Selection and characterization of DNA aptamers for egg white lysozyme.

We have selected aptamers binding to lysozyme from a DNA library using capillary electrophoresis-systematic evolution of ligands by exponential enrichment. During the selection process the dissociation constant of the ssDNA pool decreased from the micromolar to the low nanomolar range within five rounds of selection. The final aptamer had a dissociation constant of 2.8 +/- 0.3 nM, 6.1 +/- 0.5 nM, and 52.9 +/- 9.1 nM as determined by fluorescence anisotropy, surface plasmon resonance and affinity capillary electrophoresis respectively. The aptamers were successfully challenged for specificity against other egg white proteins. The high affinity aptamers open up possibilities for the development of aptamer based food and medical diagnostics.

Intragenic tandem repeat variation between Legionella pneumophila strains.

BACKGROUND: Bacterial genomes harbour a large number of tandem repeats, yet the possible phenotypic effects of those found within the coding region of genes are only beginning to be examined. Evidence exists from other organisms that these repeats can be involved in the evolution of new genes, gene regulation, adaptation, resistance to environmental stresses, and avoidance of the immune system. RESULTS: In this study, we have investigated the presence and variability in copy number of intragenic tandemly repeated sequences in the genome of Legionella pneumophila, the etiological agent of a severe pneumonia known as Legionnaires' disease. Within the genome of the Philadelphia strain, we have identified 26 intragenic tandem repeat sequences using conservative selection criteria. Of these, seven were "polymorphic" in terms of repeat copy number between a large number of L. pneumophila serogroup 1 strains. These strains were collected from a wide variety of environments and patients in several geographical regions. Within this panel of strains, all but one of these seven genes exhibited statistically different patterns in repeat copy number between samples from different origins (environmental, clinical, and hot springs). CONCLUSION: These results support the hypothesis that intragenic tandem repeats could play a role in virulence and adaptation to different environments. While tandem repeats are an increasingly popular focus of molecular typing studies in prokaryotes, including in L. pneumophila, this study is the first examining the difference in tandem repeat distribution as a function of clinical or environmental origin.

The importance of the twin-arginine translocation pathway for bacterial virulence.

The twin-arginine translocation (Tat) pathway is a prokaryotic transport system that enables the transport of folded proteins across the cytoplasmic membrane. The Tat pathway was originally thought to transport only proteins that bind cofactors in the cytoplasm and, thus, fold before transport, like many proteins related to energy metabolism. However, in recent years it has become clear that the Tat pathway has a broader role and is also an important virulence factor in different bacterial pathogens. Because the Tat pathway is well conserved among important bacterial pathogens and absent from mammalian cells, it could be a target for novel antimicrobial compounds. In this review, we highlight the importance of the Tat system for virulence in several human and plant pathogens.

A faster and more accurate assay for intracellular replication of Legionella pneumophila in amoebae hosts.

A faster, more sensitive, and more accurate method to study intracellular replication of Legionella pneumophila in amoeboid hosts is described. This assay relies on an automated plate-reader to examine the intracellular growth and release of bacteria in real-time. Our experiments using this method have already revealed new insights into the kinetics of intracellular replication of L. pneumophila in Acanthamoeba castellanii.

Evidence for the presence of Legionella bacteriophages in environmental water samples.

The existence and preliminary characterization of bacteriophages active against the Gram-negative human pathogen Legionella pneumophila, the causative agent of a very severe form of pneumonia, are reported. Four phages belonging to the family of the Myoviridae were isolated from various fresh water environments, and preliminary characterization showed that these crude preparations infect exclusively bacteria belonging to the genus Legionella. Standard phage amplification, purification, and characterization procedures were, however, not efficiently applicable making more research into these novel phages and their mechanism of infection necessary. The existence of Legionella bacteriophages is very promising for future applications such as the development of novel molecular tools, the design of new detection and typing methods, and the bioremediation of this environmental pathogen.

Differential 2-D protein gel electrophoresis analysis of Legionella pneumophila wild type and Tat secretion mutants.

