Jozef ANNE
KU Leuven, Microbiology & Immunology, Emeritus
The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans , a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S.... more
The recently discovered bacterial twin-arginine translocation (Tat) pathway was investigated in Streptomyces lividans , a gram-positive organism with a high secretion capacity. The presence of one tatC and two hcf106 homologs in the S. lividans genome together with the several precursor proteins with a twin-arginine motif in their signal peptide suggested the presence of the twin-arginine translocation pathway in the S. lividans secretome. To demonstrate its functionality, a tatC deletion mutant was constructed. This mutation impaired the translocation of the Streptomyces antibioticus tyrosinase, a protein that forms a complex with its transactivator protein before export. Also the chimeric construct pre-TorA-23K, known to be exclusively secreted via the Tat pathway in Escherichia coli , could be translocated in wild-type S. lividans but not in the tatC mutant. In contrast, the secretion of the Sec-dependent S. lividans subtilisin inhibitor was not affected. This study therefore dem...
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nudeotide sequences of the 5 S rRNAs of seven molds and a yeast and their use in studying
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Members of the genus Streptomyces are Gram-positive bacteria that are used as important cell factories to produce secondary metabolites and secrete heterologous proteins. They possess some of the largest bacterial genomes and thus... more
Members of the genus Streptomyces are Gram-positive bacteria that are used as important cell factories to produce secondary metabolites and secrete heterologous proteins. They possess some of the largest bacterial genomes and thus proteomes. Understanding their complex proteomes and metabolic regulation will improve any genetic engineering approach. Here, we performed a comprehensive annotation of the subcellular localization of the proteome of Streptomyces lividans TK24 and developed the Subcellular Topology of Polypeptides in Streptomyces database (SToPSdb) to make this information widely accessible. We first introduced a uniform, improved nomenclature that re-annotated the names of ~ 4000 proteins based on functional and structural information. Then protein localization was assigned de novo using prediction tools and edited by manual curation for 7494 proteins, including information for 183 proteins that resulted from a recent genome re-annotation and are not available in current...
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The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature... more
The gene encoding a thermostable cellulase of family 12 was previously isolated from a Rhodothermus marinus through functional screening. CelA is a protein of 260 aminoacyl residues with a 28-residue amino-terminal signal peptide. Mature CelA was poorly synthesized in some Escherichia coli strains and not at all in others. Here we present an alternative approach for its heterologous production as a secreted polypeptide in Streptomyces. CelA was successfully over-expressed as a secreted polypeptide in Streptomyces lividans TK24. To this end, CelA was fused C-terminally to the secretory signal peptide of the subtilisin inhibitor protein (Sianidis et al. in J Biotechnol. 121: 498-507, 2006) from Streptomyces venezuelae and a new cloning strategy developed. Optimal growth media and conditions that stall biomass production promote excessive CelA secretion. Under optimal growth conditions in nutrient broth medium, significant amounts of mature CelA (50-90 mg/L or 100-120 mg/g of dry cell ...
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Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of... more
Filamentous organisms of the genus Streptomyces play an important role in industrial production processes, due to their extensive secondary metabolism variability, as well as their ability to secrete efficiently large amounts of (heterologous) proteins. While genetic engineering tools are available to rapidly build up large strain libraries, the subsequent strain screening and bioprocess development still constitutes a bottleneck. This is due to the lack of reliable parallelized and accelerated cultivation techniques for morphologically challenging organisms. To address this challenge, we developed an integrated cultivation workflow for Streptomyces lividans based on a parallelized shaken 48-well microtiter-plate (MTP) cultivation device. In a first step, a feasible pre-culture method was identified and validated, revealing high comparability in subsequent main cultivations (coefficient of variation of 1.1% for in-plate replicates and 3.2% between different pre-cultures). When valid...
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Defense mechanisms of plants against phytopathogens include cationic antimicrobial peptides (CAPs). The broad-spectrum activity of these peptides has been evaluated previously against different phytopathogens. Their lack of toxicity for... more
Defense mechanisms of plants against phytopathogens include cationic antimicrobial peptides (CAPs). The broad-spectrum activity of these peptides has been evaluated previously against different phytopathogens. Their lack of toxicity for plants and animals is a promising feature for pest control. Although, some attempts have been made previously in order to increase their heterologous expressions, the employed strategies have so far proven to be ineffective. Low production yield and elevated costs are the obstacles to overcome. In this study, a strategy for CAP overexpression is presented based on the construction of an expression cassette for Streptomyces lividans TK24. This system contains elements that allow the increase of the efficiency of the peptide's expression. The main elements included in this cassette are the sequences of the promoter and signal peptide from a subtilisin inhibitor gene of Streptomyces venezuelae. This allows the efficient secretion of the peptide to t...
