Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 17 de 17
Filter
Add more filters










Publication year range
1.
Nat Commun ; 10(1): 5571, 2019 12 05.
Article in English | MEDLINE | ID: mdl-31804478

ABSTRACT

Chloroplasts are important for photosynthesis and for plant immunity against microbial pathogens. Here we identify a haustorium-specific protein (Pst_12806) from the wheat stripe rust fungus, Puccinia striiformis f. sp. tritici (Pst), that is translocated into chloroplasts and affects chloroplast function. Transient expression of Pst_12806 inhibits BAX-induced cell death in tobacco plants and reduces Pseudomonas-induced hypersensitive response in wheat. It suppresses plant basal immunity by reducing callose deposition and the expression of defense-related genes. Pst_12806 is upregulated during infection, and its knockdown (by host-induced gene silencing) reduces Pst growth and development, likely due to increased ROS accumulation. Pst_12806 interacts with the C-terminal Rieske domain of the wheat TaISP protein (a putative component of the cytochrome b6-f complex). Expression of Pst_12806 in plants reduces electron transport rate, photosynthesis, and production of chloroplast-derived ROS. Silencing TaISP by virus-induced gene silencing in a susceptible wheat cultivar reduces fungal growth and uredinium development, suggesting an increase in resistance against Pst infection.


Subject(s)
Basidiomycota/metabolism , Chloroplasts/metabolism , Fungal Proteins/metabolism , Reactive Oxygen Species/metabolism , Basidiomycota/genetics , Basidiomycota/immunology , Chloroplasts/immunology , Chloroplasts/microbiology , Disease Resistance/genetics , Disease Resistance/immunology , Fungal Proteins/genetics , Fungal Proteins/immunology , Gene Expression Regulation, Fungal/immunology , Gene Silencing , Glucans/immunology , Glucans/metabolism , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Plant Diseases/genetics , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Immunity/genetics , Plant Immunity/immunology , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/microbiology , Plant Proteins/genetics , Plant Proteins/metabolism , Protein Binding , Pseudomonas syringae/immunology , Pseudomonas syringae/physiology , Reactive Oxygen Species/immunology , Triticum/genetics , Triticum/microbiology
2.
PLoS One ; 14(7): e0218013, 2019.
Article in English | MEDLINE | ID: mdl-31265468

ABSTRACT

BACKGROUND: Paracoccidioidomycosis (PCM) is a systemic mycosis commonly found in Latin America that is caused by distinct species of Paracoccidioides genus: Paracoccidioides brasiliensis complex (S1, PS2, PS3 and PS4) and Paracoccidioides lutzii. Its pathobiology has been recently explored by different approaches to clarify the mechanisms of host-pathogen interactions underpinning PCM. The diversity of clinical forms of this disease has been attributed to both host- and fungus-related factors. METHODOLOGY/PRINCIPAL FINDINGS: For better understanding of the molecular underpinnings of host-fungus interactions, we evaluated in vivo virulence of nine Paracoccidioides brasiliensis complex isolates and correlated it to protein expression profiles obtained by two-dimensional gel electrophoresis. Based on the recovery of viable fungi from mouse organs, the isolates were classified as those having low, moderate, or high virulence. Highly virulent isolates overexpressed proteins related to adhesion process and stress response, probably indicating important roles of those fungal proteins in regulating the colonization capacity, survival, and ability to escape host immune system reaction. Moreover, highly virulent isolates exhibited enhanced expression of glycolytic pathway enzymes concomitantly with repressed expression of succinyl-CoA ligase beta chain, a protein related to the tricarboxylic acid cycle. CONCLUSIONS/SIGNIFICANCE: Our findings may point to the mechanisms used by highly virulent P. brasiliensis isolates to withstand host immune reactions and to adapt to transient iron availability as strategies to survive and overcome stress conditions inside the host.


