Biomedical subjects

Airi Palva

Publications and source records attributed to Airi Palva.

At least 19 recordsLinked to original sources

PubMed2006

Serological evidence for Borna disease virus infection in humans, wild rodents and other vertebrates in Finland.

BACKGROUND: Borna disease virus (BDV) can infect many vertebrate species, including humans. BDV infection may lead to meningoencephalomyelitis in animals. An association with human neuropsychiatric diseases has been reported, but the causal relationship between BDV and human disease remains unclear. OBJECTIVES AND STUDY DESIGN: To find out whether BDV is present in Finland and to look for a potential reservoir, we examined a large panel of blood samples from different vertebrate species with immunofluorescence assay. Samples from horses, cats, dogs, sheep, cattle, large predators, grouse, wild rodents and humans were included. Most positive results were confirmed by other specific methods and in other laboratories. RESULTS AND CONCLUSIONS: BDV-specific antibodies were detected in 10 horses, 2 cats, as well as 2 horses and 1 dog from farms housing a previously detected seropositive horse. Interestingly, BDV-specific antibodies were further detected in three wild rodents. In humans, BDV-specific antibodies were detected in a veterinarian and in two patients suspected to have a Puumala hantavirus infection. Our serological analysis suggests that BDV infects various vertebrates in Finland, including humans. Furthermore, our data indicate for the first time that BDV infects also wild rodents.

Animals↗

PubMed2006

Heterologous protein secretion in Lactococcus lactis is enhanced by the Bacillus subtilis chaperone-like protein PrsA.

The Bacillus subtilis lipoprotein PrsA enhances the yield of several homologous and heterologous exported proteins in B. subtilis by being involved in the posttranslocational stage of the secretion process. In this work, we have studied the effect of B. subtilis PrsA on the secretion of Bacillus amyloliquefaciens alpha-amylase (AmyQ), a target protein for PrsA, and Bacillus licheniformis penicillinase (PenP) a nontarget protein for PrsA, in Lactococcus lactis. Two compatible plasmids were constructed and introduced into L. lactis strain NZ9000: one high copy plasmid, expressing the AmyQ gene (amyQ) or the PenP gene (penP), and one low copy plasmid, expressing the PrsA encoding gene (prsA). When amyQ and prsA were simultaneously expressed under the nisin-inducible promoter P( nisA ), Western blotting experiments revealed a 15- to 20-fold increase in the total yield of AmyQ and a sixfold increase in secreted AmyQ activity, compared to a control strain lacking prsA. When expressed under the same induction conditions, PrsA had no effect on the secretion or total yield of PenP. These results show that the secretion yield of some heterologous proteins can be significantly increased in L. lactis when coproduced with the B. subtilis PrsA protein.

Bacillus subtilis↗

PubMed2006

Characterization of genes involved in fructose utilization by Lactobacillus fermentum.

The genes encoding phosphoglucose isomerase (fruI) and fructokinase (fruK) of Lactobacillus fermentum NRRL-B-1932 were sequenced. They constituted an operon, which is involved in fructose metabolism of this strain by channeling intracellular fructose into the phosphoketolase pathway. A third open reading frame, unkR, upstream of the operon was identified as homologous to genes of LacI/GalR family repressors. The UnkR repressor's role in transcriptional control of the fruIK operon could, however, not be established by electrophoretic mobility shift assay (EMSA) analysis. Sequence analysis revealed two putative catabolite responsive elements (cre) in the promoter region of fruIK suggesting that the fruIK operon is under negative regulatory control by carbon catabolite repression. Expression and enzyme activity data were compatible with the assumption that the fruIK operon is repressed by glucose. No sugar specific phosphoenolpyruvate sugar transferase system activity for the transport of fructose, glucose, sucrose or mannose could be detected in L. fermentum NRRL-B-1932 cells, which suggest that fructose is taken up by a permease system.

Base Sequence↗

PubMed2006

Secretion of biologically active porcine interleukin-2 by Lactococcus lactis.