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Several microbial systems are currently under investigation for the production of heterologous proteins. Because Gram-positive bacteria have no outer membrane, the proteins translocated across the cytoplasmic membrane are directly... more
Several microbial systems are currently under investigation for the production of heterologous proteins. Because Gram-positive bacteria have no outer membrane, the proteins translocated across the cytoplasmic membrane are directly released into the culture medium, in most instances in a properly folded conformation. This contrasts with proteins expressed in Gram-negative bacteria such as Escherichia coli. In these bacteria, heterologous proteins remain in the periplasm and often precipitate as inclusion bodies in a denatured form which may seriously complicate downstream processing. Faced with this problem, several genera of Gram-positive bacteria are being tested as host for the production of heterologous proteins. Among these are Streptomyces and Corynebacterium, since several of their species are known to secrete native proteins in high amounts. Because of the absence of an extensive restriction-modification system, linsited protease activity and the availability of suitable vector systems, Streptomyces lividans is the host of choice for the secretory production of heterologous proteins, when using streptomycetes as host. As Streptomyces, Corynebacterium is a non-pathogenic bacterium for which recently genetic tools have been developed. Additionally, Corynebacterium glutamicum has no broad-spectrum proteolytic activity rendering this species also a good candidate for heterologous protein production. Mycobacterium has been developed as an antigen delivery system. Taking advantage of the characteristic of Mycobacterium being a strong adjuvant for the induction of immunogenic reactions, recombinant mycobacteria expressing foreign antigens are tested to elicit specific, protective immunoresponses in animals. The current report reviews the possibilities of these GC-rich bacteria with respect to the production of heterologous proteins and the approaches to improve yield.
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Research Interests: Microbiology, Membrane Proteins, Multidisciplinary, Virulence, Humans, and 14 moreLegionella pneumophila, Escherichia coli, Animals, Bacteria, Gene, Molecular cloning, Gen, Amino Acid Sequence, Membrane Protein, Acanthamoeba, Secretion, Intracellular, Molecular Sequence Data, and Reverse transcription polymerase chain reaction
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The production of human tumor necrosis factor beta (hTNF beta) in Streptomyces lividans was studied. The expression of hTNF beta uses the transcription, translation and secretion signals of subtilisin inhibitor VSI which is naturally... more
The production of human tumor necrosis factor beta (hTNF beta) in Streptomyces lividans was studied. The expression of hTNF beta uses the transcription, translation and secretion signals of subtilisin inhibitor VSI which is naturally produced by Streptomyces venezuelae CBS762.70. In direct secretory expression cassette, hTNF beta cDNA was fused 2 amino acids after the signal peptidase cleavage site. In fusion expression cassette, hTNF beta cDNA was fused after the total vsi gene. In intracellular expression cassette, hTNF beta cDNA was fused after initiation codon ATG. The expression cassettes were subcloned into Streptomyces multi-copy plasmid pIJ486 respectively and transformed into S. lividans TK24. The recombinant strains were designated as S. lividans (pIJ486-hTNF beta), S. lividans (pIJ486-vsi-hTNF beta) and S. lividans (pIVPA-hTNF beta). The analysis of expressed proteins by Western blotting and biological activity measurements revealed hTNF beta was expressed by the recombinant strains with bioactivity. The molecular weight of directly secreted product is around 16 kDa, and the expression level at 48 hours in NB medium was 0.7 mg/L. Intact product could be obtained when hTNF beta was expressed intracellularly, although it may be degraded to lower molecular weight product when cultivation was prolonged. The intracellular expression level at 48 hours in NB medium was 25.1 mg/L.
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Protoplasts of distinct Penicillium species can be fused and their fusion products eventually give rise to interspecific heterokaryons and hybrids. The morphology, stability and segregation pattern of the heterokaryons and their nuclear... more
Protoplasts of distinct Penicillium species can be fused and their fusion products eventually give rise to interspecific heterokaryons and hybrids. The morphology, stability and segregation pattern of the heterokaryons and their nuclear fusion progenies varies depending on the species involved in the cross.
