Wilbert Bitter
Vrije Universiteit Amsterdam, Molecular Microbiology, Faculty Member
For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the... more
For application of genetically engineered fluorescent Pseudomonas spp., specific markers are required for monitoring of wild-type Pseudomonas strains and their genetically modified derivatives in natural environments. In this study, the specific siderophore receptor PupA of plant growth-promoting Pseudomonas putida WCS358 was used as a marker to monitor wild-type strain WCS358. After introduction into natural soil and rhizosphere environments, strain WCS358 could be recovered efficiently on a medium amended with 300 microM pseudobactin 358. Although low population densisties of indigenous pseudomonads (less than or equal to 10(3)/g of soil or root) were recovered on the pseudobactin 358-amended medium, subsequent agglutination assays with a WCS358-specific polyclonal antiserum enabled accurate monitoring of populations of wild-type strain WCS358 over a range of approximately 10(3) to 10(7) CFU/g of soil or root. Genetic analysis of the background population by PCR and Southern hybri...
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Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide... more
Elastase of Pseudomonas aeruginosa is synthesized as a preproenzyme. The signal sequence is cleaved off during transport across the inner membrane and, in the periplasm, proelastase is further processed. We demonstrate that the propeptide and the mature elastase are both secreted but that the propeptide is degraded extracellularly. In addition, reduction of the extracellular proteolytic activity led to the accumulation of unprocessed forms of LasA and LasD in the extracellular medium, which shows that these enzymes are secreted in association with their propeptides. Furthermore, a hitherto undefined protein with homology to a Streptomyces griseus aminopeptidase accumulated under these conditions.
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Gene expression in bacteria is mainly controlled at the level of transcription initiation. To achieve this process a number of different mechanisms have evolved, one of which is the utilization of alternative sigma factors. Sigma factors... more
Gene expression in bacteria is mainly controlled at the level of transcription initiation. To achieve this process a number of different mechanisms have evolved, one of which is the utilization of alternative sigma factors. Sigma factors are small proteins that associate with the RNA polymerase core enzyme (RNAPc) and direct it to specific promoter sequences, where they initiate gene transcription. Bacteria are able to regulate transcription initiation by synthesizing and activating different sigma factors that recognize different promoter consensus sequences. The largest group of alternative sigma factors consists of the so-called extracytoplasmic function (ECF) sigma factors that regulate gene expression in response to cell envelope stresses or environmental stimuli. The activity of ECF sigma factors is controlled by anti-sigma factors and a complex cascade of regulated (proteolytic) modifications. In gram-negative bacteria, ECF sigma factors are also controlled by cell-surface si...
Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of... more
Mycobacteria possess different type VII secretion (T7S) systems to secrete proteins across their unusual cell envelope. One of these systems, ESX-5, is only present in slow-growing mycobacteria and responsible for the secretion of multiple substrates. However, the role of ESX-5 substrates in growth and/or virulence is largely unknown. In this study, we show that esx-5 is essential for growth of both Mycobacterium marinum and Mycobacterium bovis. Remarkably, this essentiality can be rescued by increasing the permeability of the outer membrane, either by altering its lipid composition or by the introduction of the heterologous porin MspA. Mutagenesis of the first nucleotide-binding domain of the membrane ATPase EccC5 prevented both ESX-5-dependent secretion and bacterial growth, but did not affect ESX-5 complex assembly. This suggests that the rescuing effect is not due to pores formed by the ESX-5 membrane complex, but caused by ESX-5 activity. Subsequent proteomic analysis to identi...
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Over the past decade the zebrafish (Danio rerio) has become an attractive new vertebrate model organism for studying mycobacterial pathogenesis. The combination of medium-throughput screening and real-time in vivo visualization has... more
Over the past decade the zebrafish (Danio rerio) has become an attractive new vertebrate model organism for studying mycobacterial pathogenesis. The combination of medium-throughput screening and real-time in vivo visualization has allowed new ways to dissect host pathogenic interaction in a vertebrate host. Furthermore, genetic screens on the host and bacterial sides have elucidated new mechanisms involved in the initiation of granuloma formation and the importance of a balanced immune response for control of mycobacterial pathogens. This article will highlight the unique features of the zebrafish-Mycobacterium marinum infection model and its added value for tuberculosis research.
