Jozef Anne | KU Leuven (original) (raw)

Papers by Jozef Anne

Journal of Biomedical Materials Research Part A, 2014

Implant-related infections are a serious complication in prosthetic surgery, substantially jeopar... more Implant-related infections are a serious complication in prosthetic surgery, substantially jeopardizing implant fixation. As porous coatings for improved osseointegration typically present an increased surface roughness, their resulting large surface area (sometimes increasing with over 700% compared to an ideal plane) renders the implant extremely susceptible to bacterial colonization and subsequent biofilm formation. Therefore, there is particular interest in orthopaedic implantology to engineer surfaces that combine both the ability to improve osseointegration and at the same time reduce the infection risk. As part of this orthopaedic coating development, the interest of in vitro studies on the interaction between implant surfaces and bacteria/biofilms is growing. In this study, the in vitro staphylococcal adhesion and biofilm formation on newly developed porous pure Ti coatings with 50% porosity and pore sizes up to 50 μm is compared to various dense and porous Ti or Ti-6Al-4V reference surfaces. Multiple linear regression analysis indicates that surface roughness and hydrophobicity are the main determinants for bacterial adherence. Accordingly, the novel coatings display a significant reduction of up to five times less bacterial surface colonization when compared to a commercial state-of-the-art vacuum plasma sprayed coating. However, the results also show that a further expansion of the porosity with over 15% and/or the pore size up to 150 μm is correlated to a significant increase in the roughness parameters resulting in an ascent of bacterial attachment. Chemically modifying the Ti surface in order to improve its hydrophilicity, while preserving the average roughness, is found to strongly decrease bacteria quantities, indicating the importance of surface functionalization to reduce the infection risk of porous coatings. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2013.

Bioorganic Medicinal Chemistry Letters, Apr 1, 2009

Bacterial type I signal peptidase is a potential target for the development of novel antibacteria... more Bacterial type I signal peptidase is a potential target for the development of novel antibacterial agents. In this study we demonstrate that a substrate based peptide aldehyde inhibits signal peptidases with a lower IC 50 value than the lipopeptides described to date. The length of the core lipopeptide could be reduced by removing several amino acids from both termini. Conversion of this peptide to an aldehyde resulted in a molecule with an IC 50 value of 0.09 lM when tested against Saccharomyces aureus SPase I, SpsB.

European Journal of Cancer, Aug 31, 1997

Journal of Microbiological Methods, Jul 1, 2009

In the framework of the search for new antimicrobial therapies to combat resistant bacteria, the ... more In the framework of the search for new antimicrobial therapies to combat resistant bacteria, the type I signal peptidase (SPase I) serves as a potentially interesting target for the development of antibacterials with a new mode of action. Bacterial SPases I play a key role in protein secretion as they are responsible for the cleavage of signal peptides from secreted proteins. For the Gram-positive Staphylococcus epidermidis, an important source of biofilm-associated infections, three putative SPases I (denoted Sip1, Sip2, Sip3) have been described, of which Sip1 lacks the catalytic lysine. Here, we report the in vitro activity of purified Sip2 and Sip3 using pre-SceD as a native preprotein substrate of S. epidermidis and in a FRET-based assay. For the latter, a novel internally quenched fluorescent peptide substrate based on the signal peptide sequence of this native preprotein was developed and specific cleavage of this synthetic fluorogenic peptide substrate was demonstrated. The latter in vitro assay represents a rapid and reliable tool in future research for the identification and validation of potential SPase I inhibitors.

Journal of Toxicology, 2012

Solid tumour accounts for 90% of all cancers. The current treatment approach for most solid tumou... 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 the tumour microenvironment, causing significant oncolysis as well as enabling the delivery of therapeutics directly to the tumour in situ. Various strategies utilizing Clostridium are currently being investigated, and represent a novel area of emerging cancer therapy. This review provides an update review of tumour microenvironment as well as summary of the progresses and current status of Clostridial spore-based cancer therapies.