The twin-arginine translocation (Tat) pathway is a secretory pathway for translocation of folded proteins with two arginines in their signal peptide across the cytoplasmic membrane. Recently, we showed the presence of the Tat secretion pathway in Legionella pneumophila Philadelphia-1 and its role in intracellular replication and biofilm formation. To analyse the importance of the Tat pathway in protein export and its role in L. pneumophila virulence, a comparative 2-D protein gel electrophoresis analysis was performed on supernatants of the wild type and two Tat secretion mutants in order to identify possible Tat substrates. Twenty proteins were identified as differential proteins, eight of which were present in a lower quantity in the supernatant of the tat mutants. Among these, one protein with a typical twin-arginine motif in its signal peptide was identified as the 3',5'-cyclic nucleotide phosphodiesterase. Two other proteins that resulted as differential proteins from this study were flagellin and LvrE, which were studied in more detail and their Tat-dependence was further confirmed with specific antibodies. LvrE was shown to play a role in intracellular growth in differentiated U937 cells.

The role of protein secretion systems in the virulence of the intracellular pathogen Legionella pneumophila.

Legionella pneumophila is a Gram-negative facultative intracellular pathogen, which multiplies in protozoa in its natural environment and can cause Legionnaires' disease in man, following infection of alveolar macrophages. In each of the different stages of infection of host cells, virulence proteins need to be delivered to their specific place of action and therefore must cross two barriers: the inner and the outer membrane. To date, several specialized secretion machineries for transport of proteins across the inner and outer membrane have been identified in L. pneumophila. Most of these secretion pathways have been shown to affect the virulence of this pathogen. An overview will be given of all the secretion pathways and the proteins transported by these secretion systems identified so far, with special attention paid to those that play a role in the pathogenicity of L. pneumophila.

Legionella pneumophila exhibits plasminogen activator activity.

Based on their localization at the boundary of the bacterial cell and its environment, outer-membrane proteins (Omps) are important determinants for interaction of bacteria with their host cell. Therefore, they can be considered as important determinants for virulence. Looking for Legionella pneumophila Omps potentially involved in virulence, we identified a gene encoding a homologue of the plasminogen activator (Pla) of Yersinia pestis. Pla belongs to the class of omptins, a family of surface proteases/adhesins that exhibit different virulence-associated functions. In this report we describe the cloning and identification of the plasminogen activator homologue Lpa of L. pneumophila and demonstrate its outer-membrane localization. Transcriptional analysis of the Lpa region revealed expression of the gene in both exponential and stationary growth phase and showed that transcription of the lpa gene is directed by its own promoter. We also show, to our knowledge for the first time, that L. pneumophila has the capacity to convert plasminogen into plasmin by the action of the outer-membrane Lpa protein.

pspA overexpression in Streptomyces lividans improves both Sec- and Tat-dependent protein secretion.

Streptomyces is an interesting host for the secretory production of recombinant proteins because of its innate capacity to secrete proteins at high level in the culture medium. In this report, we evaluated the importance of the phage-shock protein A (PspA) homologue on the protein secretion yield in Streptomyces lividans. The PspA protein is supposed to play a role in the maintenance of the proton motive force (PMF). As the PMF is an energy source for both Sec- and Tat-dependent secretion, we evaluated the influence of the PspA protein on both pathways by modulating the pspA expression. Results indicated that pspA overexpression can improve the Tat-dependent protein secretion as illustrated for the Tat-dependent xylanase C and enhanced green fluorescent protein (EGFP). The effect on Sec-dependent secretion was less pronounced and appeared to be protein dependent as evidenced by the increase in subtilisin inhibitor (Sti-1) secretion but the lack of increase in human tumour necrosis factor (hTNFalpha) secretion in a pspA-overexpressing strain.

The twin-arginine translocation pathway is necessary for correct membrane insertion of the Rieske Fe/S protein in Legionella pneumophila.

The twin-arginine translocation (Tat) pathway translocates folded proteins across the cytoplasmic membrane. Proteins transported through this secretion system typically carry two arginine residues in their signal peptide that is cleaved off during translocation. Recently, we demonstrated the presence of the Tat pathway in Legionella pneumophila Philadelphia-1 and the Rieske Fe/S protein PetA was one of the predicted Tat substrates. Because we observed that the signal peptide of PetA is not processed and that this protein is still membrane associated in the tat mutants, correct membrane insertion was assayed using a trypsin sensitivity assay. We conclude that the Tat pathway is necessary for correct membrane insertion of L. pneumophila PetA.

Effect of iron on the expression of sirR and sitABC in biofilm-associated Staphylococcus epidermidis.

BACKGROUND: 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. RESULTS: 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 in vitro, sirR expression was high and independent of the Fe concentration. The expression of sitC was not inversely correlated to sirR expression. In vivo, expression levels of sirR and of sitABC were high during the initial phase after implantation and, after a transient decrease, remained stable over a period of two weeks. CONCLUSION: Our data suggest that the expression of sirR and the regulatory effect of sirR on the sitABC operon are different in planktonic and sessile bacteria.

