H. pylori-specific IgM antibodies was elicited by H. pylori flagellins A and B, urease B, ABC transporter binding protein, heat shock protein 70 (DnaK), and alkyl hydroperoxide reductase. Mucosal CD3, CD4, and CD8 T-cell numbers increased following infection. IgM antibody responses were detected to a range of homologous H. pylori antigens 2 to 4 weeks postchallenge. The majority of H. pylori proteins were those involved in motility and colonization and may represent targets for vaccine development.     

Heterogeneity among Helicobacter pylori strains in expression of the outer membrane protein BabA

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 approximately 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.     

幽门螺杆菌结构性群体形成的研究

目的 研究幽门螺杆菌(Helicobacter pylori,Hp)由浮游状单细胞转变成结构性群体过程中蛋白质表达差异。方法 用超微结构形态观察及双向电泳技术比较浮游状幽门螺杆菌及其结构性群体全菌蛋白表达图谱的差异，将结构性群体形成过程中表达量显著增加的蛋白斑点，进行胶内酶切，肽混合物使用基质辅助激光解吸，电离飞行时间质谱仪进行质谱分析，获得4个明确的肽质量指纹谱，通过蛋白质数据库进行检索分析。结果 与结构性群体形成密切相关的蛋白质为鞭毛丝帽蛋白、2-磷酸甘油酸醋脱水酶、3，和邻苯二酚-2-丁酮4-磷酸盐合成酶和热休克蛋白33同类物。结论 上述蛋白的鉴定将有助于发现幽门螺杆菌新的致病因子以及抗菌靶位。     

颞叶癫痫大鼠海马差异表达基因和蛋白质的筛选研究

目的：寻找颞叶癫痫大鼠海马组织的差异表达基因和蛋白质，以期为进一步探讨颞叶癫痫的发病机制，寻找新的治疗靶点和研发新的治疗手段奠定基础。方法:运用cDNA微阵列、二维电泳和MALDI-TOF-MS技术，分析氯化锂-匹罗卡品（LiCl-PILO）致痫大鼠模型海马组织的基因表达谱和蛋白质表达谱，并对发现的差异表达基因和差异表达蛋白质进行分析和鉴定结果和。结论：发现LiCl-PILO致痫大鼠海马组织中192个基因差异表达，159条可在GenBank中登陆，其中表达上调的基因84条，表达下调的基因75条；筛选到78个差异表达蛋白质斑点，其中31个在癫痫组表达下调，47个在癫痫组表达上调。有5个蛋白质最终鉴定确认。本研究结果为运用蛋白质组学方法寻找癫痫治疗新靶点研究提供实验依据。     

Proteomic analysis of fungal host factors differentially expressed by Fusarium graminearum infected with Fusarium graminearum virus-DK21

Fusarium graminearum virus–DK21 (FgV-DK21), which infects the plant pathogenic F. graminearum, perturbs host developmental processes such as sporulation, morphology, pigmentation, and attenuates the virulence (hypovirulence) of the host. To identify the differentially expressed F. graminearum proteins by FgV-DK21 infection, we have used two-dimensional electrophoresis with mass spectrometry using proteins extracted from virus-free and FgV-DK21-infected strains. A total of 148 spots showing an altered expression were identified by PDQuest™ program. Among these spots, 33 spots were exclusively analyzed including 14 spots from FgV-DK21-infected and 19 spots from virus-free strains by ESI-MS/MS analyses and successfully identified 23 proteins. Seven proteins including sporulation-specific gene SPS2, triose phosphate isomerase, nucleoside diphosphate kinase, and woronin body major protein precursor were induced or significantly up-regulated by FgV-DK21 infection. A significant decrease or down regulation of 16 proteins including enolase, saccharopine dehydrogenase, flavohemoglobin, mannitol dehydrogenase and malate dehydrogenase caused by FgV-DK21 infection was also identified. Variations of protein expression were also further investigated at the mRNA level by real-time RT-PCR analysis, which confirmed the proteomic data for 9 out of the representative 11 selected proteins including 5 proteins from up-regulated group and 6 proteins from down-regulated group. Further investigation of these differentially expressed proteins will provide novel insights into the molecular responses of F. graminearum to FgV-DK21 infection.     