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... JAN THEYS, PH.D., SANDRA NUYTS, MD, WILLY LANDUYT, PH.D., LIEVE VAN MELLAERT, PH.D., AND JOZEF ANN ´E, PH ... 24.5 Fold increase of mTNFa secretion in Clostridium acetobutylicum DSM792 pIMP-recA-mTNFa (dotted bars), pIMP-recA deleted... more
... JAN THEYS, PH.D., SANDRA NUYTS, MD, WILLY LANDUYT, PH.D., LIEVE VAN MELLAERT, PH.D., AND JOZEF ANN ´E, PH ... 24.5 Fold increase of mTNFa secretion in Clostridium acetobutylicum DSM792 pIMP-recA-mTNFa (dotted bars), pIMP-recA deleted Cheo-mTNFa ( ...
Research Interests: Hypoxia and Gene Transfer
The choice of an expression system for the meta-genomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as... more
The choice of an expression system for the meta-genomic DNA of interest is of vital importance for the detection of any particular gene or gene cluster. Most of the screens to date have used the Gram-negative bacterium Escherichia coli as a host for the meta-genomic gene libraries. However, the use of E. coli introduces a potential host bias since only 40% of the enzymatic activities may be readily recovered by random cloning in E. coli (Gabor et al., Environ Microbiol 6:879-886, 2004). To recover some of the remaining 60%, alternative cloning hosts such as Streptomyces spp. have been used (Lorenz and Eck, Nat Rev Microbiol 3:510-516, 2005). Streptomycetes are high-GC Gram-positive bacteria that belong to the Actinomycetales, and they have been studied extensively in the last 10 years as an alternative expression system (reviewed in Vrancken and Anné, Future Microbiol 4:181-188, 2009). Streptomyces is extremely well suited for the expression of DNA from other actinomycetes and genom...
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Different gene expression patterns correlate with the altered phenotype in biofilm-associated bacteria. Iron and iron-linked genes are thought to play a key-role in biofilm formation. The expression of Fe-linked genes (sirR, sitABC... more
Different gene expression patterns correlate with the altered phenotype in biofilm-associated bacteria. Iron and iron-linked genes are thought to play a key-role in biofilm formation. The expression of Fe-linked genes (sirR, sitABC operon) in Staphylococcus epidermidis, was compared in planktonic versus sessile bacteria in vitro and in vivo in a subcutaneous foreign body rat model. In vitro in a Fe-limited environment, the planktonic form of S. epidermidis produces siderophores and grows slower than in Fe-rich environment. The expression of sirR in planktonic bacteria, in vitro, was not different in medium without Fe or with 1 microM FeCl3. High Fe concentrations (25 microM FeCl3) increased expression of sirR transiently during the early phase of incubation. Expression of sitC in vitro, in planktonic bacteria, was inversely correlated with sirR expression in medium with 25 microM FeCl3: sitC expression decreased for the first 3 hours followed by an up regulation.In sessile bacteria ...
Research Interests: Biofilms, Kinetics, Biology, Medicine, Cell Division, and 15 moreBiological Sciences, Animals, Siderophores, Polymerase Chain Reaction, Iron, Gene Dosage, Rats, Analysis of Variance, Rat Model, Culture Media, Ferric Compounds, Chlorides, foreign body, reverse transcriptase polymerase chain reaction, and Medical and Health Sciences
The use of gene therapy is one of the most recent molecular strategies for the treatment of cancer. It is essential, however, to have an efficient transfer system by which the desired gene can be delivered to the correct environment. The... more
The use of gene therapy is one of the most recent molecular strategies for the treatment of cancer. It is essential, however, to have an efficient transfer system by which the desired gene can be delivered to the correct environment. The experiments described in this report investigate apathogenic Clostridium as a possible vector to transfer a specific gene product into the extracellular microenvironment of the tumour which is hypoxic/necrotic in parts, using WAG/Rij rats with transplantable rhabdomyosarcomas as a model. Our data show that Clostridium, after systemic administration of at least 10(7) spores, specifically colonises the hypoxic/necrotic areas of our tumour model, the most efficient species being C. acetobutylicum (NI-4082) and C. oncolyticum. Although spores were also detected in normal tissues for up to 4 weeks, they did not germinate in these tissues. We conclude that it seems likely that these bacteria can be used as a selective transfer system into the extracellula...