Evaluation of TatABC overproduction on Tat- and Sec-dependent protein secretion in Streptomyces lividans.

The majority of bacterial proteins are exported across the cytoplasmic membrane via the Sec pathway, but also the more recently discovered twin-arginine translocation (Tat) route seems to play an important role for protein secretion in Streptomyces lividans in whose genome tatA, tatB and tatC have been identified. In the present work we showed that simultaneous overproduction of TatABC improved the Tat-dependent secretion capacity as could be concluded from the increased amount of secreted xylanase C, an exclusive Tat-dependent substrate. This result demonstrates that next to the availability of energy to drive secretion, also the number of translocases can be rate-limiting for Tat-dependent secretion. On the other hand, tatABC overexpression was found to diminish secretion of the Sec-dependent proteins xylanase B and subtilisin inhibitor in S. lividans. These results reveal cross-talk between both pathways in S. lividans.

The type II signal peptidase of Legionella pneumophila.

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.

Detection and quantification of Legionella pneumophila in water samples using competitive PCR.

The presence of high levels of Legionella pneumophila in man-made aquatic systems correlates with the incidence of nosocomial Legionnaires' disease. This requires a rapid, reliable, and sensitive quantification of L. pneumophila concentrations in suspected water systems. In this research, a homologous competitor was developed and evaluated in a L. pneumophila competitive polymerase chain reaction (cPCR) to quantify this human pathogen in a quick, cost-effective, and reliable way. Accuracy of cPCR was evaluated by analyzing cooling tower and tap water samples spiked with known concentrations of L. pneumophila bacteria, in parallel with the standard culture method. Legionella pneumophila amounts detected and calculated from cPCR and culture correlated very well: r = 0.998, P = 0.002 for tap water and r = 0.990, P = 0.009 for cooling tower water. Nevertheless, for both kinds of water samples, mean numbers of L. pneumophila calculated from cPCR results were always higher than those obtained by culture. This study makes it clear that the rapid, sensitive, and cost-effective L. pneumophila cPCR is a promising alternative to the standard time-consuming culture method and expensive real-time PCR to enumerate L. pneumophila bacteria in environmental water samples.

Surface plasmon resonance-based interaction studies reveal competition of Streptomyces lividans type I signal peptidases for binding preproteins.

Type I signal peptidases (SPases) are responsible for the cleavage of signal peptides from secretory proteins. Streptomyces lividans contains four different SPases, denoted SipW, SipX, SipY and SipZ, having at least some differences in their substrate specificity. In this report in vitro preprotein binding/processing and protein secretion in single SPase mutants was determined to gain more insight into the substrate specificity of the different SPases and the underlying molecular basis. Results indicated that preproteins do not preferentially bind to a particular SPase, suggesting SPase competition for binding preproteins. This observation, together with the fact that each SPase could process each preprotein tested with a similar efficiency in an in vitro assay, suggested that there is no real specificity in substrate binding and processing, and that they are all actively involved in preprotein processing in vivo. Although this seems to be the case for some proteins tested, high-level secretion of others was clearly dependent on only one particular SPase demonstrating clear differences in substrate preference at the in vivo processing level. Hence, these results strongly suggest that there are additional factors other than the cleavage requirements of the enzymes that strongly affect the substrate preference of SPases in vivo.

Inactivation of the 20S proteasome in Streptomyces lividans and its influence on the production of heterologous proteins.

Proteasomes are self-compartmentalizing proteases first discovered in eukaryotes but also occurring in archaea and in bacteria belonging to the order Actinomycetales. In bacteria, proteasomes have so far no known function. In order to evaluate the influence of the 20S proteasome on the production of heterologous proteins by Streptomyces lividans TK24, the production of a number of heterologous proteins, including soluble human tumour necrosis factor receptor II (shuTNFRII) and salmon calcitonin (sCT), was compared with the wild-type TK24, a proteasome-deficient mutant designated PRO41 and a strain complemented for the disrupted proteasome genes (strain PRO41R). S. lividans cells lacking intact proteasome genes are phenotypically indistinguishable from the wild-type or the complemented strain containing functional proteasomes. Using the expression and secretion signals of the subtilisin inhibitor of Streptomyces venezuelae CBS762.70 (Vsi) for shuTNFRII and those of tyrosinase of Streptomyces antibioticus (MelC1) for the production of sCT, both proteins were secreted in significantly higher amounts in the strain PRO41 than in the wild-type S. lividans TK24 or the complemented strain PRO41R. However, the secretion of other heterologous proteins such as shuTNFRI was not enhanced in the proteasome-deficient strain. This suggests that S. lividans TK24 can degrade some heterologous proteins in a proteasome-dependent fashion. The proteasome-deficient strain may therefore be useful for the efficient production of these heterologous proteins.