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FomA is a major non-specific porin of Fusobacterium nucleatum with no sequence similarity to other known porins. According to the topology model, the protein consists of 16 transmembrane beta-strands, connected by eight surface-exposed... more
FomA is a major non-specific porin of Fusobacterium nucleatum with no sequence similarity to other known porins. According to the topology model, the protein consists of 16 transmembrane beta-strands, connected by eight surface-exposed loops and seven periplasmic turns. In this study, the insertion mutagenesis approach was applied to probe the topology model. A Semliki Forest Virus (SFV) epitope was successfully inserted at 11 different sites of the FomA protein and a 6-aa insertion was successfully inserted at two different sites. Correct folding of the mutant proteins and proper incorporation into the outer membrane were assessed by heat modifiability and by an in vivo porin activity assay. Immunofluorescence microscopy analysis of intact cells, using mAbs directed against the inserted SFV epitope, revealed that three of the eight putative extracellular loops are indeed surface-exposed. Trypsin accessibility experiments confirmed the cell surface exposure of two additional loops. ...
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The Fox system of Pseudomonas aeruginosa is a cell-surface signalling (CSS)(2) pathway employed by the bacterium to sense and respond to the presence of the heterologous siderophore ferrioxamine in the environment. This regulatory pathway... more
The Fox system of Pseudomonas aeruginosa is a cell-surface signalling (CSS)(2) pathway employed by the bacterium to sense and respond to the presence of the heterologous siderophore ferrioxamine in the environment. This regulatory pathway controls the transcription of the foxA ferrioxamine receptor gene through the extracytoplasmic function sigma factor σ(FoxI). In the absence of ferrioxamine the activity of σ(FoxI) is inhibited by the transmembrane anti-sigma factor FoxR. Upon binding of ferrioxamine by the FoxA receptor, FoxR is processed by a complex proteolytic cascade leading to the release and activation of σ(FoxI). Interestingly, we have recently shown that FoxR undergoes self-cleavage between the periplasmic Gly-191 and Thr-192 residues independent of the perception of ferrioxamine. This autoproteolytic event, which is widespread among CSS anti-sigma factors, produces two distinct domains that interact and function together to transduce the presence of the signal. In this wo...
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Cell-surface signalling (CSS) enables Gram-negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti-sigma factor in... more
Cell-surface signalling (CSS) enables Gram-negative bacteria to transduce an environmental signal into a cytosolic response. This regulatory cascade involves an outer membrane receptor that transmits the signal to an anti-sigma factor in the cytoplasmic membrane, allowing the activation of an extracytoplasmic function (ECF) sigma factor. Recent studies have demonstrated that RseP-mediated proteolysis of the anti-sigma factors is key to σ(ECF) activation. Using the Pseudomonas aeruginosa FoxR anti-sigma factor, we show here that RseP is responsible for the generation of an N-terminal tail that likely contains pro-sigma activity. Furthermore, it has been reported previously that this anti-sigma factor is processed in two separate domains prior to signal recognition. Here, we demonstrate that this process is common in these types of proteins and that the processing event is probably due to autoproteolytic activity. The resulting domains interact and function together to transduce the C...
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Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific... more
Tuberculous meningitis (TBM) is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish-M. marinum model) to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain ...
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The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular... more
The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes elastase, in Pseudomonas putida did not result in extracellular elastase activity, it has been suggested that the enzyme is not recognized by the Xcp secretion machinery of the heterologous host. Here, it is demonstrated that the proenzyme is normally processed in P. putida and that it is indeed not actively secreted by the Xcp machinery. Nevertheless, substantial amounts of the enzyme were detected in the extracellular medium. Co-immunoprecipitations revealed that the extracellular enzyme was associated with the propeptide, which explains the lack of enzymic activity. Since the propeptide-enzyme complex in P. putida apparently does not dissociate spontaneously, it is concluded that a host-speci...