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Attenuated Salmonella typhimurium has been demonstrated as a potential gene delivery vector. Previous findings induce the necessity to optimize tumor selectivity and bacterial dosing in relation to tumor volume and intratumoral... more
Attenuated Salmonella typhimurium has been demonstrated as a potential gene delivery vector. Previous findings induce the necessity to optimize tumor selectivity and bacterial dosing in relation to tumor volume and intratumoral therapeutic gene expression. Attenuated Salmonella VNP20009 and VNP20047 (expressing cytosine deaminase) were systemically administered to tumor-bearing rats. The bacteria were quantified in tumor and normal organs. Conversion of 5-fluorocytosine to 5-fluorouracil was evaluated using thin layer chromatography. Tumor colonization efficiency was dependent on Salmonella density, administration route and tumor volume. Colonization of normal tissues gradually decreased with time, while intratumoral proliferation of bacteria remained high during the follow-up period. The Optimal Therapeutic Dose (OTD) was found to be 5.10(7) cfu/rat. Intratumoral VNP20047-expressed CDase leading to the conversion of 5-FC to 5-FU was detected in vivo. Our results indicate the need t...
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Four adjacent genes (sipW, sipX, sipY and sipZ) encoding different type I signal peptidases, were isolated on a 7860 bp DNA fragment from Streptomyces lividans TK21. Three of the sip genes constitute an operon and the fourth is the first... more
Four adjacent genes (sipW, sipX, sipY and sipZ) encoding different type I signal peptidases, were isolated on a 7860 bp DNA fragment from Streptomyces lividans TK21. Three of the sip genes constitute an operon and the fourth is the first gene of another operon encompassing three additional, unrelated genes. A DNA fragment containing the four sip genes complemented an Escherichia coli type I signal peptidase mutant when cloned in a multicopy plasmid. Clustering of four different type I signal peptidase genes seems, so far, to be a unique feature of Streptomyces.
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In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis... more
In order to evaluate the expression and secretion signals of the highly secreted subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (VSI) for the production of heterologous proteins by Streptomyces lividans, mouse tumor necrosis factor alpha (mTNF) was chosen as a model protein. The mTNF cDNA was fused to the vsi signal sequence. The analysis of secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and biological activity measurements revealed an efficient translocation of mTNF. Up to 300 mg of secreted biologically active mTNF per liter could be obtained in shaken-flask cultures. By analyzing the effects of mutations in the N region of the VSI signal peptide on secretion, we found that decreasing the +3 charge of the wild-type protein to +2 resulted in a 3- to 10-fold increase in secretion.
Research Interests: Multidisciplinary, Protein synthesis, Signal Transduction, Streptomyces, Mice, and 15 moreSecretory Pathway, Applied, Animals, Applied Environmental Microbiology, Enzyme, Molecular cloning, Genetic Recombination, Amino Acid Profile, SIGNAL PEPTIDE, Biological activity, Polyacrylamide Gel Electrophoresis, Immunoblotting, Sodium Dodecyl Sulfate, Mutation analysis, and Electrostatic interaction
The development cycle of the temperate actinophage VWB was investigated. Adsorption of most phage particles occurred within 30 min and the adsorption constant was 0.6 x 10(-8) ml min-1. The latent and rise periods were 140 and 100 min,... more
The development cycle of the temperate actinophage VWB was investigated. Adsorption of most phage particles occurred within 30 min and the adsorption constant was 0.6 x 10(-8) ml min-1. The latent and rise periods were 140 and 100 min, respectively, and the burst size was estimated to be 130-250 p.f.u. Although phage VWB could infect only Streptomyces venezuelae ETH 14630 (ATCC 40755), of six different S. venezuelae strains tested, phage DNA could be introduced by transfection into most non-infectible strains. Upon transfection, phage DNA was propagated in these non-infectible strains and phage particles were released. In addition, the transfected strains could be lysogenized. By comparison of restriction fragments of VWB DNA, either free or integrated in the chromosomal DNA of the S. venezuelae ETH 14630 lysogen, the attachment site was localized. PAGE of the phage proteins revealed at least 17 different proteins with three major bands estimated as 16.5, 27.2 and 43 kDa in size. Th...