Subject(s)
Fungal Proteins/immunology , Gene Expression Regulation, Fungal/immunology , Paracoccidioides , Paracoccidioidomycosis/immunology , Animals , Male , Mice , Mice, Inbred BALB C , Paracoccidioides/immunology , Paracoccidioides/isolation & purification , Paracoccidioides/pathogenicity , Paracoccidioidomycosis/pathology
3.
Plant Physiol ; 175(4): 1853-1863, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29070517

ABSTRACT

RNA interference (RNAi) is a powerful genetic tool to accelerate research in plant biotechnology and control biotic stresses by manipulating target gene expression. However, the potential of RNAi in wheat to efficiently and durably control the devastating stripe rust fungus Puccinia striiformis f. sp. tritici (Pst) remained largely under explored so far. To address this issue, we generated transgenic wheat (Triticum aestivum) lines expressing dsRNA targeting PsFUZ7 transcripts of Pst We analyzed expression of PsFUZ7 and related genes, and resistance traits of the transgenic wheat lines. We show that PsFUZ7 is an important pathogenicity factor that regulates infection and development of Pst A PsFUZ7 RNAi construct stably expressed in two independent transgenic wheat lines confers strong resistance to PstPst hyphal development is strongly restricted, and necrosis of plant cells in resistance responses was significantly induced. We conclude that trafficking of RNA molecules from wheat plants to Pst may lead to a complex molecular dialogue between wheat and the rust pathogen. Moreover, we confirm the RNAi-based crop protection approaches can be used, to our knowledge, as a novel control strategy against rust pathogens in wheat.


Subject(s)
Fungal Proteins/metabolism , Gene Expression Regulation, Fungal/immunology , Gene Expression Regulation, Plant/immunology , Mitogen-Activated Protein Kinase Kinases/metabolism , Triticum/microbiology , Basidiomycota/physiology , Fungal Proteins/genetics , Plant Diseases/microbiology , RNA Interference
4.
J Proteome Res ; 15(5): 1418-34, 2016 05 06.
Article in English | MEDLINE | ID: mdl-27048922

ABSTRACT

Macrophages may induce fungal apoptosis to fight against C. albicans, as previously hypothesized by our group. To confirm this hypothesis, we analyzed proteins from C. albicans cells after 3 h of interaction with macrophages using two quantitative proteomic approaches. A total of 51 and 97 proteins were identified as differentially expressed by DIGE and iTRAQ, respectively. The proteins identified and quantified were different, with only seven in common, but classified in the same functional categories. The analyses of their functions indicated that an increase in the metabolism of amino acids and purine nucleotides were taking place, while the glycolysis and translation levels dropped after 3 h of interaction. Also, the response to oxidative stress and protein translation were reduced. In addition, seven substrates of metacaspase (Mca1) were identified (Cdc48, Fba1, Gpm1, Pmm1, Rct1, Ssb1, and Tal1) as decreased in abundance, plus 12 proteins previously described as related to apoptosis. Besides, the monitoring of apoptotic markers along 24 h of interaction (caspase-like activity, TUNEL assay, and the measurement of ROS and cell examination by transmission electron microscopy) revealed that apoptotic processes took place for 30% of the fungal cells, thus supporting the proteomic results and the hypothesis of macrophages killing C. albicans by apoptosis.


Subject(s)
Apoptosis/immunology , Candida albicans/cytology , Macrophages/chemistry , Animals , Biomarkers/analysis , Gene Expression Regulation, Fungal/immunology , Host-Pathogen Interactions/immunology , Macrophages/immunology , Macrophages/microbiology , Mice , Proteomics/methods
5.
Cell Immunol ; 284(1-2): 60-7, 2013.
Article in English | MEDLINE | ID: mdl-23928558

ABSTRACT

We investigated the features of Dectin-2 expression both at transcriptional and translational levels during Aspergillus fumigatus infection in human lung. Simultaneously, the expression of CD206 was assayed as an activated marker of alveolar macrophages. The characteristic of Dectin-2 expression were then confirmed in Monocyte-derived macrophages (MDM) after A. fumigatus stimulation by Flow Cytometry. We found that the expression of Dectin-2 was low in normal lung, while it revealed a markedly up-regulation during A. fumigatus invasion. Dectin-2 expression was predominantly restricted to CD206 positive cells. There was salient positive correlation between Dectin-2 expression and CD206. We conclude that Dectin-2 expression is largely restricted to alveolar macrophages in human lung. The conspicuous expression of Dectin-2 during A. fumigatus invasion suggests its notable contribution to antifungal defenses in pulmonary aspergillosis.


