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PubMed2005

Concerted evolution in the repeats of an immunomodulating cell surface protein, SOWgp, of the human pathogenic fungi Coccidioides immitis and C. posadasii.

Genome dynamics that allow pathogens to escape host immune responses are fundamental to our understanding of host-pathogen interactions. Here we present the first population-based study of the process of concerted evolution in the repetitive domain of a protein-coding gene. This gene, SOWgp, encodes the immunodominant protein in the parasitic phase of the human pathogenic fungi Coccidioides immitis and C. posadasii. We sequenced the entire gene from strains representing the geographic ranges of the two Coccidioides species. By using phylogenetic and genetic distance analyses we discovered that the repetitive part of SOWgp evolves by concerted evolution, predominantly by the mechanism of unequal crossing over. We implemented a mathematical model originally developed for multigene families to estimate the rate of homogenization and recombination of the repetitive array, and the results indicate that the pattern of concerted evolution is a result of homogenization of repeat units proceeding at a rate close to the nucleotide point mutation rate. The release of the SOWgp molecules by the pathogen during proliferation may mislead the host: we speculate that the pathogen benefits from concerted evolution of repeated domains in SOWgp by an enhanced ability to misdirect the host's immune system.

Antigens, Fungal↗

PubMed2006

Development of a novel three-dimensional in vitro model of oral Candida infection.

Oropharyngeal candidiasis is emerging as a serious health problem in the elderly as well as other chronically immunosuppressed patient populations. Several approaches have been used to study the interactions between Candida and the oral mucosa in vitro. Recently, three-dimensional organotypic systems of the oral mucosa have been developed, which provide an organizational complexity that is between the culture of single cell types and organ cultures in vivo. In this report we describe the development of a novel three-dimensional system of the human oral mucosa based on an immortalized oral keratinocyte cell line. Unlike the commercially available cell line systems, this system also contains a connective tissue cell component, which ensures the quality and resemblance of the tissue model to the human oral masticatory mucosa and submucosa. Using a panel of Candida albicans strains with variable virulence in vivo, we showed that the extent of tissue damage, fungal invasion and host inflammatory response in this system was proportional to the well-documented in vivo virulence potential of these strains. Therefore, this investigation has added another useful tool in the study of host pathogen interactions in oral candidiasis.

Candida albicans↗

PubMed2001

Two novel insect defensins from larvae of the cupreous chafer, Anomala cuprea: purification, amino acid sequences and antibacterial activity.

A humoral immune response in larvae of the coleopteran insect, Anomala cuprea has been examined for exploring the molecular basis of host-pathogen interactions. The antibacterial activity against the Gram-positive strain, Micrococcus luteus was detected at a low level in absence of injection. The activity increased strikingly in the hemolymph of the larvae challenged with Escherichia coli, showing the fluctuating profile through a time course, which consists of the static induction phase, the production phase rising to a maximum level, and the reduction phase extending over a long duration. Two peptides were purified and characterized by reverse-phase HPLC, Edman degradation and mass spectrometry. They were isoforms, composed of similar sequences with two amino acid substitutions in 43 residues, and novel members of the insect defensins, cysteine-rich antibacterial peptides. Anomala defensins A and B showed potent activity against Gram-positive bacteria, with slight differences in activity against a few strains of tested bacteria. Anomala defensin B was active at high concentration of 40 microM against the Gram-negative strain, Xenorhabdus japonicus, a pathogen toward the host, A. cuprea larvae.

Amino Acid Sequence↗

PubMed2003

Disruption of mptpB impairs the ability of Mycobacterium tuberculosis to survive in guinea pigs.

Protein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier. MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell. To determine the role of MptpB in the pathogenesis of M. tuberculosis, we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host-pathogen interaction. Infection of guinea pigs with the mutant strain resulted in a 70-fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain. These observations demonstrate a role of MptpB in the pathogenesis of M. tuberculosis.

Animals↗

PubMed2005

The Mycobacterium tuberculosis cell wall component mycolic acid elicits pathogen-associated host innate immune responses.

