Brian Berg Vandahl

Proteome analysis of the Chlamydia pneumoniae elementary body.

Chlamydia pneumoniaeis an obligate intracellular human pathogen that causes acute and chronic respiratory tract diseases and that has been implicated as a possible risk factor in the development of atherosclerotic heart disease. C. pneumoniaecultivated in Hep‐2 cells were 35S‐labeled and infectious elementary bodies (EB) were purified. The EB proteins were separated by two‐dimensional gel electrophoresis. Excised protein spots were in‐gel digested with trypsin and peptides were concentrated on reverse‐phase chromatographic beads for identification analysis by matrix‐assisted laser desorption/ionization‐mass spectrometry. In the pH range from 3–11, 263 C. pneumoniaeprotein spots encoded from 167 genes were identified. These genes constitute 15 % of the genome. The identified proteins include 31 hypothetical proteins. It has recently been suggested that EB should be able to synthesize ATP. This view may be strengthened by the identification of several proteins involved in energy metabolism. Furthermore, proteins have been found which are involved in the type III secretion apparatus important for pathogenesis of intracellular bacteria. Proteome maps and a table of all identified proteins have been made available on the world wide web at www.gram.au.dk.

Characterization of a secreted Chlamydia protease

Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D‐PAGE profiles of [35S]‐labelled Chlamydia proteins from whole lysates of infected cells to 2D‐PAGE profiles of proteins from purified Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the ‘chlamydial protease‐ or proteasome‐like activity factor’ (CPAF) and were clearly present in 2D‐PAGE profiles of whole lysates of infected cells but absent from purified Chlamydia. CPAF was recently identified by Zhong and colleagues as a secreted protease which cleaves host cell transcription factors essential for MHC class I and II antigen presentation. The identification of CPAF in this paper verifies the applicability of the described method for the identification of secreted proteins. We extend the findings by Zhong et al. by proteome studies of expression and turnover of C. trachomatis CPAF showing that the degradation of C. trachomatis D CPAF in the host cell is very limited. Furthermore, we show that two fragments of CPAF exist in C. pneumoniae as well as in C. trachomatis.

The expression, processing and localization of polymorphic membrane proteins in Chlamydia pneumoniae strain CWL029

BackgroundChlamydiae are obligate intracellular bacteria, which are important human pathogens. Genome sequences of C. trachomatis and C. pneumoniae have revealed the presence of a Chlamydia specific gene family encoding polymorphic outer membrane proteins, Pmps. In C. pneumoniae the family comprises twenty-one members, which are all transcribed. In the present study, the expression, processing and localisation of the sixteen full-length Pmps in C. pneumoniae strain CWL029 have been further investigated by two-dimensional gel electrophoresis and immunofluorescence microscopy.ResultsTen Pmps were identified in elementary bodies (EBs). Eight of these were investigated with respect to time dependent expression and all were found to be up-regulated between 36 and 48 hours post infection. Antibodies against Pmp6, 8, 10, 11 and 21 reacted with chlamydiae when infected cells were formalin fixed. Pmp6, Pmp20 and Pmp21 were found in cleaved forms, and the cleavage sites of Pmp6 and Pmp21 were identified.ConclusionsThe Pmps are heavily up-regulated at the time of conversion of RB to EB, and at least ten Pmps are present in EBs. Due to their reaction in formalin fixation it is likely that Pmp6, 8, 10, 11 and 21 are surface exposed. The identified cleavage sites of Pmp6 and Pmp21 are in agreement with the theory that the Pmps are autotransporters.

Glucagon Fibril Polymorphism Reflects Differences in Protofilament Backbone Structure

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril classes differ in terms of protofilament backbone regions, secondary structure, chromophore alignment along the fibril axis, and fibril superstructure. Straight fibrils show a conventional beta-sheet-rich far-UV circular dichroism spectrum whereas that of twisted fibrils is dominated by contributions from beta-turns. Fourier transform infrared spectroscopy confirms this and also indicates a more dense backbone with weaker hydrogen bonding for the twisted morphology. According to linear dichroism, the secondary structural elements and the aromatic side chains in the straight fibrils are more highly ordered with respect to the alignment axis than the twisted fibrils. A series of highly periodical reflections in the diffractogram of the straight fibrils can be fitted to the diffraction pattern expected from a cylinder. Thus, the highly integrated structural organization in the straight fibril leads to a compact and highly uniform fibril with a well-defined edge. Prolonged proteolytic digestion confirmed that the straight fibrils are very compact and stable, while parts of the twisted fibril backbone are much more readily degraded. Differences in the digest patterns of the two morphologies correlate with predictions from two algorithms, suggesting that the polymorphism is inherent in the glucagon sequence. Glucagon provides a striking illustration of how the same short sequence can be folded into two remarkably different fibrillar structures.