Research Interests: Microbiology, Protein Folding, Multidisciplinary, Western blotting, Protein-Protein Interaction, and 10 moreMutation, Pseudomonas putida, Plasmids, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Pseudomonas aeruginosa, Enzyme, Enzyme activity, Host Specificity, Pseudomonas Aeruginosa, and Protein Binding
Trypanosoma brucei is a unicellular parasite transmitted between African mammals by tsetse flies. T. brucei multiplies freely in the bloodstream of many different mammals, and survives by antigenic variation of the main component of its... more
Trypanosoma brucei is a unicellular parasite transmitted between African mammals by tsetse flies. T. brucei multiplies freely in the bloodstream of many different mammals, and survives by antigenic variation of the main component of its surface coat, variant surface glycoprotein (VSG). Trypanosomes take up transferrin through a heterodimeric transferrin receptor, the genes for which are expressed in telomeric expression sites along with the VSG gene. There are up to 20 of these expression sites per trypanosome nucleus, but usually only one is active at a time. Different expression sites encode transferrin receptors that are similar but not identical. Here we show that these small differences between transferrin receptors can have profound effects on the binding affinity for transferrins from different mammals, and on the ability of trypanosomes to grow in the sera of these mammals. Our results suggest that the ability to switch between different transferrin-receptor genes allows T. ...
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We review here antigenic variation in African trypanosomes with emphasis on genetic mechanisms and on the expression sites in which the genes for Variant Surface Glycoproteins (VSGs) are expressed. There are multiple expression sites in a... more
We review here antigenic variation in African trypanosomes with emphasis on genetic mechanisms and on the expression sites in which the genes for Variant Surface Glycoproteins (VSGs) are expressed. There are multiple expression sites in a trypanosome, but only one of these is active at a time. We discuss recent experiments that provide new information on expression site regulation, i.e., how inactive sites are kept inactive and how the trypanosome switches from expression of one site to expression of another one. Trypanosomes can also change the gene expressed by replacing the gene in an active expression site by another VSG gene. This replacement involves the duplicative transposition of a silent VSG gene into the expression site. We present a model for the mechanism of this transposition that incorporates new features and that explains several unusual characteristics of the transposition process. We also discuss how new knowledge of nutrient uptake, notably uptake of host transfer...
Research Interests: Immune response, Membrane Proteins, Biological Sciences, Humans, Animals, and 15 moreP-glycoprotein, Transferrin, Host-parasite interactions, Gene Conversion, Glycoproteins, Trypanosoma, Trypanosoma brucei, Trypanosoma brucei brucei, Trypanosomiasis, Protozoan Proteins, Amino Acid Sequence, Mobile Genetic Elements, Antigens, Gene Expression Regulation, and Antigenic Variation
The outer membrane protein PupB of Pseudomonas putida WCS358 facilitates transport of iron complexed to the siderophores pseudobactin BN8 and pseudobactin BN7 into the cell. Its synthesis is induced by the presence of these specific... more
The outer membrane protein PupB of Pseudomonas putida WCS358 facilitates transport of iron complexed to the siderophores pseudobactin BN8 and pseudobactin BN7 into the cell. Its synthesis is induced by the presence of these specific siderophores under iron limitation. The signal transduction pathway regulating siderophore-dependent expression of pupB was shown to consist of two regulatory proteins, PupI and PupR, and the PupB receptor itself. Mutational analysis of the regulatory genes suggested that PupI acts as a positive regulator of pupB transcription, whereas PupR modifies PupI activity dependent on the presence of pseudobactin BN8. PupI and PupR do not share homology with the classical bacterial two-component systems but display significant similarity to the FecI and FecR proteins of Escherichia coli involved in regulation of ferric dicitrate transport. The function of the PupB receptor in pupB regulation was studied by the use of chimeric receptor proteins composed of PupB an...
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In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is... more
In the bloodstream of the mammalian host, Trypanosoma brucei takes up host transferrin by means of a high-affinity uptake system, presumably a transferrin receptor. Transferrin-binding activity is seen in the flagellar pocket and is absent in insect form trypanosomes. By transfection we have reconstituted a transferrin-binding complex in insect form trypanosomes. Formation of this complex requires the products of two genes that are part of a variant surface glycoprotein expression site, expression site-associated gene (ESAG) 6 (encoding a protein with GPI-anchor) and ESAG 7 (encoding a protein without any obvious membrane attachment). This complex can be precipitated by transferrin-Sepharose and by an antibody directed only against the ESAG 6 protein. Transfection of ESAG 6 or 7 alone did not result in transferrin binding. In the transfected trypanosomes, the products of ESAG 6 alone and the combination of ESAG 6 and 7 did not exclusively localize to the flagellar pocket, but were p...