蛋白质组学检测结直肠腺瘤及早期恶变患者血清的差异蛋白质变化及意义

目的： 通过比较分析结直肠腺瘤及早期恶变患者血清蛋白质的表达差异,寻找结直肠癌早期诊断的血清标记物。方法：建立结直肠腺瘤及腺瘤早期恶变患者血清蛋白双向电泳图谱,比较分析差异蛋白质斑点,切取酶解行MALDI-TOF／TOF质谱分析鉴定。结果：成功建立结直肠腺瘤及早期恶变患者血清蛋白双向电泳图谱,其平均蛋白质点分别为1672±73和1732±46,早期恶变组表达差异大于1.5倍的蛋白质斑点有28个,其中15个下调,13个上升;质谱分析鉴定出23个蛋白质,鉴定率达82.14%;合并重复鉴定的蛋白质,共获得的差异蛋白13种,其中8种为上调蛋白,5种为下调蛋白,分为6类,包括蛋白酶抑制剂、补体系统、免疫球蛋白、角质素、信号转导蛋白及未知蛋白。结论：蛋白质组学技术能灵敏分析结直肠腺瘤及早期恶变患者血清蛋白质的异常改变,本研究鉴定的蛋白质可能成为结直肠癌早期诊断的血清肿瘤标记物。     

Identification and immunogenicity of group A Streptococcus culture supernatant proteins

Extracellular proteins made by group A Streptococcus (GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed in Escherichia coli. Western blot analysis with these recombinant proteins and sera from GAS-infected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.     

克罗恩病患者与健康人血清蛋白质组学比较初步研究

目的：应用蛋白质组学方法在患者血清中寻找与克罗恩病相关的蛋白质。方法: 采取克罗恩病患者以及正常成人血清蛋白样本各4例，用不同的CyDye荧光染料标记后进行胶内差异双向凝胶电泳(2-D DIGE)，并对获得的图谱进行分析及对差异蛋白质进行基质辅助激光解吸离子化飞行时间质谱(MALDI-TOF-MS) 鉴定和生物学信息分析。结果: 通过2-D DIGE分析，发现了克罗恩病患者中存在29个表达异常蛋白质点，质谱分析和数据库检索共鉴定出22种蛋白质，包括SER/THR，CD45，APC等。结论: 蛋白质组学能很好显示克罗恩病患者与正常人血清蛋白质表达差异，本研究鉴定的蛋白质可能为研究克罗恩病的生物学行为提供新的分子标记物。     

Comparative studies on the proteomic expression patterns in the third- and fifth-stage larvae of Angiostrongylus cantonensis

Angiostrongylus cantonensis is an important zoonotic parasite causing eosinophilic meningitis and eosinophilic meningoencephalitis in humans. In this study, the protein expression profiles of the infective third- and pathogenic fifth-stage larvae (L3 and L5) of this parasite were compared by proteomic techniques. Isolated protein samples were separated by two-dimensional gel electrophoresis (2-DE), stained with silver nitrate, and analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Proteins from L5 were mainly at pH 5–7 and with molecular weight (MW) 40–100 kDa, whereas those from L3 were at pH 5–6 and with 5–35 kDa. Of 100 protein spots identified, 33 were from L3 whereas 67 from L5 and 63 had known identities, whereas 37 were hypothetical proteins. There were 15 spots of stress proteins, and HSP60 was the most frequently found heat stress proteins in L5. More binding and protein transport-related proteins were found in L5 including peptidylprolyl isomerase (cyclophilin)-like 2, serum albumin, preproalbumin precursor, and dilute class unconventional myosin. L3 had a higher expression of cytoskeleton and membrane proteins than L5. In addition, four protein spots were identified in the sera of the rat host by Western blot analysis. The present proteomic study revealed different protein expression profiles in L3 and L5 of A. cantonensis. These changes may reflect the development of L3 from the poikilothermic snails to L5 in the homoeothemic rats. This information may be useful for the finding of stage-specific proteins and biomarker for diagnosis of angiostrongyliasis.     