Subject(s)
Aspergillus fumigatus/immunology , Gene Expression Regulation, Fungal/immunology , Lectins, C-Type/immunology , Macrophages, Alveolar/immunology , Pulmonary Aspergillosis/immunology , Adult , Aged , Aspergillus fumigatus/genetics , Blotting, Western , Female , Flow Cytometry , Humans , Immunohistochemistry , Lectins, C-Type/genetics , Macrophages, Alveolar/microbiology , Male , Middle Aged , Pulmonary Aspergillosis/microbiology , RNA, Fungal/chemistry , RNA, Fungal/genetics , Real-Time Polymerase Chain Reaction , Statistics, Nonparametric
6.
PLoS Pathog ; 7(6): e1002074, 2011 Jun.
Article in English | MEDLINE | ID: mdl-21731485

ABSTRACT

Candida albicans yeast cells are found in the intestine of most humans, yet this opportunist can invade host tissues and cause life-threatening infections in susceptible individuals. To better understand the host factors that underlie susceptibility to candidiasis, we developed a new model to study antifungal innate immunity. We demonstrate that the yeast form of C. albicans establishes an intestinal infection in Caenorhabditis elegans, whereas heat-killed yeast are avirulent. Genome-wide, transcription-profiling analysis of C. elegans infected with C. albicans yeast showed that exposure to C. albicans stimulated a rapid host response involving 313 genes (124 upregulated and 189 downregulated, ~1.6% of the genome) many of which encode antimicrobial, secreted or detoxification proteins. Interestingly, the host genes affected by C. albicans exposure overlapped only to a small extent with the distinct transcriptional responses to the pathogenic bacteria Pseudomonas aeruginosa or Staphylococcus aureus, indicating that there is a high degree of immune specificity toward different bacterial species and C. albicans. Furthermore, genes induced by P. aeruginosa and S. aureus were strongly over-represented among the genes downregulated during C. albicans infection, suggesting that in response to fungal pathogens, nematodes selectively repress the transcription of antibacterial immune effectors. A similar phenomenon is well known in the plant immune response, but has not been described previously in metazoans. Finally, 56% of the genes induced by live C. albicans were also upregulated by heat-killed yeast. These data suggest that a large part of the transcriptional response to C. albicans is mediated through "pattern recognition," an ancient immune surveillance mechanism able to detect conserved microbial molecules (so-called pathogen-associated molecular patterns or PAMPs). This study provides new information on the evolution and regulation of the innate immune response to divergent pathogens and demonstrates that nematodes selectively mount specific antifungal defenses at the expense of antibacterial responses.


Subject(s)
Caenorhabditis elegans/microbiology , Candida albicans/immunology , Gene Expression Regulation, Fungal/immunology , Immunity, Innate/genetics , Animals , Biological Evolution , Fungi/immunology , Gene Expression Profiling , Genes, Bacterial , Genes, Fungal , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/immunology , Staphylococcus aureus/genetics , Staphylococcus aureus/immunology
7.
J Immunol ; 187(2): 999-1005, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21685325

ABSTRACT

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α-converting enzyme (TACE), critical for the cleavage of membrane-bound pro-TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.