In this study, secretion of two functional recombinant porcine interleukin-2 (rIL-2) proteins by Lactococcus lactis was studied. Two secretion cassettes were constructed in which the secretion was achieved by gene fusion between the lactococcal usp45 secretion signal, a synthetic propeptide and the sequence encoding the mature IL-2. In addition, one of the two secretion cassettes contained the H-domains of L. lactis PrtP. Both of the constructed recombinant IL-2 proteins were found to be secreted in the same quantities, approximately 0.5mg/l. According to a cell proliferative assay using CTLL-2 cell line the specific biological activities of both purified rIL-2 proteins were found to be of similar levels.

Amino Acid Sequence↗

PubMed2005

Production of xylitol from D-xylose by recombinant Lactococcus lactis.

The D-xylose reductase from Pichia stipitis CBS 5773 and the xylose transporter from Lactobacillus brevis ATCC 8287 were expressed in active form in Lactococcus lactis NZ9800. Xylitol production was investigated using non-growing recombinant cells in high cell-density under microaerobic conditions in the presence of xylose and glucose. Besides xylose, the recombinant strain with xylose reductase activity reduced l-arabinose and D-ribose in significant extent to the corresponding pentitols. The ratio of xylitol produced per glucose consumed was almost 10-fold higher under glucose limitation than the ratio in the presence of excess initial glucose. The co-expression of the xylose transporter with the xylose reductase did not increase the efficiency of xylitol production appreciably when compared to the strain in which only the xylose reductase gene was expressed. A fed-batch experiment with high initial xylose concentration (160 gl(-1)) under glucose limitation was carried out using the strain co-expressing xylose reductase and xylose transporter genes. The xylitol yield from xylose was 1.0 mol mol(-1) and the ratio of xylitol produced per glucose consumed was 2.5 mol mol(-1). The volumetric productivity was 2.72 gl(-1)h(-1) at 20 h. Of the xylose initially present, 34% was consumed. Analysis of the fermentation metabolites revealed a shift from homolactic to mixed acid fermentation at early stages of the experiment.

Aldehyde Reductase↗

PubMed2005

Effect of heat-shock and bile salts on protein synthesis of Bifidobacterium longum revealed by [35S]methionine labelling and two-dimensional gel electrophoresis.

Experimental conditions for efficient protein radiolabelling and two-dimensional gel electrophoresis were developed for Bifidobacterium longum. Using these tools, protein synthesis in cells before and after heat-shock and bile salts treatment was investigated. Following heat-stress, 13 proteins were upregulated, of which HtrA, DnaK and GroEL were also moderately induced by bile salts, indicating close relationship between the heat and bile salts responses in bifidobacteria. Our work indicated that, as a consequence of prolonged heat-stress, HtrA undergoes sequential modification and proteolysis, and that this mechanism could be employed by bifidobacteria to respond to heat-stress.

Bacterial Proteins↗

PubMed2005

Composition and temporal stability of gastrointestinal microbiota in irritable bowel syndrome--a longitudinal study in IBS and control subjects.

Irritable bowel syndrome (IBS) is a common intestinal disorder that includes continuous or recurrent intestinal pain and discomfort and altered bowel habits. The pathophysiology of IBS is incompletely understood, but it may involve an altered intestinal microbiota. The aim of the present study was to compare the composition and temporal stability of faecal microbiota of IBS patients and healthy controls by applying culture-based techniques and PCR-DGGE analysis. No difference in the prevalence or mean culturable manners of bacteroides, bifidobacteria, spore-forming bacteria, lactobacilli, enterococci or yeasts were observed between the IBS and the control groups, whereas slightly higher numbers of coliforms as well as an increased aerobe:anaerobe ratio was observed in the IBS group. PCR-DGGE revealed more temporal instability in the predominant bacterial population of IBS subjects than in controls. In 9 out of 21 IBS subjects and 5 out of 17 controls the PCR-DGGE profiles obtained from the samples of the same individual on different occasions (sampling points 0, 3 and 6 months) were clearly different. However, the instability in some of the IBS subjects could partly be explained by the antibiotic consumption during the study. The present study suggests that instability of intestinal microbiota may be involved in IBS. However, further studies are needed to associate the instability with specific IBS symptoms or with specific bacterial groups and species.