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Protein secretion is an essential determinant of mycobacterial virulence. Mycobacterium tuberculosis has a unique cell envelope consisting of two lipid bilayers, which requires dedicated protein secretion pathways. The conserved general... more
Protein secretion is an essential determinant of mycobacterial virulence. Mycobacterium tuberculosis has a unique cell envelope consisting of two lipid bilayers, which requires dedicated protein secretion pathways. The conserved general Sec and Tat translocation systems are responsible for protein transport across the inner membrane and are both essential. Additionally, the accessory Sec pathway specifically contributes to virulence. How transport of Sec/Tat substrates across the outer membrane is accomplished is currently an enigma. In addition to these pathways, M. tuberculosis also developed specialized secretion systems for protein transport across both membranes, the type VII or ESX secretion systems. Here, we discuss our current knowledge about the mechanisms and substrates of these different protein translocation systems and their role in mycobacterial physiology and virulence.
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ABSTRACT Next to the two-component and quorum sensing systems, cell-surface signalling (CSS) has been recently identified as an important regulatory system in Pseudomonas. CSS senses signals from outside the cell and transmits them into... more
ABSTRACT Next to the two-component and quorum sensing systems, cell-surface signalling (CSS) has been recently identified as an important regulatory system in Pseudomonas. CSS senses signals from outside the cell and transmits them into the cytoplasm. This regulatory system generally consists of three components, an extracytoplasmic function (ECF) sigma factor in the cytoplasm, a sigma factor regulator (or anti-sigma factor) located in the cytoplasmic membrane, and an outer membrane receptor. The outer membrane receptor belongs to the TonB-dependent receptor family, which are mostly involved in the transport of iron-siderophore complexes across the outer membrane. To accomplish this task these receptors need to be energized by the TonB-ExbBD complex. Fluorscent pseudomonads are entangled in a fierce competition for iron. To fulfil their iron requirements, most species produce the yellow–green siderophores pyoverdine, but they are also highly specialized in the utilization of heterologous siderophores. For this they have an impressive amount of different TonB-dependent receptors that are not only produced under low iron conditions but are also dependent on the presence of the heterologous siderophores in the direct surrounding of the bacterium. CSS systems are responsible for this extra level of regulation. This signal transduction pathway starts with binding of the inducing signal, which is usually the iron-siderophore complex, to the TonB-dependent receptor. The signal is then transduced to the cytoplasmic membrane protein, which leads to the activation of ECF sigma factor. The activated sigma factor directs the RNA polymerase to the promoter region of gene(s) under control of the CSS system. One of the genes that is normally activated by the CSS pathway is the one encoding the TonB receptor, which is used for both the transport of the iron siderophore complex and the CSS signal transduction pathway that upregulates its synthesis. In addition, also other genes can be upregulated such as periplasmic transport system for the siderophore complex. In this chapter we review current knowledge of the better characterized CSS system of pseudomonads and discuss the role that other not yet experimentally analyzed CSS systems may have.
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Mycobacteria, such as the major human pathogen Mycobacterium tuberculosis, have a highly unusual and characteristic diderm cell envelope that protects them against harmful conditions. Protein secretion across this hydrophobic barrier... more
Mycobacteria, such as the major human pathogen Mycobacterium tuberculosis, have a highly unusual and characteristic diderm cell envelope that protects them against harmful conditions. Protein secretion across this hydrophobic barrier requires specialized secretion systems. Recently, a type VII secretion (T7S) pathway has been identified that fulfills this function. Pathogenic mycobacteria have up to five different T7S systems, some of which play a crucial role in virulence. The interactions between secreted substrates and host molecules are only starting to become clear and will help in furthering our understanding of the persistence of these enigmatic pathogens. In this review, we discuss current knowledge on the role of T7S systems in mycobacterial virulence.