Subject(s)
Aspergillus fumigatus/enzymology , Aspergillus fumigatus/immunology , Gene Expression Regulation, Fungal/immunology , Mucin 5AC/biosynthesis , Mucins/biosynthesis , Respiratory Mucosa/immunology , Respiratory Mucosa/microbiology , Serine Proteases/metabolism , ADAM Proteins/physiology , ADAM17 Protein , Animals , Aspergillus fumigatus/genetics , Cell Line, Tumor , Cells, Cultured , Enzyme Activation/genetics , Enzyme Activation/immunology , ErbB Receptors/physiology , Gene Expression Regulation, Enzymologic/immunology , Humans , Mice , Mice, Inbred C57BL , Mucin 5AC/genetics , Mucins/genetics , Respiratory Mucosa/enzymology , Transforming Growth Factor alpha/physiology
8.
Infect Immun ; 78(7): 3097-102, 2010 Jul.
Article in English | MEDLINE | ID: mdl-20421379

ABSTRACT

CD8+ T-cell immunity has been shown to play an important role in the protective immune response against Encephalitozoon cuniculi. Although earlier studies suggest that dendritic cells (DC) are important for the induction of this response, the factors responsible for initiation of the dendritic cell response against this pathogen have not been evaluated. In the current study, we demonstrate that E. cuniculi infection causes strong Toll-like receptor 4 (TLR4)-dependent dendritic cell activation and a blockade of this molecule reduces the ability of DC to prime an antigen-specific CD8+ T-cell response. Pretreatment of DC with anti-TLR4 antibody causes a defect in both in vitro and in vivo CD8+ T-cell priming. These findings, for the first time, emphasize the contribution of TLR4 in the induction of CD8+ T-cell immunity against E. cuniculi infection.


Subject(s)
CD8-Positive T-Lymphocytes/physiology , Dendritic Cells/physiology , Encephalitozoon cuniculi/immunology , Encephalitozoonosis/immunology , Toll-Like Receptor 4/physiology , Animals , Autoantibodies/immunology , CD8-Positive T-Lymphocytes/immunology , Dendritic Cells/chemistry , Dendritic Cells/immunology , Encephalitozoonosis/microbiology , Gene Expression Regulation, Fungal/immunology , Gene Expression Regulation, Fungal/physiology , Immunity, Cellular/immunology , Immunity, Cellular/physiology , Immunoprecipitation , Interleukin-12/analysis , Interleukin-12/immunology , Lymphocyte Activation/immunology , Lymphocyte Activation/physiology , Mice , Mice, Inbred C57BL , Toll-Like Receptor 4/biosynthesis , Toll-Like Receptor 4/immunology , Up-Regulation/physiology
9.
J Immunol ; 183(7): 4609-18, 2009 Oct 01.
Article in English | MEDLINE | ID: mdl-19734205

ABSTRACT

Chronic granulomatous disease (CGD) is a primary immunodeficiency characterized by life-threatening bacterial and fungal infections and hyperinflammation. The susceptibility to aspergillosis in experimental CGD (p47(phox-/-) mice) is associated with the failure to control the inherent inflammatory response to the fungus and to restrict the activation of inflammatory Th17 cells. We assessed whether pentraxin (PTX)3, a member of a family of multimeric pattern-recognition proteins with potent anti-Aspergillus activity, could limit pathogenic inflammation in p47(phox-/-) mice by curbing the IL-23/Th17 inflammatory axis in response to the fungus. We found that the production of PTX3 was delayed in CGD mice in infection but exogenous administration of PTX3 early in infection restored antifungal resistance and restrained the inflammatory response to the fungus. This occurred through down-regulation of IL-23 production by dendritic cells and epithelial cells which resulted in limited expansion of IL-23R+ gammadelta+ T cells producing IL-17A and the emergence of Th1/Treg responses with minimum pathology. Thus, PTX3 could be therapeutically used for the exploitation of NADPH-independent mechanism(s) of antifungal immune protection with limited immunopathology in CGD.


