RESUMEN
BACKGROUND: In 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. METHODS: Naturally 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. RESULTS: IgG4 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. CONCLUSIONS: In 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.
Asunto(s)
Anticuerpos Antiprotozoarios/sangre , Malaria Falciparum/inmunología , Plasmodium falciparum/inmunología , Adulto , Antígenos de Protozoos/análisis , Antígenos de Protozoos/química , Antígenos de Protozoos/inmunología , Western Blotting , Niño , Preescolar , Electroforesis en Gel de Poliacrilamida , Humanos , Inmunoglobulina G/sangre , Lactante , Recién Nacido , Peso Molecular , Plasma/inmunología , Plasmodium falciparum/químicaRESUMEN
BACKGROUND: Chlamydiae 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. RESULTS: Ten 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. CONCLUSIONS: The 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.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/metabolismo , Chlamydophila pneumoniae/metabolismo , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Chlamydophila pneumoniae/genética , ADN Bacteriano/genética , Electroforesis en Gel de Poliacrilamida , Regulación Bacteriana de la Expresión Génica , Humanos , Espectrometría de Masas , Microscopía Fluorescente , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de Proteína , Células Tumorales CultivadasRESUMEN
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.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Linfocitos T CD4-Positivos/inmunología , Infecciones por Chlamydophila/inmunología , Chlamydophila pneumoniae/química , Animales , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Recuento de Linfocito CD4 , Chlamydophila pneumoniae/genética , Chlamydophila pneumoniae/inmunología , Genoma Bacteriano , Ratones , Ratones Endogámicos BALB CRESUMEN
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.
Asunto(s)
Amiloide/química , Amiloide/metabolismo , Glucagón/química , Glucagón/metabolismo , Multimerización de Proteína , Dicroismo Circular , Péptido Hidrolasas/metabolismo , Pliegue de Proteína , Estabilidad Proteica , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , Difracción de Rayos XAsunto(s)
Proteínas Bacterianas/química , Chlamydophila pneumoniae/química , Proteoma , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Infecciones por Chlamydia/microbiología , Chlamydophila pneumoniae/genética , Electroforesis en Gel Bidimensional/métodos , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Indicadores y Reactivos , Transcripción GenéticaRESUMEN
BACKGROUND: It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed. METHODS AND FINDINGS: Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge. CONCLUSION: CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.
Asunto(s)
Adyuvantes Inmunológicos/química , Cationes , Glucolípidos/química , Glucolípidos/inmunología , Liposomas/química , Liposomas/inmunología , Mycobacterium/metabolismo , Compuestos de Amonio Cuaternario/química , Vacunas/inmunología , Animales , Chlamydia trachomatis/metabolismo , Femenino , Humanos , Malaria/parasitología , Ratones , Ratones Endogámicos C57BL , Mycobacterium tuberculosis/metabolismo , Plasmodium yoelii/metabolismo , Linfocitos T/inmunologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Chlamydophila pneumoniae/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Infecciones por Chlamydia/microbiología , Chlamydophila pneumoniae/genética , Cricetinae , Citoplasma/metabolismo , Electroforesis en Gel Bidimensional , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Pulmón/microbiología , Ratones , Ratones Endogámicos C57BL , Microscopía Fluorescente , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Transporte de Proteínas , Proteoma/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Unsaturated fatty acids play an important role in the prevention of human diseases such as diabetes, obesity, cancer, and neurodegeneration. However, their oxidation in vivo by acyl-CoA dehydrogenases (ACADs) that catalyze the first step of each cycle of mitochondrial fatty acid beta-oxidation is not entirely understood. Recently, a novel ACAD (ACAD-9) of unknown function that is highly homologous to human very-long-chain acyl-CoA dehydrogenase was identified by large-scale random sequencing. To characterize its enzymatic role, we have expressed ACAD-9 in Escherichia coli, purified it, and determined its pattern of substrate utilization. The N terminus of the mature form of the enzyme was identified by in vitro mitochondrial import studies of precursor protein. A 37-amino acid leader peptide was cleaved sequentially by two mitochondrial peptidases to yield a predicted molecular mass of 65 kDa for the mature subunit. Submitochondrial fractionation studies found native ACAD-9 to be associated with the mitochondrial membrane. Gel filtration analysis indicated that, like very-long-chain acyl-CoA dehydrogenase, ACAD-9 is a dimer, in contrast to the other known ACADs, which are tetramers. Purified mature ACAD-9 had maximal activity with long-chain unsaturated acyl-CoAs as substrates (C16:1-, C18:1-, C18:2-, C22:6-CoA). These results suggest a previously unrecognized role for ACAD-9 in the mitochondrial beta-oxidation of long-chain unsaturated fatty acids. Because of the substrate specificity and abundance of ACAD-9 in brain, we speculate that it may play a role in the turnover of lipid membrane unsaturated fatty acids that are essential for membrane integrity and structure.
Asunto(s)
Acil-CoA Deshidrogenasa de Cadena Larga/fisiología , Ácidos Grasos/metabolismo , Mitocondrias/metabolismo , Oxígeno/metabolismo , Acil-CoA Deshidrogenasa de Cadena Larga/metabolismo , Secuencia de Aminoácidos , Animales , Encéfalo/metabolismo , Membrana Celular/metabolismo , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/metabolismo , Humanos , Cinética , Metabolismo de los Lípidos , Lípidos/química , Modelos Moleculares , Datos de Secuencia Molecular , Músculos/metabolismo , Mutagénesis , Péptidos/química , Reacción en Cadena de la Polimerasa , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Análisis de Secuencia de ADN , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
It has been difficult to study the molecular biology of the obligate intracellular bacterium Chlamydia due to lack of genetic transformation systems. Therefore, genome sequencing has greatly expanded the information concerning the biology of these pathogens. Comparing the genomes of seven sequenced Chlamydia genomes has provided information of the common gene content and gene variation. In addition, the genome sequences have enabled global investigation of both transcript and protein content during the developmental cycle of chlamydiae. During this cycle Chlamydia alternates between an infectious extracellular form and an intracellular dividing form surrounded by a phagosome membrane termed the chlamydial inclusion. Proteins secreted from the chlamydial inclusion into the host cell may interact with host cell proteins and modify the host cell's response to infection. However, identification of such proteins has been difficult because the host cell cytoplasm of Chlamydia infected cells cannot be purified. This problem has been circumvented by comparative proteomics.
Asunto(s)
Chlamydia/genética , Genoma Bacteriano , Proteoma , Fenómenos Fisiológicos Bacterianos , Chlamydia/metabolismo , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Electroforesis en Gel Bidimensional , Modelos Biológicos , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
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.