Research Interests: Electron Microscopy, Biology, Bacteriophages, Medicine, Streptomyces, and 10 moreGeneral, General Microbiology, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Molecular Characterization, Transfection, Amino Acid Sequence, Lysogeny, DNA sequence, Molecular Sequence Data, and Restriction Mapping
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Insu⁄cient blood supply of rapidly growing tumors leads to the presence of hypoxia, a well-known feature in solid tumors. Hypox- ia is known to decrease the e⁄ciency of currently used anti-can- cer modalities like surgery, chemotherapy... more
Insu⁄cient blood supply of rapidly growing tumors leads to the presence of hypoxia, a well-known feature in solid tumors. Hypox- ia is known to decrease the e⁄ciency of currently used anti-can- cer modalities like surgery, chemotherapy and radiotherapy. Therefore, hypoxia seems to be a major limitation in current anti- cancer therapy. The use of non-pathogenic clostridia to deliver toxic agents
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The recent development of the alternating current electrophoretic deposition (AC-EPD) technique has rendered it possible to deposit material from aqueous suspensions while preventing the electrochemical reactions associated with the... more
The recent development of the alternating current electrophoretic deposition (AC-EPD) technique has rendered it possible to deposit material from aqueous suspensions while preventing the electrochemical reactions associated with the application of high voltages on such systems. This does not only allow for more economical and ecological processes but also opens up electrophoretic deposition as a processing technique to a whole range of materials sensitive to either electrochemical reactions or non-aqueous solvents. Living cells can be considered as one class of such materials. In this paper the deposition of two types of bacteria, the Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli, and one type of yeast cells, Saccharomyces cerevisiae, is demonstrated.
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Page 1. 71 Emerging Cancer Therapy: Microbial Approaches and Biotechnological Tools, Edited by Arsénio M. Fialho and Ananda M. Chakrabarty Copyright © 2010 John Wiley & Sons, Inc. 4 LIVE CLOSTRIDIA: A POWERFUL TOOL IN TUMOR... more
Page 1. 71 Emerging Cancer Therapy: Microbial Approaches and Biotechnological Tools, Edited by Arsénio M. Fialho and Ananda M. Chakrabarty Copyright © 2010 John Wiley & Sons, Inc. 4 LIVE CLOSTRIDIA: A POWERFUL TOOL IN TUMOR BIOTHERAPY ...
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Research Interests: British, Apoptosis, Cell Division, Genetic Engineering, Adipose tissue, and 15 moreCell line, Female, Animals, Enzyme, Aziridines, Gene Delivery, Cancer cells, Cancers, Cell Survival, Antineoplastic Agents, Clostridium, Gene Transfer, Colorectal Neoplasms, Combined Modality Therapy, and Genetic Therapy
Solid tumour accounts for 90% of all cancers. The current treatment approach for most solid tumours is surgery, however it is limited to early stage tumours. Other treatment options such as chemotherapy and radiotherapy are non-selective,... more
Solid tumour accounts for 90% of all cancers. The current treatment approach for most solid tumours is surgery, however it is limited to early stage tumours. Other treatment options such as chemotherapy and radiotherapy are non-selective, thus causing damage to both healthy and cancerous tissue. Past research has focused on understanding tumour cells themselves, and conventional wisdom has aimed at targeting these cells directly. Recent research has shifted towards understanding the tumour microenvironment and it's differences from that of healthy cells/tissues in the body and then to exploit these differences for treatmeat of the tumour. One such approach is utilizing anaerobic bacteria. Several strains of bacteria have been shown to selectively colonize in solid tumours, making them valuable tools for selective tumour targeting and destruction. Amongst them, the anaerobic Clostridium has shown great potential in penetration and colonization of the hypoxic and necrotic areas of...
Research Interests: Toxicology and Medicine
Research Interests: Gene Therapy and Medicine
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The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of... more
The 45/47 kDa APA protein (Rv1860) of Mycobacterium tuberculosis was produced by Streptomyces lividans. The recombinant protein could be recovered from the culture medium of an S. lividans clone containing the apa gene under control of the promoter and signal sequence of the Streptomyces coelicolor agarase gene. The recombinant protein production was further scaled-up using fermentation conditions. The APA protein was subsequently purified from the culture supernatant by means of immunochromatography. About 80 mg of recombinant protein were obtained per liter of culture media. In vivo tests with the APA protein purified from S. lividans TK24/pRGAPA1 revealed that the recombinant protein was antigenic and could induce high titers of specific antibodies in the mouse biological model. Results obtained concerning heterologous production of APA, its immunogenic and antigenic capacity, demonstrated the potential of S. lividans as a valuable host for the production of recombinant proteins from M. tuberculosis.