Subject(s)
Antifungal Agents/administration & dosage , C-Reactive Protein/administration & dosage , Drug Resistance, Fungal/immunology , Granulomatous Disease, Chronic/pathology , Granulomatous Disease, Chronic/prevention & control , Inflammation Mediators/administration & dosage , Pulmonary Aspergillosis/pathology , Pulmonary Aspergillosis/prevention & control , Serum Amyloid P-Component/administration & dosage , Animals , Antifungal Agents/metabolism , Antifungal Agents/therapeutic use , Aspergillus fumigatus/immunology , Aspergillus fumigatus/pathogenicity , C-Reactive Protein/biosynthesis , C-Reactive Protein/genetics , C-Reactive Protein/therapeutic use , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , Drug Resistance, Fungal/genetics , Gene Expression Regulation, Fungal/immunology , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/immunology , Inflammation Mediators/metabolism , Inflammation Mediators/therapeutic use , Mice , Mice, Inbred C57BL , Mice, Knockout , Pulmonary Aspergillosis/genetics , Pulmonary Aspergillosis/immunology , Serum Amyloid P-Component/biosynthesis , Serum Amyloid P-Component/genetics , Serum Amyloid P-Component/therapeutic use
10.
Int Immunol ; 20(12): 1527-41, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18927317

ABSTRACT

Glucuronoxylomannan (GXM) is the major component of Cryptococcus capsular polysaccharide, which represents an essential virulence factor for this yeast. Cryptococcus neoformans infections in immunocompetent rats are associated with inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production by macrophages. This study demonstrates in vitro and in vivo that GXM promotes iNOS expression with NO production in rat macrophages. GXM also induced macrophage apoptosis after 48 h of culture, with this phenomenon being prevented by the iNOS inhibitor, aminoguanidine. The NO-induced macrophage apoptosis triggered by GXM was dependent on interactions with CD18, Fcgamma receptor II and protein kinase C activation, without participation of tyrosine kinases or mitogen-activated protein kinases. Furthermore, this study reveals that GXM down-regulates the macrophage caspase-3 activity, induces a caspase-independent cell death and promotes depolarization of mitochondria membrane potential with increased cytosolic expression of the apoptosis-inducing factor. Taken together, this study describes the pathways and mechanisms involved in the macrophage apoptosis promoted by GXM through NO generation. These findings indicate new mechanisms of immunomodulation for the main capsular polysaccharide of C. neoformans.


Subject(s)
Caspase 3/metabolism , Cryptococcus neoformans/metabolism , Macrophages, Peritoneal/immunology , Macrophages, Peritoneal/pathology , Nitric Oxide/genetics , Polysaccharides/immunology , Animals , Apoptosis/drug effects , Apoptosis/immunology , Cells, Cultured , Cryptococcus neoformans/immunology , Female , Gene Expression Regulation, Fungal/immunology , Macrophages, Peritoneal/drug effects , Membrane Potential, Mitochondrial , Nitric Oxide/biosynthesis , Polysaccharides/genetics , Polysaccharides/metabolism , Polysaccharides/pharmacology , Rats , Rats, Wistar , Signal Transduction/immunology , Species Specificity
11.
Microb Pathog ; 42(4): 138-47, 2007 Apr.
Article in English | MEDLINE | ID: mdl-17306958

ABSTRACT

Oral candidiasis is a common opportunistic infection, with Candida albicans being the most prevalent etiologic agent and Candida glabrata emerging as an important pathogen. C. glabrata is frequently co-isolated with C. albicans from oral lesions. Although C. albicans has been shown to trigger significant cytokine responses and cell damage, C. glabrata has not been systematically studied yet. The purpose of this study was to characterize the ability of C. glabrata to induce proinflammatory cytokine responses and host damage as a single infecting organism and in combination with C. albicans, using in vitro models of the oral mucosa. In monolayer oral epithelial cell cultures, C. glabrata failed to induce a significant interleukin-1alpha and interleukin-8 cytokine response and showed lower cytotoxicity, compared to C. albicans. However, C. glabrata triggered a significantly higher granulocyte macrophage colony stimulating factor response than C. albicans. C. glabrata strains showed a strain-dependent tissue damaging ability and a superficial invasion of the mucosal compartment in a three-dimensional (3-D) in vitro model of the human oral mucosa and submucosa. In the 3-D system, co-infection failed to promote host damage beyond the levels of infection with C. albicans alone. These studies indicate that C. glabrata induces cytokines in human oral epithelium in a strain-specific manner, but its tissue/cell damaging ability, compared to C. albicans, is low. Synergy between C. glabrata and C. albicans in cytokine induction and host damage was not observed with the strains tested.