Adult↗

PubMed2005

Improved mannitol production by a random mutant of Leuconostoc pseudomesenteroides.

A mutant of Leuconostoc pseudomesenteroides ATCC12291 that was unable to grow on fructose was constructed by chemical mutagenesis. The fructose uptake of this mutant, designated as BPT143, was unaltered and allowed fructose still to be converted into mannitol when glucose was present in the growth medium. The mutant grew and consumed fructose faster than the parent strain when grown in a medium containing both glucose and fructose. The specific activity of fructokinase, the enzyme involved in phosphorylation of fructose to fructose-6-phosphate, was decreased to about 10% of that of the parent strain, and resulted in a reduced leakage of fructose into the phosphoketolase (PK) pathway. The yield of mannitol from fructose was improved from 74 to 86 mol%. The increased fructose consumption rate and higher mannitol yield of the mutant also resulted in improvement of volumetric mannitol productivity. In addition, isolation and characterization of the wild type L. pseudomesenteroides fructokinase gene (fruK) was performed. DNA sequence analysis of the fruK gene region of BPT143 revealed only one silent mutation which does not explain the highly reduced fructokinase activity of the mutant. The genetic characterization of fruK was completed by analyzing the expression, size and 5' end of fruK transcripts. Expression data with BPT143, revealing absence of fruK transcripts, was in accordance with the reduced fructokinase activity of the mutant.

Cell Proliferation↗

PubMed2005

Lactobacillus surface layers and their applications.

Surface (S-) layers are crystalline arrays of proteinaceous subunits present as the outermost component of cell wall in several species of the genus Lactobacillus, as well as in many other bacteria and Archaea. Despite the high similarity of the amino acid composition of all known S-layer proteins, the overall sequence similarity is, however, surprisingly small even between the Lactobacillus S-layer proteins. In addition, the typical characteristics of Lactobacillus S-layer proteins, distinguishing them from other S-layer proteins, are small size and high-predicted pI value. Several lactobacilli possess multiple S-layer protein genes, which can be differentially or simultaneously expressed. To date, the characterized functions of Lactobacillus S-layers are involved in mediating adhesion to different host tissues. A few applications for the S-layer proteins of lactobacilli already exist, including their use as antigen delivery vehicles.

Bacterial Outer Membrane Proteins↗

PubMed2005

Analysis of the fecal microbiota of irritable bowel syndrome patients and healthy controls with real-time PCR.

OBJECTIVE: The gut microbiota may contribute to the onset and maintenance of irritable bowel syndrome (IBS). In this study, the microbiotas of patients suffering from IBS were compared with a control group devoid of gastrointestinal (GI) symptoms. METHODS: Fecal microbiota of patients (n = 27) fulfilling the Rome II criteria for IBS was compared with age- and gender-matched control subjects (n = 22). Fecal samples were obtained at 3 months intervals. Total bacterial DNA was analyzed by 20 quantitative real-time PCR assays covering approximately 300 bacterial species. RESULTS: Extensive individual variation was observed in the GI microbiota among both the IBS- and control groups. Sorting of the IBS patients according to the symptom subtypes (diarrhea, constipation, and alternating predominant type) revealed that lower amounts of Lactobacillus spp. were present in the samples of diarrhea predominant IBS patients whereas constipation predominant IBS patients carried increased amounts of Veillonella spp. Average results from three fecal samples suggested differences in the Clostridium coccoides subgroup and Bifidobacterium catenulatum group between IBS patients (n = 21) and controls (n = 15). Of the intestinal pathogens earlier associated with IBS, no indications of Helicobacter spp. or Clostridium difficile were found whereas one case of Campylobacter jejuni was identified by sequencing. CONCLUSIONS: With these real-time PCR assays, quantitative alterations in the GI microbiota of IBS patients were found. Increasing microbial DNA sequence information will further allow designing of new real-time PCR assays for a more extensive analysis of intestinal microbes in IBS.