Recognition of conserved pathogen-associated molecular patterns constitutes a crucial step in the initiation of innate immune responses. We studied the contribution to the host-pathogen interaction of mycolic acid (MA), a major lipid component of the cell envelope of the macrophage intracellular pathogen Mycobacterium tuberculosis and other mycobacteria. MA administered to the peritoneal cavity or to the airways induced a unique macrophage morphotype, similar to the foamy macrophage derivatives observed in tuberculous granulomas and characterized by intracellular accumulation of neutral lipids and entry into mitosis. When assayed for production of inflammatory mediators, a conditioning rather than a direct activation of the MA-elicited foamy macrophages was observed. MA enabled production of IFN-gamma and myeloperoxidase, enhanced TNF-alpha production and suppressed IL-10 upon renewed exposure to innate triggers. Intratracheal instillation of MA mimicked additional features of the airway response to M. tuberculosis infection, namely a rapid but transient neutrophil influx and IL-6 production and a chronic IL-12 production. These MA-elicited cellular innate defenses and the accompanying formation of foamy macrophages identify for the first time the foamy macrophage morphotype as part of the host response to a pathogen-associated structure. Furthermore, these results characterize MA as a direct trigger of innate immunity, distinct from Toll-like receptor ligands.

Animals↗

PubMed2005

Subproteomes of soluble and structure-bound Helicobacter pylori proteins analyzed by two-dimensional gel electrophoresis and mass spectrometry.

Helicobacter pylori is one of the most common bacterial pathogens and causes a variety of diseases, such as peptic ulcer or gastric cancer. Despite intensive study of this human pathogen in the last decades, knowledge about its membrane proteins and, in particular, those which are putative components of the type IV secretion system encoded by the cag pathogenicity island (PAI) remains limited. Our aim is to establish a dynamic two-dimensional electrophoresis-polyacrylamide gel electrophoresis (2-DE-PAGE) database with multiple subproteomes of H. pylori (http://www.mpiib-berlin.mpg.de/2D-PAGE) which facilitates identification of bacterial proteins important in pathogen-host interactions. Using a proteomic approach, we investigated the protein composition of two H. pylori fractions: soluble proteins and structure-bound proteins (including membrane proteins). Both fractions differed markedly in the overall protein composition as determined by 2-DE. The 50 most abundant protein spots in each fraction were identified by peptide mass fingerprinting. We detected four cag PAI proteins, numerous outer membrane proteins (OMPs), the vacuolating cytotoxin VacA, other potential virulence factors, and few ribosomal proteins in the structure-bound fraction. In contrast, catalase (KatA), gamma-glutamyltranspeptidase (Ggt), and the neutrophil-activating protein NapA were found almost exclusively in the soluble protein fraction. The results presented here are an important complement to genome sequence data, and the established 2-D PAGE maps provide a basis for comparative studies of the H. pylori proteome. Such subproteomes in the public domain will be effective instruments for identifying new virulence factors and antigens of potential diagnostic and/or curative value against infections with this important pathogen.

Amino Acid Sequence↗

PubMed2006

Protein array staining methods for undefined protein content, manufacturing quality control, and performance validation.

Methods to assess the quality and performance of protein microarrays fabricated from undefined protein content are required to elucidate slide-to-slide variability and interpolate resulting signal intensity values after an interaction assay. We therefore developed several simple total- and posttranslational modification-specific, on-chip staining methods to quantitatively assess the quality of gel element protein arrays manufactured with whole-cell lysate in vitro protein fractions derived from two-dimensional liquid-phase fractionation (PF2D) technology. A linear dynamic range of at least 3 logs was observed for protein stains and immobilized protein content, with a lower limit of detection at 8 pg of protein per gel element with Deep Purple protein stain and a field-portable microarray imager. Data demonstrate the successful isolation, separation, transfer, and immobilization of putative transmembrane proteins from Yersinia pestis KIM D27 with the combined PF2D and gel element array method. Internal bovine serum albumin standard curves provided a method to assess on-chip PF2D transfer and quantify total protein immobilized per gel element. The basic PF2D array fabrication and quality assurance/quality control methods described here therefore provide a standard operating procedure and basis for developing whole-proteome arrays for interrogating host-pathogen interactions, independent of sequenced genomes, affinity tags, or a priori knowledge of target cell composition.