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... Titre du document / Document title. Type VII secretion in mycobacteria: classification in line with cell envelope structure. Auteur(s) / Author(s). BITTER Wilbert ; HOUBEN Edith NG ; LUIRINK Joen ; APPELMELK Ben J. ; Revue / Journal... more
... Titre du document / Document title. Type VII secretion in mycobacteria: classification in line with cell envelope structure. Auteur(s) / Author(s). BITTER Wilbert ; HOUBEN Edith NG ; LUIRINK Joen ; APPELMELK Ben J. ; Revue / Journal Title. Trends in microbiology ISSN 0966-842X ...
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Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as... more
Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.
Research Interests: Developmental Biology, Tissue Engineering, Immune response, Computational Biology, Pattern Formation, and 17 moreInflammation, Drug development, Biological Sciences, Humans, Automation, High throughput screening, Animals, The, Scaling up, Cost effectiveness, Phenotype, Immune system, Body Patterning, Basic Research, Zebrafish, Amino Acid Sequence, and Embryos
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Research Interests: Microbiology, Immunology, Medical Microbiology, Fluorescence Microscopy, Immunohistochemistry, and 16 moreDanio rerio, Protein Stability, Homology Modeling, Humans, Animals, Quorum Sensing, Iron, Pseudomonas aeruginosa, Microarray Analysis, Reproducibility of Results, Virulence factor, Zebrafish, Pseudomonas Aeruginosa, Virulence Factors, Embryos, and Cell Surface Markers
Mycobacterium marinum causes tuberculosis in fish and can cause skin infections in humans who swim in contaminated water or who have direct contact with infected fish. We report the case study of an 18-month-old girl with M. marinum... more
Mycobacterium marinum causes tuberculosis in fish and can cause skin infections in humans who swim in contaminated water or who have direct contact with infected fish. We report the case study of an 18-month-old girl with M. marinum abscesses, who acquired the infection through indirect contact with a contaminated bucket. Appropriate cleaning of aquarium equipment is very important, especially with young children in the household.
Research Interests: Pediatrics, Fish Diseases, Humans, Transmission, Female, and 7 moreAnimals, Bacteria, Fishes, Infant, Mycobacterium marinum, Abscess, and Nino
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Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high... more
Catechol-cephalosporins are siderophore-like antibiotics which are taken up by cells of Pseudomonas putida WCS358 via the ferric-siderophore transport pathway. Mutants of strain WCS358 were isolated that are resistant to high concentrations of these antibiotics. These mutants failed to grow under iron-limiting conditions, and could not utilize different ferric-siderophores. The mutants fall in three complementation groups. The nucleotide sequence determination identified three contiguous open reading frames, which were homologous to the exbB, exbD and tonB genes of Escherichia coli respectively. The deduced amino acid sequence of P. putida ExbB showed 58.6% homology with its E. coli homologue, but, unlike the E. coli protein, it has a N-terminal extension of 91 amino acids. The ExbD proteins are 64.8% homologous, whereas the TonB proteins only show 27.7% homology. The P. putida exbB gene could complement an E. coli exbB mutation, but the TonB proteins were not interchangeable between the species. It is concluded that P. putida WCS358 contains an energy-coupling system between the membranes, for active transport across the outer membrane, which is comprised of a TonB-like energy-transducing protein and two accessory proteins. This system is similar to, but not completely compatible with, the E. coli system.
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The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting... more
The zebrafish genomic sequence database was analysed for the presence of genes encoding members of the Toll-like receptors (TLR) and interleukin receptors (IL-R) and associated adaptor proteins containing a TIR domain. The resulting predictions show the presence of one or more counterparts for the human TLR1, TLR2, TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, IL-1R and IL-18R genes and one copy of the adaptor genes MyD88, MAL, TRIF and SARM. In contrast to data for the pufferfish Fugu rubripes, zebrafish has two genes that are highly similar to human TLR4. In addition, one fish-specific TLR group can be distinguished that is closely related to the Drosophila melanogaster Toll-9 gene. The sequence of cloned cDNAs for TLR4, TLR2 and MyD88 show the same intron-exon organisation as in the human counterparts. Expression analysis using reverse transcriptase-PCR (RT-PCR) shows that 17 of the predicted zebrafish TLR genes and all the genes encoding adaptor proteins are expressed in the adult stage. A subset of the TLR genes are expressed at higher levels in fish infected with the pathogen Mycobacterium marinum. The induced genes include the homologues of the human TLR1 and TLR2 genes, whose functions are associated with mycobacterial infections, underscoring the suitability of zebrafish as a model for analysis of the vertebrate innate immune system.