Identification of an in vivo CD4+ T cell-mediated response to polymorphic membrane proteins of Chlamydia pneumoniae during experimental infection

Chlamydia pneumoniae is an obligate intracellular bacterium that causes upper and lower respiratory tract infection in humans. C. pneumoniae harbors the polymorphic membrane protein (Pmp) family with 21 different proteins with a molecular mass around 100 kDa. The Pmps are species-specific, abundant and, together with major outer membrane protein and outer membrane protein 2, the dominant proteins in the C. pneumoniae outer membrane complex. Nevertheless, it is unknown whether Pmps are recognized by the cell-mediated immune response. To address this issue, C57BL/6J mice were infected intranasally with C. pneumoniae and the immune response to primary infection was investigated. We demonstrate, as expected, that the primary response is of the Th1 type by IgG2a- and IgG1-specific sELISA (Medac) on serum. In vivo-primed spleen lymphocytes were found to be reactive to Pmp8, Pmp20 and Pmp21 in an interferon-gamma ELISpot assay. The responses were shown to be mediated by CD4(+) T cells. To our knowledge, this is the first identification of antigens recognized by CD4(+) T cells during murine C. pneumoniae infection.

Secretion of Cpn0796 from Chlamydia pneumoniae into the host cell cytoplasm by an autotransporter mechanism

By comparison of proteome profiles of purified Chlamydia pneumoniae and whole lysates of C. pneumoniae infected HEp‐2 cells, an N‐terminal fragment of the previously uncharacterized chlamydial protein Cpn0796 was identified as a secreted protein. A 38 kDa cleavage product of Cpn0796 was present in infected cells, whereas only the 65 kDa full‐length Cpn0796 could be detected in purified Chlamydia. Confocal immunofluorescence microscopy demonstrated that Cpn0796 was localized in the Chlamydia membrane in young inclusions. However, at 36 h post infection and later Cpn0796 was detected in the cytoplasm of C. pneumoniae infected HEp‐2 and BHK cells. Furthermore, Cpn0796 was detected in the cytoplasm of infected cells in the lungs of C. pneumoniae infected C57Bl mice. When cleavage was inhibited, Cpn0796 was retained in the chlamydiae. We propose that Cpn0796 is an autotransporter the N‐terminal of which is translocated to the host cell cytoplasm. This is the first example of secretion of a Chlamydia autotransporter passenger domain into the host cell cytoplasm. Cpn0796 is specific for C. pneumoniae, where five homologous proteins are encoded by clustered genes. None of these five proteins were found to be secreted.

Potential Relevance of Chlamydia pneumoniae Surface Proteins to an Effective Vaccine

The surface of Chlamydia pneumoniae is covered with proteins but their exact identification is not known probably because of the presence of conformational epitopes. A family of 21 pmp genes has been found by DNA sequencing. In common, these genes have the capacity to encode the amino acid motif GGAI. Several of the genes have the capacity to encode outer membrane proteins of about 100 kDa. Thus, they are candidate genes to encode the protein(s) present in the 98-kDa protein band of the C. pneumoniae outer membrane complex. The production of recombinant GGAI proteins is described as is the use of polyclonal antibodies raised against the recombinant GGAI proteins to determine their expression in C. pneumoniae elementary bodies. At least three of the proteins, Omp4, 5, and 11, are expressed.

Proteome analysis of Chlamydia pneumoniae

Publisher Summary This chapter describes proteome analysis of bacteria particularly, Chlamydia pneumoniae. Bacteria are especially well suited for proteome analysis, because of the availability of high quality genomes, the relatively small size of the genomes, and the low level of functional redundancy. All Chlamydia species are obligate intracellular gram-negative bacteria sharing a characteristic biphasic developmental cycle in which they alternate between two morphologically and metabolically distinct forms, the infectious elementary bodies (EB) and the replicative reticular bodies (RB). The described proteome comparison is a novel method to identify candidate secreted proteins for obligate intracellular bacteria. Identification and further investigation of the proteins differing between whole cell lysates and purified bacteria may reveal important Chlamydia pathogenicity factors and vaccine candidates and contribute to the understanding of the intracellular life of Chlamydia.