Animals↗

PubMed1998

Cloning and characterization of PRA1, a gene encoding a novel pH-regulated antigen of Candida albicans.

Candida albicans is an opportunistic fungal pathogen of humans. The cell wall of the organism defines the interface between the pathogen and host tissues and is likely to play an essential and pivotal role in the host-pathogen interaction. The components of the cell wall critical to this interaction are undefined. Immunoscreening of a lambda expression library with sera raised against mycelial cell walls of C. albicans was used to identify genes encoding cell surface proteins. One of the positive clones represented a candidal gene that was differentially expressed in response to changes in the pH of the culture medium. Maximal expression occurred at neutral pH, with no expression detected below pH 6.0. On the basis of the expression pattern, the corresponding gene was designated PRA1, for pH-regulated antigen. The protein predicted from the nucleotide sequence was 299 amino acids long with motifs characteristic of secreted glycoproteins. The predicted surface localization and N glycosylation of the protein were directly demonstrated by cell fractionation and immunoblot analysis. Deletion of the gene imparted a temperature-dependent defect in hypha formation, indicating a role in morphogenesis. The PRA1 protein was homologous to surface antigens of Aspergillus spp. which react with serum from aspergillosis patients, suggesting that the PRA1 protein may have a role in the host-parasite interaction during candidal infection.

Amino Acid Sequence↗

PubMed2006

Deletion of the genes encoding the type III secretion system and cytotoxic enterotoxin alters host responses to Aeromonas hydrophila infection.

In our previous study, we deleted the gene encoding Aeromonas outer membrane protein B (AopB), a structural component of the type III secretion system (T3SS) from a cytotoxic enterotoxin gene (act)-minus diarrheal isolate SSU of Aeromonas hydrophila. Our laboratory also molecularly characterized the cytotoxic enterotoxin (Act), which is secreted by the bacterium utilizing the type II secretion system (T2SS). The act/aopB mutant exhibited significantly reduced cytotoxicity to cultured cells (e.g. RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells) and was avirulent in mice. In this study, we developed additional A. hydrophila mutants in which T3SS-associated ascV and acrV genes were deleted, either individually or in combination with that of the act gene, to examine host-pathogen interactions. A significant reduction in the induction of inflammatory cytokines and chemokines was noted in the sera of mice infected with these mutants when compared to animals infected with wild-type (WT) A. hydrophila. After infection with the WT and act/aopB mutant, we performed microarray analyses on RNA from the above-mentioned murine macrophages and human colonic epithelial cells to examine global cellular transcriptional responses. Based on three independent experiments, WT A. hydrophila altered the expression of 434 genes in RAW 264.7 cells and 80 genes in HT-29 cells. Alteration in the expression of 209 macrophage and 32 epithelial cell genes was reduced when the act/aopB mutant was used, compared to when cells were infected with the WT bacterium, indicating the involvement of Act and/or AopB in transcriptional regulation of these genes. We verified up-regulation of 15 genes by real-time reverse transcriptase-polymerase chain reaction and confirmed A. hydrophila WT-versus mutant-induced production of cytokines/chemokines in supernatants from RAW 264.7 and HT-29 cells. This is the first description of host cell transcriptional responses to A. hydrophila infection.

Aeromonas hydrophila↗

PubMed1990

Implication of Pectic Components in Cell Surface Interactions between Tomato Root Cells and Fusarium oxysporum f. sp. radicis-lycopersici: A Cytochemical Study by Means of a Lectin with Polygalacturonic Acid-Binding Specificity.