Functional analysis of TatA and TatB in Streptomyces lividans.

Recently, genes encoding TatA, TatB, and TatC homologues were identified in Streptomyces lividans and the functionality of the twin-arginine translocation (Tat) pathway was demonstrated. Previously, we have shown that TatC is indispensable for Tat-dependent secretion in S. lividans. In the present work, we demonstrate that as TatB, S. lividans TatA is important but not essential for efficient secretion of xylanase C and tyrosinase. The results presented here indicate that in the presence of TatC, still partially functional translocation systems composed of TatAC or TatBC can be formed, suggesting that TatA and TatB have at least partially overlapping activities. However, the dissimilar effect caused by a tatA deletion or a tatB deletion on Tat-dependent secretion together with the fact that TatA cannot fully functionally substitute TatB and vice versa indicates that in S. lividans TatA and TatB are not functionally equivalent. Interestingly, soluble GST-tagged TatA and TatB were able to specifically bind Tat-dependent preproteins. The ability to bind Tat-dependent preproteins together with their cytoplasmic localization in S. lividans strongly suggests that both TatA and TatB, independently or associated, serve to recruit Tat-dependent preproteins to the translocase.

Legionella pneumophila Philadelphia-1 tatB and tatC affect intracellular replication and biofilm formation.

Legionella pneumophila is a facultative intracellular human pathogen and an important cause of Legionnaires' disease, a severe form of pneumonia. Recently, we showed the presence of a putative twin-arginine translocation (Tat) pathway in L. pneumophila Philadelphia-1. This secretion pathway is used to transport completely folded proteins across the cytoplasmic membrane. The importance of the Tat pathway in L. pneumophila was investigated by constructing a tatB and a tatC mutant. Functionality of the Tat pathway was shown using a proven heterologous Tat substrate. It was shown that tatB and tatC are involved in intracellular replication in Acanthamoeba castellanii and differentiated U937 cells, and in biofilm forming ability. A putative Legionella Tat substrate was identified via 2D gel electrophoresis.

Secretory production of biologically active rat interleukin-2 by Clostridium acetobutylicum DSM792 as a tool for anti-tumor treatment.

The search for effective means of selectively delivering high therapeutic doses of anti-cancer agents to tumors has explored a variety of systems in the last decade. The ability of intravenously injected clostridial spores to infiltrate and thence selectively germinate in the hypoxic regions of solid tumors is exquisitely specific, making this system an interesting addition to the anti-cancer therapy arsenal. To increase the number of therapeutic proteins potentially useful for cancer treatment we have tested the possibility of Clostridium acetobutylicum to secrete rat interleukin-2 (rIL2). Therefore, rIL2 cDNA was placed under the control of the endo-beta-1,4-glucanase promoter and signal sequence of C. saccharobutylicum. Recombinant C. acetobutylicum containing the relevant construct secreted up to 800 microgl(-1) biologically active rIL2. The obtained yield should be sufficient to provoke in vivo effects.

Structural organization of the twin-arginine translocation system in Streptomyces lividans.

The twin-arginine translocation (Tat) system exports folded proteins across bacterial cytoplasmic membranes. Recently, genes encoding TatA, TatB and TatC homologues were identified in Streptomyces lividans and the functionality of the Tat pathway was demonstrated. Here, we have examined the localization and structural organization of the Tat components in S. lividans. Interestingly, besides being membrane-associated proteins, S. lividans TatA and TatB were also detected in the cytoplasm. TatC could only be detected in isolated membrane fractions. Whereas all TatC was found to be stably inserted in the membrane, part of membrane-associated TatA and TatB could be extracted following high salt, sodium carbonate or urea treatment suggesting a more loose association with the membrane. Finally, we have analyzed Tat complexes that could be purified from an S. lividans TatABC overproducing strain. From the cytoplasmic membrane, two types of high molecular mass Tat complexes could be isolated having a similar composition as those isolated from Escherichia coli. In the cytoplasm, TatA and TatB were detected as monomer or as homo-oligomeric complexes.