Adult↗

PubMed2005

Characterization of a mobile clpL gene from Lactobacillus rhamnosus.

Two genes encoding ClpL ATPase proteins were identified in a probiotic Lactobacillus rhamnosus strain, E-97800. Sequence analyses revealed that the genes, designated clpL1 and clpL2, share 80% identity. The clpL2 gene showed the highest degree of identity (98.5%) to a clpL gene from Lactobacillus plantarum WCFSI, while it was not detected in three other L. rhamnosus strains studied. According to Northern analyses, the expression of clpL1 and the clpL2 were induced during heat shock by > 20- and 3-fold, respectively. The functional promoter regions were determined by primer extension analyses, and the clpL1 promoter was found to be overlapped by an inverted repeat structure identical to the conserved CIRCE element, indicating that clpL1 belongs to the HrcA regulon in L. rhamnosus. No consensus binding sites for HrcA or CtsR could be identified in the clpL2 promoter region. Interestingly, the clpL2 gene was found to be surrounded by truncated transposase genes and flanked by inverted repeat structures nearly identical to the terminal repeats of the ISLpl1 from L. plantarum HN38. Furthermore, clpL2 was shown to be mobilized during prolonged cultivation at elevated temperature. The presence of a gene almost identical to clpL2 in L. plantarum and its absence in other L. rhamnosus strains suggest that the L. rhamnosus E-97800 has acquired the clpL2 gene via horizontal transfer. No change in the stress tolerance of the ClpL2-deficient derivative of E-97800 compared to the parental strain was observed.

Adenosine Triphosphatases↗

PubMed2004

Receptor binding domain of Escherichia coli F18 fimbrial adhesin FedF can be both efficiently secreted and surface displayed in a functional form in Lactococcus lactis.

Adherence of F18 fimbrial Escherichia coli to porcine intestinal epithelial cells is mediated by the adhesin (FedF) of F18 fimbriae. In a previous study, we demonstrated the specificity of the amino acid residues between 60 and 109 as the receptor binding domain of FedF. In this study, different expression, secretion, and anchoring systems for the receptor binding domain of the FedF adhesin in Lactococcus lactis were evaluated. Two partially overlapping receptor binding domains (42 and 62 amino acid residues) were expressed as fusions with L. lactis subsp. cremoris protein PrtP for evaluation of secretion efficiency. To evaluate the cell surface display of these FedF-PrtP fusions, they were further combined with different lengths of PrtP spacers fused with either the L. lactis AcmA anchor or the PrtP cell wall binding domain. An HtrA-defective L. lactis NZ9000 mutant was constructed to determine its effect on the level of secreted or anchored fusion proteins. Recombinant L. lactis clones secreting the receptor binding domain of F18 fimbriae as a fusion with the H domains of L. lactis protein PrtP were first constructed by using two different signal peptides. FedF-PrtP fusions, directed by the signal sequence of L. brevis SlpA, were throughout found to be secreted at significantly higher quantities than corresponding fusions with the signal peptide of L. lactis Usp45. In the surface display systems tested, the L. lactis AcmA anchor performed significantly better, particularly in the L. lactis NZ9000DeltahtrA strain, compared to the L. lactis PrtP anchor region. Of the cell surface display constructs with the AcmA anchor, only those with the longest PrtP spacer regions resulted in efficient binding of recombinant L. lactis cells to porcine intestinal epithelial cells. These results confirmed that it is possible to efficiently produce the receptor binding domain of the F18 adhesin in a functionally active form in L. lactis.

Adhesins, Bacterial↗

PubMed2003

Metabolic engineering of Lactobacillus fermentum for production of mannitol and pure L-lactic acid or pyruvate.