Subject(s)
Candida glabrata/immunology , Candida glabrata/pathogenicity , Candidiasis, Oral/microbiology , Cytokines/biosynthesis , Gene Expression Regulation, Fungal/immunology , Candida albicans/immunology , Candida albicans/pathogenicity , Candida glabrata/classification , Cell Line , Cytokines/analysis , Cytokines/genetics , DNA Primers/chemistry , Enzyme-Linked Immunosorbent Assay , Humans , Keratinocytes/cytology , Keratinocytes/pathology , Mouth Mucosa/microbiology , Reverse Transcriptase Polymerase Chain Reaction , Time Factors
12.
FEMS Immunol Med Microbiol ; 38(3): 231-9, 2003 Oct 15.
Article in English | MEDLINE | ID: mdl-14522458

ABSTRACT

Threatening virus infections constantly illustrate the growing need for novel vaccines that specifically induce efficient T cell-mediated immune responses. In this study, we used a human whole blood assay to determine the activation of antigen-specific human T lymphocytes by a viral antigen of human cytomegalovirus (HCMV). The major HCMV tegument protein pp65, recombinantly expressed in fission yeast (Schizosaccharomyces pombe), specifically activated antigen-specific CD4- and CD8-positive memory T cells in blood of HCMV seropositive donors. Moreover, the immune response against recombinant pp65, in particular that of CD8 class I major histocompatibility complex-restricted cytotoxic T cells, was similar to the response against the intact HCMV. Since fission yeast cells per se did not activate a significant number of human T lymphocytes ex vivo, the system described here might represent a novel approach in vaccine development as well as in the identification of vaccine candidates directly from human whole blood.


Subject(s)
Cytomegalovirus/immunology , Lymphocyte Activation , Phosphoproteins/immunology , Schizosaccharomyces/genetics , T-Lymphocytes/immunology , Viral Matrix Proteins/immunology , Antibodies, Viral/blood , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Gene Expression Regulation, Fungal/genetics , Gene Expression Regulation, Fungal/immunology , Humans , Immunologic Memory , Lymphocyte Count , Phagocytosis , Phosphoproteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/immunology , Schizosaccharomyces/growth & development , Schizosaccharomyces/immunology , Schizosaccharomyces/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , T-Lymphocytes, Cytotoxic/cytology , T-Lymphocytes, Cytotoxic/immunology , Viral Matrix Proteins/genetics
13.
Vaccine ; 21(15): 1650-7, 2003 Apr 02.
Article in English | MEDLINE | ID: mdl-12639486

ABSTRACT

In previously published studies, Saccharomyces cerevisiae recombinant protein expression systems have been employed to express the malaria parasite antigen Pfs25, a candidate transmission-blocking vaccine antigen against Plasmodium falciparum malaria. However, despite having been in two Phase 1 trials, the recombinant Pfs25 so produced (previously called TBV25H) exists as a mixture of two monomeric protein conformational forms, Pfs25H-A and Pfs25H-B. In this study, we optimized the expression and purification of the two Pfs25H conformers in S. cerevisiae, and characterized their biochemical and antigenic properties, immunogenicities, and transmission-blocking activities. Pfs25H-A is apparently homogeneous, and has the correct conformation as measured by monoclonal antibody recognition. It is, however, expressed at a low yield of only 0.19mg/l. By contrast, Pfs25H-B is produced as a heterogeneous population of molecules that do not seem to have the correct conformation. Nonetheless, both forms appear equally effective in their ability to produce transmission-blocking antibodies in mice. To address the low yield seen with S. cerevisiae, we also expressed Pfs25 in Pichia pastoris. P. pastoris is apparently superior to S. cerevisiae in producing higher yield, immunologically more potent, biologically more active Pfs25H-A.