Distinct patterns of blood-stage parasite antigens detected by plasma IgG subclasses from individuals with different level of exposure to Plasmodium falciparum infections

BackgroundIn endemic regions naturally acquired immunity against Plasmodium falciparum develops as a function of age and exposure to parasite infections and is known to be mediated by IgG. The targets of protective antibodies remain to be fully defined. Several immunoepidemiological studies have indicated an association of cytophilic anti-parasite IgG with protection against malaria. It has been hypothesized that the initial antibody responses against parasite antigens upon first few Plasmodium falciparum infections is dominated by non-protective IgG2/IgG4 and IgM antibodies, which then gradually develop into protective response dominated by cytophilic IgG1 and IgG3 antibodies.MethodsNaturally occurring IgG antibodies against P. falciparum blood-stage antigens were analysed from plasma samples collected from four groups of individuals differing in age and level of exposure to P. falciparum infections. Western Blot profiling of blood-stage parasite antigens displaying reactivity with individual plasma samples in terms of their subclass specificities was conducted. Parasite antigens detected by IgG were grouped based on their apparent molecular sizes resolved by SDS-PAGE as high molecular weight (≥ 70 kDa) or low molecular weight (< 70 kDa). The number of discernable low molecular weight parasite antigens detected by different IgG subclass antibodies from each plasma sample was recorded. Using Wilcoxons rank sum test these reactivities were compared amongst groups of individuals with different levels of exposure to P. falciparum infections.ResultsIgG4 and IgM antibodies in plasma samples from all groups detected very few parasite antigens. IgG2 antibodies from all groups detected a common pattern of high molecular weight parasite antigens. Cytophilic IgG subclasses in plasma samples from individuals with higher levels of exposure to P. falciparum infections distinctly detected higher numbers of low molecular weight parasite antigens.ConclusionsIn the present study, there was no evidence for switching of antibody responses from non-cytophilic to cytophilic subclasses against blood-stage parasite antigens as a likely mechanism for induction of protective immunity against malaria.

Preparation of Bacterial Samples for 2-D PAGE

Recent developments in the field of proteomics have revolutionized the way that proteins, and their contribution to cellular functions, are studied. The subsequent increased understanding of the mechanisms of cellular function and misfunction will have particular impact in the area of medical research, where disease processes will be better understood, many new (protein) therapeutic targets identified, and novel therapeutic agents developed. At the basic research level, phenotype will be explained in terms of cellular mechanisms. The completion of the sequences of an ever-widening range of genomes—not least of all, the human genome—has provided the molecular biologist with a wealth of data that needs to be analyzed and interpreted. For a variety of reasons (including alternative mRNA splicing, varying translational stop/start sites, frameshifting, and the inability to deduce posttranslational modifications), complete sequences of genomes are insufficient to elucidate the protein components of cells. The focus of attention has therefore turned to directly examining these protein components as the means of understanding cell function, as well as the cellular changes involved in disease states. However, the wealth of gene sequencing data now available has produced a glut of information that challenges the protein chemist to develop new tools to utilize this flood of genomic data. From the beginning, the cornerstone of proteomics has been the use of twodimensional gel electrophoresis to compare proteomes of different tissues (for example, normal and diseased tissue) with the subsequent identification of protein differences by the use of mass spectrometry and database searching. These still remain valuable techniques and receive appropriate coverage in this book. However, the term proteomics now encompasses a range of newly developed methodologies for determining the structure and function of a protein. I have therefore included in The Proteomics Protocols Handbook a number of novel mass spectrometry and LC-MS techniques, protein array technology, new bioinformatics tools, and the range of techniques central to structural and functional proteomics that are needed to deduce the function of newly discovered protein sequences. The use of these techniques, and no doubt further ones that will be developed in the coming years, will lead to achieving the ultimate goal of proteomics, namely to catalog the identity and function of all proteins in living organisms. The Proteomics Protocols Handbook should prove a valuable resource for molecular biologists, protein chemists, clinical/medical researchers, structural chemists/biochemists, and microbiologists, as well as those involved in bioinformatics and structural/ functional genomics.