For production of mannitol in combination with pure L-lactic acid or pyruvate, the D- and L-lactate dehydrogenase genes (ldhD and ldhL) of a mannitol-producing Lactobacillus fermentum strain were cloned and stepwise inactivated. For inactivation of both ldh genes by a gene replacement technique, deletion constructs removing a 0.4-kb fragment from the promoter and the 5' end region of the ldh genes were used. The first inactivation mutant, designated L. fermentum GRL1030, carried the deletion in ldhD (DeltaldhD). A double mutant, DeltaldhD-DeltaldhL, was constructed by the inactivation of the ldhL gene of strain GRL1030, resulting in strain L. fermentum GRL1032. The correctness of the both mutants was confirmed at the DNA level by polymerase chain reaction, as shown by the absence of ldh transcripts by northern blotting and as a lack of the corresponding enzyme activity. In bioreactor cultivations, the single mutant GRL1030 produced mannitol and L-lactic acid as expected. Mannitol and lactic acid yields and productivities were practically unaffected by deletion of the ldhD gene. The double mutant GRL1032 produced mannitol and pyruvate as expected. However, although the yield of mannitol from fructose remained high, its volumetric productivity was reduced. The double mutation negatively affected the glucose consumption rate, resulting in reduced cellular growth. In addition to pyruvate, the double mutant produced 2,3-butanediol. More surprisingly, some lactic acid was still produced.

Bioreactors↗

PubMed2003

Probiotic and milk technological properties of Lactobacillus brevis.

Two Lactobacillus brevis strains ATCC 8287 and ATCC 14869(T), were evaluated for their applicability as putative probiotics in dairy products. The strains expressed good in vitro adherence to human Caco-2 and Intestine 407 cells and tolerated well low pH, bile acids and pancreatic fluid under in vitro conditions. In antimicrobial activity assays, strain ATCC 8287 showed inhibitory properties toward selected potential harmful microorganisms, particularly against Bacillus cereus. Both L. brevis strains were resistant to vancomycin, which is typical for the genus Lactobacillus. The L. brevis strains were not able to acidify milk to yoghurt but were suitable as supplement strains in yoghurts. This was shown by producing a set of yoghurt products and analysing their rheological and sensory properties during a cold storage period of 28 days. Survival of the strains through human intestine was examined in 1-week feeding trials. Despite its human origin, L. brevis ATCC 14869(T) could not survive through the gastrointestinal (GI) tract, whereas L. brevis ATCC 8287 was detected in the faecal samples taken during and immediately after ingestion of the strain. In conclusion, L. brevis ATCC 8287 is a promising candidate as a probiotic supplement in dairy products.

Animals↗

PubMed2003

Heat and DNA damage induction of the LexA-like regulator HdiR from Lactococcus lactis is mediated by RecA and ClpP.

The SOS response is a paradigm for bacterial cells response to DNA damage. Yet some bacteria lack a homologue of the SOS regulator, LexA, including the Gram-positive, Lactococcus lactis. In this organism we have identified a negative transcriptional regulator, HdiR that induces target gene expression both upon DNA damage and heat shock. Gel mobility shift assays revealed that the binding site for HdiR is located within an inverted repeat structure. HdiR is able to carry out a self-cleavage reaction in vitro at high pHs, while in vivo it undergoes RecA-dependent self-cleavage in the presence of a DNA-damaging agent. Intriguingly, the N-terminal cleavage product of HdiR retains DNA binding activity, and only when degraded by the Clp protease, is gene expression induced. Thus, the activity of HdiR in response to DNA damage is controlled by sequential proteolysis, involving self-cleavage and Clp-dependent degradation of HdiR. During heat-stress, limited self-cleavage occurs; however, recA and clpP are still required for full induction of target gene expression. Thus, our data show that common elements are involved in both the DNA damage and the heat-mediated induction of the HdiR regulon.

Adenosine Triphosphatases↗

PubMed2003

Comparison of real-time PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria.