Archives of Microbiology, 2010

Twitching motility, a flagella-independent type of translocation of bacteria over moist surfaces,... more Twitching motility, a flagella-independent type of translocation of bacteria over moist surfaces, requires an array of proteins, including FimV. To investigate the role of this protein in twitching motility in Legionella pneumophila, we have generated a knockout mutant of fimV and characterized its phenotypic effects. In addition to a major reduction in twitching motility, deletion of the fimV gene caused a number of other phenotypic effects including decreased protective pigment formation, and it also affected cell morphology. Since fimV contains a variable number of tandem repeats, which can vary according to the origin of a given strain, we have examined the importance of this variability found within the coding region of this gene. By complementing the knockout strain with constructs containing a different number of this tandem repeat, we have been able to also show that repeat copy number is important in the functioning of this gene.

Microbiology Sgm, Dec 1, 1998

Mi n d e r b roede rsst raa t 1 0, B-3000 Leuven, Belgium

Cancer Investigation, 2001

Potential anticancer therapy with the fumagillin analog TNP-470 was investigated in the present p... more Potential anticancer therapy with the fumagillin analog TNP-470 was investigated in the present project using subcutaneously growing rhabdomyosarcomas in rats. Specifically, influences of different tumor sizes at the start of treatment as well as dose/schedules were evaluated with this angiogenesis inhibitor. The results show a significant (p = < or = 0.01) reduction of the growth rate, even for relatively large-sized (> 7 cm3) tumors, when 50 mg/kg TNP-470 was used every other day for up to 3 or 5 injections. With 30 mg/kg TNP-470 injections, effects were seen only with tumors measuring < 7 cm3. The histologic examinations demonstrate an increase in necrosis, both in the center and in the peripheral part of TNP-470-treated tumors. Overall, both tumor volume and drug dose determine treatment outcome with the rat rhabdomyosarcoma. The results suggest that angiogenesis inhibitors could represent a valid component in the treatment of progressive tumor growth, also of large tumors as often encountered in clinics. The antivasculature therapy might also improve hypoxia/necrosis-related therapeutic approaches.

Anticancer Research, 2002

Attenuated Salmonella typhimurium has been demonstrated as a potential gene delivery vector. Prev... 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 to define an OTD, probably for each species, when using genetically engineered Salmonella as a tumor- and species-selective vector in cancer therapy.

Aids Res Hum Retrovirus, 1998

ABSTRACT We compared the development of resistance toward BI-RG-587 (nevirapine) and alpha-APA R8... more ABSTRACT We compared the development of resistance toward BI-RG-587 (nevirapine) and alpha-APA R89439 (loviride) starting from the wild-type HIV-1 strain IIIB and the 3TC-resistant HIV-1 strain containing the M184V mutation. The reverse transcriptase of the M184V mutant has been reported to have a higher fidelity. Our experiments showed that there was no significant delay in virus breakthrough of the M184V mutant as compared with the wild-type virus. We therefore conclude that the reported higher fidelity of the M184V mutant does not lead to a delay in the development of resistance to the nonnucleoside reverse transcriptase inhibitors nevirapine and loviride.

Communications in Agricultural and Applied Biological Sciences, Feb 1, 2004

Research in Microbiology, Nov 30, 2006

Legionella pneumophila is a facultative intracellular Gram-negative bacterium that has become an ... more Legionella pneumophila is a facultative intracellular Gram-negative bacterium that has become an important cause of community-acquired and nosocomial pneumonia. Recent studies concerning the unravelling of bacterial virulence have suggested the involvement of protein secretion systems in bacterial pathogenicity. In this respect, the type II signal peptidase (LspA), which is specifically required for the maturation of lipoproteins, is of particular interest. This paper reports the cloning and functional characterization of the L. pneumophila lspA gene encoding the type II signal peptidase (SPase II). Activity of the L. pneumophila LspA was demonstrated using a globomycin sensitivity assay in Escherichia coli. In L. pneumophila, the lspA gene is flanked by the isoleucyl-tRNA synthetase (ileS) gene and the gene encoding a 2-hydroxy-3-deoxy-phosphogluconate aldolase. Although there is no apparent physiological connection, transcriptional analysis demonstrated that, as in some other Gram-negative bacteria, lspA is cotranscribed with ileS in L. pneumophila. Finally, in silico analysis revealed that several proteins known to be crucial for virulence and intracellular growth of L. pneumophila are predicted to be lipoproteins. These include, in particular, proteins involved in protein secretion and motility. Results obtained strongly suggest an important role for LspA in the pathogenicity of L. pneumophila, making it a promising new target for therapeutic intervention.