Subject(s)
Clinical Trials as Topic , Malaria Vaccines/genetics , Malaria Vaccines/therapeutic use , Malaria, Falciparum/immunology , Malaria, Falciparum/parasitology , Pichia/metabolism , Protozoan Proteins/biosynthesis , Amino Acid Sequence , Animals , Clinical Trials as Topic/methods , Culicidae/immunology , Culicidae/parasitology , Female , Gene Expression Regulation, Fungal/immunology , Humans , Malaria Vaccines/biosynthesis , Malaria Vaccines/immunology , Malaria, Falciparum/transmission , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Pichia/immunology , Plasmids/immunology , Plasmodium falciparum/immunology , Protozoan Proteins/immunology , Protozoan Proteins/isolation & purification , Saccharomyces cerevisiae/immunology , Saccharomyces cerevisiae/isolation & purification , Saccharomyces cerevisiae/metabolism , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/immunology , Vaccines, Synthetic/therapeutic use
14.
Am J Respir Crit Care Med ; 167(7): 991-8, 2003 Apr 01.
Article in English | MEDLINE | ID: mdl-12493647

ABSTRACT

The most common form of hypersensitivity pneumonitis in Japan is summer-type hypersensitivity pneumonitis (SHP), which is caused by the inhalation of Trichosporon asahii or Trichosporon mucoides. To seek protein antigens relevant to the immunopathogenesis of SHP, we constructed a cDNA expression library of T. asahii, a major causative yeast species of SHP. Using the immunoscreening method, we identified and cloned a novel gene encoding a 19-kD protein, named TA-19, which proved to be specifically recognized in the bronchoalveolar lavage (BAL) fluids and sera of patients with SHP. IgG, IgA, and IgM antibodies to the recombinant TA-19 protein were significantly elevated in the sera as well as in the BAL fluids from SHP patients compared with those from non-SHP groups. This protein also induced SHP-specific proliferation of the mononuclear cells from both the peripheral blood and BAL. These results reveal that TA-19 derived from T. asahii may play a relevant role in specific cellular and humoral immune responses in patients with SHP.


Subject(s)
Alveolitis, Extrinsic Allergic/etiology , Antigens, Fungal/adverse effects , Antigens, Fungal/immunology , Trichosporon/immunology , Administration, Inhalation , Adult , Aged , Alveolitis, Extrinsic Allergic/blood , Alveolitis, Extrinsic Allergic/genetics , Antibodies, Fungal/genetics , Antibodies, Fungal/immunology , Antibody Specificity/immunology , Antigens, Fungal/administration & dosage , Base Sequence , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/immunology , Female , Gene Expression Regulation, Fungal/genetics , Gene Expression Regulation, Fungal/immunology , Gene Library , Humans , Immunoglobulin A/immunology , Immunoglobulin A/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunoglobulin M/immunology , Immunoglobulin M/metabolism , Japan , Lymphocytosis/blood , Lymphocytosis/etiology , Male , Middle Aged , Molecular Sequence Data , Nucleic Acid Amplification Techniques , Predictive Value of Tests , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Seasons , Trichosporon/genetics
15.
J Biochem ; 134(6): 911-7, 2003 Dec.
Article in English | MEDLINE | ID: mdl-14769881

ABSTRACT

Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, there is no such universal system available up to now. A vector system was constructed based on pPIC9- Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of mouse IgG1 and His-tag were cloned into the Pichia expression vector pPIC9. A model ScFv was introduced into pPIC9-Fc, which can bind Glutathione-S-transferase (GST) from Schistosoma japonicum, to yield the expression cassette pPIC9-ScFv-Fc. Following fermentation in a 5-liter reactor, the fusion was expressed at high levels in the methylotrophic yeast Pichia Pastoris, secreted as a dimeric form in the culture, and purified by Ni2+-NTA column chromatography. The expression yield can reach 10-30 mg/liter of culture medium. The ScFv-Fc fusion retains the biological binding ability of the parent ScFv, and can be applied as anti-GST antibodies for the detection of GST and GST-fusion proteins. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-mouse antibodies.