Comparative genomics of Helicobacter pylori: analysis of the outer membrane protein families

The two complete genomic sequences of Helicobacter pylori J99 and 26695 were used to compare the paralogous families (related genes within one genome, likely to have related function) of genes predicted to encode outer membrane proteins which were present in each strain. We identified five paralogous gene families ranging in size from 3 to 33 members; two of these families contained members specific for either H. pylori J99 or H. pylori 26695. Most orthologous protein pairs (equivalent genes between two genomes, same function) shared considerable identity between the two strains. The unusual set of outer membrane proteins and the specialized outer membrane may be a reflection of the adaptation of H. pylori to the unique gastric environment where it is found. One subfamily of proteins, which contains both channel-forming and adhesin molecules, is extremely highly related at the sequence level and has likely arisen due to ancestral gene duplication. In addition, the largest paralogous family contained two essentially identical pairs of genes in both strains. The presence and genomic organization of these two pairs of duplicated genes were analyzed in a panel of independent H. pylori isolates. While one pair was present in every strain examined, one allele of the other pair appeared partially deleted in several isolates.     

Development of two PCR-based techniques for detecting helical and coccoid forms of Helicobacter pylori

The primary mode of transmission of Helicobacter pylori, a human pathogen carried by more than half the population worldwide, is still unresolved. Some epidemiological data suggest water as a possible transmission route. H. pylori in the environment transforms into a nonculturable, coccoid form, which frequently results in the failure to detect this bacterium in environmental samples by conventional culture techniques. To overcome limitations associated with culturing, molecular approaches based on DNA amplification by PCR have been developed and used for the detection of H. pylori in clinical and environmental samples. Our results showed the glmM gene as the most promising target for detection of H. pylori by PCR amplification. Under optimal amplification conditions, glmM-specific primers generated PCR-amplified products that were specific for H. pylori and some other Helicobacter species. Genome sequence analysis revealed the existence of a conserved region linked to a hypervariable region upstream of the 16S rRNA gene of H. pylori. Selective PCR primer sets targeting this sequence were evaluated for the specific detection of H. pylori. One primer set, Cluster2 and B1J99, were shown to be highly specific for H. pylori strains and did not produce any PCR products when other Helicobacter species and other bacterial species were analyzed. In tests with 32 strains of H. pylori, 6 strains of other Helicobacter species, 8 strains of Campylobacter jejuni, and 21 strains belonging to different genera, the primers for glmM were selective for the Helicobacter genus and the primers containing the region flanking the 16S rRNA gene were selective for H. pylori species only. The combination of two sensitive PCR-based methods, one targeting the glmM gene and the other targeting a hypervariable flanking region upstream of the 16S rRNA gene, are complementary to each other. Whereas the glmM-specific primers provide a rapid, sensitive presumptive assay for the presence of H. pylori and closely related Helicobacter spp., the primers for sequences flanking the 16S rRNA gene can confirm the presence of H. pylori and locate the potential source of this bacterium.     

Two-dimensional gel electrophoresis and immunoblotting of Campylobacter pylori proteins.