Journal of Bacteriology, 2002

Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from se... more Most bacteria contain one type I signal peptidase (SPase) for cleavage of signal peptides from secreted proteins. The developmental complex bacterium Streptomyces lividans has the ability to produce and secrete a significant amount of proteins and has four different type I signal peptidases genes (sipW, sipX, sipY, and sipZ) unusually clustered in its chromosome. Functional analysis of the four SPases was carried out by phenotypical and molecular characterization of the different individual sip mutants. None of the sip genes seemed to be essential for bacterial growth. Analysis of total extracellular proteins indicated that SipY is likely to be the major S. lividans SPase, since the sipY mutant strain is highly deficient in overall protein secretion and extracellular protease production, showing a delayed sporulation phenotype when cultured in solid medium.

Mathematical Biosciences, 2015

(13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central ca... more (13)C-based metabolic flux analysis is an excellent technique to resolve fluxes in the central carbon metabolism but costs can be significant when using specialized tracers. This work presents a framework for cost-effective design of (13)C-tracer experiments, illustrated on two different networks. Linear and non-linear optimal input mixtures are computed for networks for Streptomyces lividans and a carcinoma cell line. If only glucose tracers are considered as labeled substrate for a carcinoma cell line or S. lividans, the best parameter estimation accuracy is obtained by mixtures containing high amounts of 1,2-(13)C2 glucose combined with uniformly labeled glucose. Experimental designs are evaluated based on a linear (D-criterion) and non-linear approach (S-criterion). Both approaches generate almost the same input mixture, however, the linear approach is favored due to its low computational effort. The high amount of 1,2-(13)C2 glucose in the optimal designs coincides with a high experimental cost, which is further enhanced when labeling is introduced in glutamine and aspartate tracers. Multi-objective optimization gives the possibility to assess experimental quality and cost at the same time and can reveal excellent compromise experiments. For example, the combination of 100% 1,2-(13)C2 glucose with 100% position one labeled glutamine and the combination of 100% 1,2-(13)C2 glucose with 100% uniformly labeled glutamine perform equally well for the carcinoma cell line, but the first mixture offers a decrease in cost of $ 120 per ml-scale cell culture experiment. We demonstrated the validity of a multi-objective linear approach to perform optimal experimental designs for the non-linear problem of (13)C-metabolic flux analysis. Tools and a workflow are provided to perform multi-objective design. The effortless calculation of the D-criterion can be exploited to perform high-throughput screening of possible (13)C-tracers, while the illustrated benefit of multi-objective design should stimulate its application within the field of (13)C-based metabolic flux analysis.

Focus on Biotechnology, 2001

The increasing demand of recombinant compounds in bioscience and bioindustries requires the furth... more The increasing demand of recombinant compounds in bioscience and bioindustries requires the further exploration and improvement of production systems including bacteria, fungi, insect and human cells. For compounds that do not require glycosylation for biological activity, microbial systems are most favourable hosts because of high level expression and relatively inexpensive culture systems. Traditionally, Escherichia coli was and still is most often the host of choice. However, the major drawbacks of this Gram-negative organism are the periplasmic location of the secreted proteins of interest due to the presence of an outer membrane and the frequent occurrence of cytoplasmically or periplasmically located inclusion bodies consisting of denaturated recombinant proteins. Gram-positive bacteria secrete the protein of interest directly into the culture media, thereby greatly facilitating downstream processing and protein recovery. As a consequence, they are being extensively explored for recombinant protein production. This report reviews the possible applications of Gram-positive bacteria as host cells for the production of proteins of biopharmaceutical interest. It will also highlight eventual advantages of Gram-positive bacteria compared to Gram-negative organisms. Although successful in some cases, several bottlenecks in the secretion of heterologous proteins remain. Approaches undertaken to improve the yield of secreted recombinant proteins in these Gram-positive bacteria will be summarised. Finally, results obtained so far regarding the production of biopharmaceutical compounds in a soluble active form and with respect to the cell surface display of recombinant proteins by Gram-positive bacteria will be discussed.

Methods in molecular biology (Clifton, N.J.), 2010

The choice of an expression system for the meta-genomic DNA of interest is of vital importance fo... 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...

BMC microbiology, Jan 19, 2006

Different gene expression patterns correlate with the altered phenotype in biofilm-associated bac... 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 ...

Anaerobe, 1998

The use of gene therapy is one of the most recent molecular strategies for the treatment of cance... 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...

Anticancer research