Aplysia gonad lectin, a polygalacturonic acid-binding lectin isolated from the sea mollusc Aplysia depilans, was complexed to colloidal gold and used for localizing polygalacturonic-acid-containing molecules in tomato root tissues infected with Fusarium oxysporum f. sp. radicis-lycopersici (FORL). Colonization of host tissues by FORL was associated with striking wall modifications including disruption and even loss of middle lamellae. According to the labeling pattern observed in host wall areas adjacent to fungal penetration channels, it is likely that FORL pectolytic enzymes act through localized wall degradation. The release of polygalacturonic acid-rich wall fragments and the accumulation of polygalacturonic acid-containing molecules in some altered phloem cells were frequently observed and considered to be specific host reactions to fungal attack. The heavy deposition of such molecules at strategic sites such as wall oppositions and intercellular spaces provides support to their implication in the plant defense system. The possible interrelation between polygalacturonic acid-containing molecules and other polymers such as lignin and phenolic compounds remains to be investigated further. The role of these molecules in host-pathogen interactions is discussed in relation to plant defense.

Journal Article↗

PubMed2026

Structure-resolved virus-host interactomics by cross-linking mass spectrometry.

Viruses depend on host protein networks to replicate, assemble progeny, and spread between cells and organisms. Defining these virus-host protein interactions is challenging because they are highly dependent on infection stage, cell type, species, and because mechanistic interpretation requires information about structural interfaces and conformational states. Cross-linking mass spectrometry (XL-MS) addresses these challenges by adding a spatial and structural dimension to virus-host interactomics in native systems. In this review, we discuss how XL-MS has advanced from targeted analysis of viral protein complexes to structure-resolved mapping of virion architecture and infected-cell virus-host interactomes. We highlight how XL-MS complements AP-MS, cryo-EM/cryo-ET, quantitative proteomics, genetic perturbation, and structure prediction to connect physical proximity with molecular mechanisms. Finally, we discuss current limitations in sensitivity, chemical coverage, temporal resolution, and model interpretation, and outline how future quantitative and integrative XL-MS workflows may enable systems-level structural virology.

Mass Spectrometry↗

PubMed2005

Acute-phase concentrations of lipopolysaccharide (LPS)-binding protein inhibit innate immune cell activation by different LPS chemotypes via different mechanisms.

The chain length of bacterial lipopolysaccharide (LPS) is a crucial factor for host-pathogen interaction during bacterial infection. While rough (R)-type and smooth (S)-type LPSs have been shown to differ in their ability to interact with the bactericidal/permeability-increasing protein, little is known about the differential mode of interaction with the acute-phase reactant LPS-binding protein (LBP). At lower concentrations, LBP catalyzes the binding of LPS to CD14 and enhances LPS-induced cellular activation via Toll-like receptor 4. In humans, however, concentrations of LBP in serum increase during an acute-phase response, and these LBP concentrations exhibit inhibitory effects in terms of cellular activation. The mechanisms of inhibition of LPS effects by LBP are not completely understood. Here, we report that human high-dose LBP (hd-LBP) suppresses binding of both R-type and S-type LPS to CD14 and inhibits LPS-induced nuclear translocation of NF-kappaB, although cellular uptake of R-type LPS was found to be increased by hd-LBP. In contrast, we found that hd-LBP enhanced the binding and uptake of S-type LPS only under serum-free conditions, whereas in the presence of serum, hd-LBP inhibited cellular binding and uptake. This inhibitory effect of serum could be mimicked by the addition of purified high-density lipoprotein (HDL) to serum-free medium, indicating an LBP-mediated transfer of preferentially S-type LPS to plasma lipoproteins such as HDL. A complete understanding of the host's mechanisms to modulate the proinflammatory effects of LPS will most likely help in the understanding of inflammation and infection and may lead to novel therapeutic intervention strategies.

Acute-Phase Proteins↗

PubMed2005

Differential effects of interferon-gamma and tumor necrosis factor-alpha on Toxoplasma gondii proliferation in organotypic rat brain slice cultures.