Research Interests: Immunology, Drosophila melanogaster, Innate immunity, Toll like receptor signaling, Toll Like Receptors, and 12 moreMolecular, Phylogeny, Molecular Immunology, Sequence alignment, Animals, Protein Sequence Analysis, Zebrafish, Genome sequence, Reverse Transcriptase, Expression analysis, Gene expression profiling, and Innate Immune System
Research Interests: Immunology, Immune response, Innate immunity, Gene expression, Signal Transduction, and 17 moreMolecular, Tuberculosis, Molecular Immunology, Adaptive Immunity, Chronic Disease, Animals, Transcription Factor, Mycobacterium marinum, Immune system, Host Pathogen Interaction, Disease Progression, Zebrafish, Granuloma, Embryos, MHC class I, Gene expression profiling, and Matrix Metalloproteinase
Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas... more
Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.
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Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins... more
Folding of lipases that are secreted by Pseudomonads and other gram-negative bacteria via the type II secretion pathway is facilitated by dedicated chaperones, called lipase-specific foldases (Lifs). Lifs are membrane-anchored proteins with a large periplasmic domain. The functional interaction between the Lif and its cognate lipase is specific, since the Pseudomonas aeruginosa Lif was found not to substitute for Lifs from Burkholderia glumae or Acinetobacter calcoaceticus. However, the P. aeruginosa Lif was able to activate the lipase from the closely related species P. alcaligenes. Hybrid proteins constructed from parts of the P. aeruginosa and B. glumae Lifs revealed that the C-terminal 138 amino acids of the B. glumae Lif determine the specificity of the interaction with the cognate lipase. Furthermore, the periplasmic domain of the B. glumae Lif was functional when cloned in frame with a cleavable signal sequence, which demonstrates that the membrane anchor is not essential for Lif function in vivo. However, the recombinant Lif was released into the medium, indicating that the function of the membrane anchor is to prevent secretion of the Lif together with the lipase.
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Trypanosoma brucei escapes destruction by the host immune system by regularly replacing its Variant Surface Glycoprotein (VSG) coat. The VSG is expressed in a VSG expression site, together with expression site associated gene (ESAG) 6 and... more
Trypanosoma brucei escapes destruction by the host immune system by regularly replacing its Variant Surface Glycoprotein (VSG) coat. The VSG is expressed in a VSG expression site, together with expression site associated gene (ESAG) 6 and 7, encoding the heterodimeric transferrin receptor (Tf-R). There are around 20 VSG expression sites, and trypanosomes can change the site that is active. Since ESAG6 and 7 in different expression sites differ somewhat in sequence, expression site switching results in the production of a slightly different Tf-R. We have studied the physiological relevance of Tf-R variation for the survival of T. brucei in mammalian sera. Trypanosomes with an active 221 expression site, encoding a Tf-R with a very low affinity for canine Tf (Kd>1 microM), were cultured in canine serum based medium. This resulted in selection of trypanosomes that had switched to the VO2, the 223 or the bR-2 expression site, each encoding a Tf-R with higher affinity for canine Tf than the 221 site Tf-R. Adding bovine Tf to the medium could prevent the switch, indicating that the low uptake of Tf provided the selection against 221 trypanosomes. Horse serum based medium also induced switching to the VO2 expression site, but this was not prevented by bovine Tf. In the presence of physiological concentrations of anti-Tf-R antibody, only a high-affinity Tf allowed the growth of 221 Tf-R expressing trypanosomes. Our results suggest that a high-affinity Tf-R not only ensures efficient Tf uptake, but is also required to allow sufficient iron uptake by the trypanosome in the presence of anti-Tf-R antibodies.