Subject(s)
Immunoglobulin Fc Fragments/biosynthesis , Immunoglobulin Fc Fragments/genetics , Pichia/genetics , Recombinant Fusion Proteins/biosynthesis , Animals , Binding Sites, Antibody/genetics , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Fungal/immunology , Genetic Vectors , Immunoglobulin Fc Fragments/metabolism , Mice , Mutagenesis, Insertional , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism
16.
J Infect Dis ; 186(3): 389-96, 2002 08 01.
Article in English | MEDLINE | ID: mdl-12134235

ABSTRACT

The host inflammatory response to the opportunistic pathogen Candida albicans determines susceptibility to disseminated infection. This study used immunohistochemical analysis to correlate microbiologic findings and pathologic changes with local expression of cytokines and leukocyte adhesion molecules in mice with disseminated candidiasis. After being inoculated intravenously as blastospores, the organisms filamented extensively in the kidneys while remaining predominantly as blastospores or short germ tubes in the lung, liver, and spleen. Very few leukocytes accumulated around the invading organisms until 24 h after inoculation. The leukocytes at the infection site appeared to amplify the inflammatory response. They expressed interleukin-1beta, tumor necrosis factor-alpha, intercellular adhesion molecule 1, and platelet-endothelial cell adhesion molecule 1. Therefore, the morphology of the organism varies with the infection site. Furthermore, leukocyte recruitment occurs relatively late in the infection, and this recruitment is likely amplified by proinflammatory mediators produced by the leukocytes themselves.


Subject(s)
Candida albicans/immunology , Candidiasis/immunology , Cell Adhesion Molecules/biosynthesis , Cytokines/biosynthesis , Gene Expression Regulation, Fungal/immunology , Animals , Candidiasis/metabolism , Candidiasis/pathology , Cell Adhesion Molecules/analysis , Cytokines/blood , Histocytochemistry , Kidney/microbiology , Kidney/pathology , Liver/microbiology , Liver/pathology , Lung/microbiology , Lung/pathology , Male , Mice , Mice, Inbred BALB C , Microscopy, Confocal , Microscopy, Fluorescence , Spleen/microbiology , Spleen/pathology
17.
Zh Mikrobiol Epidemiol Immunobiol ; (10): 109-12, 1990 Oct.
Article in Russian | MEDLINE | ID: mdl-2150140

ABSTRACT

The regulation of the synthesis of factor inhibiting the migration of macrophages in response to C. albicans antigen in CBA (H-2k) and C57BL/6 (H-2b) mice has been studied. The low level of macrophage migration inhibition factor in response to this antigen is due to the existence of cyclophosphamide-inhibited specific suppressors. Differences between various strains of mice ensue from different activity of suppressors of thymic origin, whose nature has been revealed as the result of the transfer of marrow cells treated with anti-Thy-1 serum.


Subject(s)
Antigens, Fungal/immunology , Candida albicans/immunology , Gene Expression Regulation, Fungal/immunology , Genes, Regulator/immunology , Macrophage Migration-Inhibitory Factors/biosynthesis , Tuberculin/immunology , Animals , Antigens, Fungal/genetics , Candida albicans/genetics , Genes, Regulator/genetics , Hypersensitivity, Delayed/genetics , Hypersensitivity, Delayed/immunology , Immunization , Macrophage Migration-Inhibitory Factors/genetics , Macrophage Migration-Inhibitory Factors/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Phenotype , T-Lymphocytes, Regulatory/immunology , Tuberculin/genetics
SELECTION OF CITATIONS
SEARCH DETAIL
...