Organotypic slice culture explants of rat cortical tissue infected with Toxoplasma gondii tachyzoites were applied as an in vitro model to investigate host-pathogen interactions in cerebral toxoplasmosis. The kinetics of parasite proliferation and the effects of interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha) in infected organotypic cultures were monitored by light microscopy, transmission electron microscopy (TEM), and quantitative polymerase chain reaction (PCR) assay. As assessed by the loss of the structural integrity of the glial fibrillary acidic protein-intermediate filament network, tachyzoites infected and proliferated mainly within astrocytes, whereas neurons and microglia remained largely unaffected. Toxoplasma gondii proliferation was severely inhibited by IFN-y. However, this inhibition was not linked to tachyzoite-to-bradyzoite stage conversion. In contrast, TNF-alpha treatment resulted in a dramatically enhanced proliferation rate of the parasite. The cellular integrity in IFN-gamma-treated organotypic slice cultures was severely impaired compared with untreated and TNF-alpha-treated cultures. Thus, on infection of organotypic neuronal cultures, IFN-gamma and TNF-alpha exhibit largely detrimental effects, which could contribute to either inhibition or acceleration of parasite proliferation during cerebral toxoplasmosis.

Animals↗

PubMed1997

Activation, binding, and processing of complement component 3 (C3) by Blastomyces dermatitidis.

Complement plays a key role in phagocyte recognition and killing of Blastomyces dermatitidis, but little is known about how complement components interact with the yeast. We report the characteristics of activation, binding, and processing of C3 by B. dermatitidis. In pooled normal human serum (NHS), deposition of C3 on the yeast was detectable within 2 min, whereas in NHS containing MgEGTA, deposition was delayed by 6 min, indicating that the yeast activates C3 by both classical and alternative pathways. When both pathways were operative, maximal binding of 4.5 x 10(6) C3 molecules/cell was achieved in less than 30 min. In the absence of the classical pathway, yeast cells bound 80% of the maximum C3, indicating that the yeast intrinsically activates the alternative pathway. Delayed deposition of C3 in NHS-MgEGTA was similar to that in NHS preabsorbed by the yeast or by immobilized protein A/G to remove serum immunoglobulin. Purified immunoglobulin restored C3 binding to NHS preabsorbed by the yeast, suggesting that antibody in nonimmune serum initiates the classical pathway. beta-Glucan absorption of NHS abolished the classical pathway, suggesting that cell wall beta-glucan is a target of initiating antibodies. Hydroxylamine treatment of NHS-opsonized yeast cells showed that 76% of C3 was bound to yeast cells by ester linkage, supporting a role for hydroxyl groups on cell wall polysaccharides. Hydroxylamine-cleaved fragments were chiefly C3b and iC3b; 70% of hydroxylamine-sensitive C3b was converted to iC3b within 1 min of opsonization, and the ratio was stable over 1 h. Our data predict that C3b and iC3b on opsonized yeast cells direct binding to CR1 and CR3 receptors on human phagocytes, which, in turn, may influence the fate of this host-pathogen interaction.

Blastomyces↗

PubMed2005

Characterization of a temperature-sensitive mouse middle ear epithelial cell line.

CONCLUSION: Temperature-sensitive mouse middle ear epithelial cells have been successfully established and characterized. OBJECTIVE: Temperature-sensitive middle ear epithelial cell lines are essential for pathophysiologic studies of otitis media. They are useful for studying the pathogen-host interaction, receptor identification, signal transduction, cytokine/mucin production and cellular responses, especially for cell proliferation and differentiation. The purpose of this study was to establish a large T-antigen [simian virus 40 A-gene (SV40)] mutant-immortalized mouse middle ear epithelial cell line for otitis media studies. MATERIAL AND METHODS: Primary culture of middle ear epithelial cells was established from the middle ear mucosa of an Immortomouse. The cells were transduced by a temperature-sensitive large T-antigen mutant and cultured for >50 passages. The expression of mRNA transcripts and proteins for epithelial cells was characterized by reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry. The temperature-sensitive properties of cells cultured at 33 degree C and 39 degree C were evaluated using 3H-thymidine incorporation, Trypan blue exclusion and peroxisome proliferator-activated receptor-gamma activity. RESULTS: Immortalized middle ear epithelial cells demonstrated a cobblestone-like monolayer culture. The cells expressed mucosal cell markers such as mucins, keratins and collagens. They proliferated at 33 degree C when the SV40 antigen was active and differentiated at 39 degree C when the SV40 antigen was inactive.