PCR primers and hybridization probes were designed for the 16S rRNA genes of six bacterial species or groups typically present in human faeces or used in the dairy industry. The primers and probes were applied for quantification of the target bacterial genomes added in artificial DNA mixtures or faecal DNA preparations, using dot-blot hybridization and real-time PCR with SYBR Green I and TaqMan chemistries. Dot-blot hybridization with (33)P-labelled oligonucleotide probes was shown to detect a 10 % target DNA fraction present in mixed DNA samples. Applicability of the rDNA-targeted oligonucleotide probes without pre-enrichment of the 16S gene pool by PCR was thus limited to the detection of the predominant microbial groups. Real-time PCR was performed using a 96-well format and was therefore feasible for straightforward analysis of large sample amounts. Both chemistries tested could detect and quantify a subpopulation of 0.01 % from the estimated number of total bacterial genomes present in a population sample. The linear range of amplification varied between three and five orders of magnitude for the specific target genome while the efficiency of amplification for the individual PCR assays was between 88.3 and 104 %. Use of a thermally activated polymerase was required with the SYBR Green I chemistry to obtain a similar sensitivity level to the TaqMan chemistry. In comparison to dot-blot hybridization, real-time PCR was easier and faster to perform and also proved to have a superior sensitivity. The results suggest that real-time PCR has a great potential for analysis of the faecal microflora.

Bacteria↗

PubMed2003

Surface display of the receptor-binding region of the Lactobacillus brevis S-layer protein in Lactococcus lactis provides nonadhesive lactococci with the ability to adhere to intestinal epithelial cells.

Lactobacillus brevis is a promising lactic acid bacterium for use as a probiotic dietary adjunct and a vaccine vector. The N-terminal region of the S-layer protein (SlpA) of L. brevis ATCC 8287 was recently shown to mediate adhesion to various human cell lines in vitro. In this study, a surface display cassette was constructed on the basis of this SlpA receptor-binding domain, a proteinase spacer, and an autolysin anchor. The cassette was expressed under control of the nisA promoter in Lactococcus lactis NZ9000. Western blot assay of lactococcal cell wall extracts with anti-SlpA antibodies confirmed that the SlpA adhesion domain of the fusion protein was expressed and located within the cell wall layer. Whole-cell enzyme-linked immunosorbent assay and immunofluorescence microscopy verified that the SlpA adhesion-mediating region was accessible on the lactococcal cell surface. In vitro adhesion assays with the human intestinal epithelial cell line Intestine 407 indicated that the recombinant lactococcal cells had gained an ability to adhere to Intestine 407 cells significantly greater than that of wild-type L. lactis NZ9000. Serum inhibition assay further confirmed that adhesion of recombinant lactococci to Intestine 407 cells was indeed mediated by the N terminus-encoding part of the slpA gene. The ability of the receptor-binding region of SlpA to adhere to fibronectin was also confirmed with this lactococcal surface display system. These results show that, with the aid of the receptor-binding region of the L. brevis SlpA protein, the ability to adhere to gut epithelial cells can indeed be transferred to another, nonadhesive, lactic acid bacterium.

Bacterial Adhesion↗

PubMed2003

ClpE from Lactococcus lactis promotes repression of CtsR-dependent gene expression.

The heat shock response in bacterial cells is characterized by rapid induction of heat shock protein expression, followed by an adaptation period during which heat shock protein synthesis decreases to a new steady-state level. In this study we found that after a shift to a high temperature the Clp ATPase (ClpE) in Lactococcus lactis is required for such a decrease in expression of a gene negatively regulated by the heat shock regulator (CtsR). Northern blot analysis showed that while a shift to a high temperature in wild-type cells resulted in a temporal increase followed by a decrease in expression of clpP encoding the proteolytic component of the Clp protease complex, this decrease was delayed in the absence of ClpE. Site-directed mutagenesis of the zinc-binding motif conserved in ClpE ATPases interfered with the ability to repress CtsR-dependent expression. Quantification of ClpE by Western blot analysis revealed that at a high temperature ClpE is subjected to ClpP-dependent processing and that disruption of the zinc finger domain renders ClpE more susceptible. Interestingly, this domain resembles the N-terminal region of McsA, which was recently reported to interact with the CtsR homologue in Bacillus subtilis. Thus, our data point to a regulatory role of ClpE in turning off clpP gene expression following temporal heat shock induction, and we propose that this effect is mediated through CtsR.

Adenosine Triphosphatases↗