Whole-cell, outer-membrane protein, flagellum-associated antigens and partially purified urease of Campylobacter pylori were analyzed by two-dimensional gel electrophoresis. C. pylori strains were readily distinguished from strains of Campylobacter jejuni, C. coli, and C. fetus by absence of major outer membrane proteins with Mrs of 41,000 to 45,000. C. pylori strains also lacked the acidic surface-array proteins at Mr 100,000 to 149,000 identified previously in serum-resistant strains of C. fetus. Surface labeling of intact C. pylori cells with 125I revealed two common major proteins, which we have designated protein 2 (pI 5.6 to 5.8, Mr 66,000) and protein 3 (pI 5.2 to 5.5, Mr 63,000). Proteins 2 and 3 were also the major components (subunits) observed in partially purified urease. Partially purified preparations of flagella consistently contained proteins 2 and 3. Thus, urease appears to be associated with both outer membranes and flagella of C. pylori. C. pylori strains also possessed an antigen at Mr 59,000 which was cross-reactive with antiserum against flagella of C. jejuni. However, the antigen did not appear to be associated with flagella per se in C. pylori. Protein 2 was unique to C. pylori among the Campylobacter species studied. It was not recognized by antibody against whole cells of C. jejuni or C. fetus or flagella of C. jejuni. Protein 3 was cross-reactive with antiserum against whole cells of C. jejuni and C. fetus, as were several other major protein antigens. Because protein 2 is a major outer membrane protein that is apparently unique to C. pylori, development of monospecific antibodies against this antigen may be useful for the identification of C. pylori in tissues, and purified antigen may be useful for serologic tests for specific diagnosis of C. pylori infections.     

Molecular analysis of urease genes from a newly identified uncultured species of Helicobacter.

"Gastrospirillum hominis" is an uncultured gastric spiral bacterium that has recently been shown by 16S rDNA sequence analysis to be a newly recognized species of Helicobacter that infects humans, and it has been provisionally designated "Helicobacter heilmannii." We used PCR to directly amplify the urease structural genes of "H. heilmannii" from infected gastric tissue. DNA sequence analysis identified two open reading frames, ureA and ureB, which code for polypeptides with predicted molecular weights of 25,729 and 61,831, respectively. The urease subunit genes from "H. heilmannii" were cloned and expressed in Escherichia coli. Western blot (immunoblot) analysis showed that antiserum directed against the ureA and ureB gene products from H. pylori was cross-reactive with the corresponding polypeptides from "H. heilmannii." Analysis of the derived amino acid sequences of "H. heilmannii" UreA and UreB demonstrated that "H. heilmannii" urease is more highly related to the urease from H. felis (found in the stomachs of cats and dogs) than to the urease from H. pylori. These data are consistent with 16S rDNA sequence analysis and suggest that "H. heilmannii" is phylogenetically most closely related to H. felis.     

Proteomic analysis of Leishmania mexicana differentiation

We have resolved the proteome of axenically differentiated Leishmania mexicana parasites by two-dimensional gel electrophoresis (2DE), employing optimised, robust and reproducible procedures, and visualised (by silver staining) approximately 2000 protein species in each of three developmental stages: procyclic promastigotes, metacyclic promastigotes and amastigotes. This analysis has used homogeneous populations of these parasite stages, characterised according to their morphology, protease and nuclease activity profiles and expression of stage-specific antigens. Following comparison of the whole proteome profiles between stages, 47 spots were found to be stage-specific, while a further 100 spots changed in intensity during differentiation. The majority of "unique" spots were expressed during the infective stages of parasite differentiation, metacyclic promastigotes and amastigotes. CapLC-QTOF mass spectrometry has allowed the identification of 47 protein species to date, including a number which are only detected in the amastigote stage. Proteins identified are members of eight functionally related groupings, some of which are implicated in infectivity and host-parasite interactions.     

First proteomic analysis of Legionella pneumophila based on its developing genome sequence

The first proteomic analysis of the respiratory pathogen Legionella pneumophila ATCC 33152 is presented in this report. Two-dimensional gel electrophoresis of total cell extracts was carried out. In total, 130 protein spots were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MS) or by quadruple time-of-flight tandem MS, including proteins correlated with virulence. For the first time, proteins of L. pneumophila were identified using mass spectrometric methods and mapped on a two-dimensional gel; this will be of considerable use for comparison of protein expression profiles of L. pneumophila wild-type and knock-out mutant strains and of L. pneumophila grown under different conditions.