Animals↗

PubMed2001

Development of antimicrobial agents in the era of new and reemerging infectious diseases and increasing antibiotic resistance.

During the past 2 decades, new infectious diseases have appeared and old ones previously thought to be controlled have reemerged. New and reemerging infectious agents will continue to pose serious threats well into the 21st century. The prediction that the threat of infectious disease may not diminish is supported by evidence that infectious agents cause many chronic diseases and cancer of previous unknown etiology. Moreover, the utility of existing antimicrobial agents is rapidly eroding, tipping the balance in favor of multidrug-resistant pathogens, and there appear to be few, if any, new classes of drugs currently in clinical development. The need for research directed toward development of new antibiotics has never been greater. Advances in research technologies and microbial genome sequencing in the past decade have led to identification of a large number of new targets. Functional genomics and integrative biology should validate these targets and provide the best opportunity for developing effective new therapies, improved diagnostic techniques, and better tools to understand host-pathogen interactions.

Animals↗

PubMed1999

Actin cytoskeleton in plants: from transport networks to signaling networks.

The plant actin cytoskeleton is characterized by a high diversity in regard to gene families, isoforms, and degree of polymerization. In addition to the most abundant F-actin assemblies like filaments and their bundles, G-actin obviously assembles in the form of actin oligomers composed of a few actin molecules which can be extensively cross-linked into complex dynamic meshworks. The role of the actomyosin complex as a force generating system - based on principles operating as in muscle cells - is clearly established for long-range mass transport in large algal cells and specialized cell types of higher plants. Extended F-actin networks, mainly composed of F-actin bundles, are the structural basis for this cytoplasmic streaming of high velocities On the other hand, evidence is accumulating that delicate meshworks built of short F-actin oligomers are critical for events occurring at the plasma membrane, e.g., actin interventions into activities of ion channels and hormone carriers, signaling pathways based on phospholipids, and exo- and endocytotic processes. These unique F-actin arrays, constructed by polymerization-depolymerization processes propelled via synergistic actions of actin-binding proteins such as profilin and actin depolymerizing factor (ADF)/cofilin are supposed to be engaged in diverse aspects of plant morphogenesis. Finally, rapid rearrangements of F-actin meshworks interconnecting endocellular membranes turn out to be especially important for perception-signaling purposes of plant cells, e.g., in association with guard cell movements, mechano- and gravity-sensing, plant host-pathogen interactions, and wound-healing.

Actins↗

PubMed1998

Human epidermal Langerhans cells express the mannose-fucose binding receptor.

Sugar receptors are being increasingly implicated in host-pathogen interactions because of their specific recognition of carbohydrates of microorganisms. The aim of this study was to identify sugar receptors expressed on the surface of human epidermal Langerhans cells (LC). To this end, binding of a panel of fluorescent neoglycoproteins to human epidermal LC was analyzed by quantitative flow cytofluorometry after standardization with calibrated beads. We demonstrate that fresh human LC are the only cells isolated from healthy epidermis which express a membrane receptor specific for fucose-bovine serum albumin (BSA) and mannose-BSA. Quantitative analysis of mannose-BSA or fucose-BSA binding showed non-linear Scatchard plots, denoting the presence of high and moderate affinity binding on the LC surface. The binding parameters of these two ligands were not significantly different. Mannan, the yeast mannose-rich polysaccharide, fucose-BSA, mannose-BSA and free fucose are strong competitors of the three known ligands of the mannose receptor, i.e. fucose-BSA, mannose-BSA and fluorescein isothiocyanate dextran. The amount of mannose-BSA and fucose-BSA bound to LC was 1.5-fold higher at 37 degrees C than at 4 degrees C, suggesting an internalization process. Antibodies raised against the human macrophage mannose receptor strongly stained CD1a-positive LC but not CD1a-negative population. Taken together, our data demonstrate that fresh human LC are the only cells in the epidermis to express a fucose-mannose receptor on their surface.

Animals↗