Intra-operative biopsy in chronic sinusitis detects pathogenic Escherichia coli that carry fimG/H, fyuA and agn43 genes coding biofilm formation.

The aim of this study was to investigate whether or not surgical biopsy of sinus tissue in chronic sinusitis, not responsive to treatment, would detect E. coli. We intended to evaluate E. coli virulence genes, therefore dispute the causal role of such an unusual microorganism in chronic sinusitis, as well as consider effective pathogen-targeted therapy. Patients with E. coli isolated by intra-operative puncture biopsy were included in the study. Genetic analysis of E. coli isolates, including phylogenetic grouping and virulence factor characteristics, were done by multiplex PCR. We identified 26 patients with chronic sinusitis, in which 26 E. coli isolates were cultured. The E. coli isolates belonged mainly to pathogenic phylogenetic group B2, and carried multiple virulence genes. Three genes in particular were present in all (100%) of examined isolates, they were (1) marker agn43 gene for forming biofilm, (2) type 1 fimbriae (fimG/H gene) and (3) yersiniabactin receptor (fyuA). Furthermore, a pseudo-phylogenetic tree of virulence genes distribution revealed possible cooperation between agn43, fimG/H, and fyuA in the coding of biofilm formation. Intra-operative-biopsy and culture-based therapy, targeting the isolated E. coli, coincided with long-term resolution of symptoms. This is the first report demonstrating an association between a highly pathogenic E. coli, chronic sinus infection, and resolution of symptoms upon E. coli targeted therapy, a significant finding due to the fact that E. coli has not been considered to be a commensal organism of the oropharynx or sinuses. We postulate that the simultaneous presence of three genes, each coding biofilm formation, may in part account for the chronicity of E. coli sinusitis.

Recurrent bowel-blood translocations of Escherichia coli with the unique virulence characteristics over three-year period in the patient with acute myeloid leukaemia - case report.

In patients with haematological malignancies, the bowel remains the main source of Escherichia coli bloodstream infections. We present the clinical example of recurrent bowel-blood translocations of E. coli with the unique virulence characteristics in a 55-year-old male with the diagnosis of acute myeloid leukaemia. The virulent factors profile of examined strains confirmed that the co-existence of genes papC, sfa, usp and cnf1, encoding virulence factors, predisposes E. coli to translocation from the gastrointestinal tract to the vascular bed. The close cooperation between haematologists and microbiologists is essential to improve the outcome of patients colonised with highly pathogenic strains.

Modified DNA polymerases for PCR troubleshooting.

PCR has become an essential tool in biological science. However, researchers often encounter problems with difficult targets, inhibitors accompanying the samples, or PCR trouble related to DNA polymerase. Therefore, PCR optimization is necessary to obtain better results. One solution is using modified DNA polymerases with desirable properties for the experiments. In this article, PCR troubleshooting, depending on the DNA polymerase used, is shown. In addition, the reasons that might justify the need for modification of DNA polymerases, type of modifications, and links between modified DNA polymerases and PCR efficiency are described.

Structural studies of a cold-adapted dimeric ß-D-galactosidase from Paracoccus sp. 32d.

The crystal structure of a novel dimeric ß-D-galactosidase from Paracoccus sp. 32d (ParßDG) was solved in space group P212121 at a resolution of 2.4 Šby molecular replacement with multiple models using the BALBES software. This enzyme belongs to glycoside hydrolase family 2 (GH2), similar to the tetrameric and hexameric ß-D-galactosidases from Escherichia coli and Arthrobacter sp. C2-2, respectively. It is the second known structure of a cold-active GH2 ß-galactosidase, and the first in the form of a functional dimer, which is also present in the asymmetric unit. Cold-adapted ß-D-galactosidases have been the focus of extensive research owing to their utility in a variety of industrial technologies. One of their most appealing applications is in the hydrolysis of lactose, which not only results in the production of lactose-free dairy, but also eliminates the `sandy effect' and increases the sweetness of the product, thus enhancing its quality. The determined crystal structure represents the five-domain architecture of the enzyme, with its active site located in close vicinity to the dimer interface. To identify the amino-acid residues involved in the catalytic reaction and to obtain a better understanding of the mechanism of action of this atypical ß-D-galactosidase, the crystal structure in complex with galactose (ParßDG-Gal) was also determined. The catalytic site of the enzyme is created by amino-acid residues from the central domain 3 and from domain 4 of an adjacent monomer. The crystal structure of this dimeric ß-D-galactosidase reveals significant differences in comparison to other ß-galactosidases. The largest difference is in the fifth domain, named Bgal_windup domain 5 in ParßDG, which contributes to stabilization of the functional dimer. The location of this domain 5, which is unique in size and structure, may be one of the factors responsible for the creation of a functional dimer and cold-adaptation of this enzyme.

A novel chemiluminescent immunoassay for detection of Toxoplasma gondii IgG in human sera.

This study describes Toxoplasma gondii IgG chemiluminescent immunoassay (CLIA) based on the use of a novel immunochemical reagents in the form of the conjugates of original acridinium ester (AE) labels attached to antibodies and SAG2-GRA1-ROP1L chimeric antigen and shows that this test is useful for diagnostic purposes.

Principles and applications of Ligation Mediated PCR methods for DNA-based typing of microbial organisms.

A significant number of DNA-based techniques has been introduced into the field of microorganisms' characterization and taxonomy. These genomic fingerprinting methods were developed to detect DNA sequence polymorphisms by using general principles, such as restriction endonuclease analysis, molecular hybridization, and PCR amplification. In recent years, some alternative techniques based on ligation of oligonucleotide adapters before DNA amplification by PCR, known as Ligation-Mediated PCR methods (LM PCR), have been successfully applied for the typing of microorganisms below the species level. These molecular methods include: Amplified Fragment Length Polymorphism (AFLP), Amplification of DNA fragments Surrounding Rare Restriction Sites (ADSRRS), PCR Melting Profiles (PCR MP), Ligation Mediated PCR/Shifter (LM PCR/Shifter), Infrequent-Restriction-Site Amplification (IRS PCR), double digestion Ligation Mediated Suppression PCR (ddLMS PCR). These techniques are now applied more and more often because they involve less time, are comparably inexpensive, and require only standard lab equipment. Here, we present a general review of this group of methods showing their possibilities and limitations. We also identify questions and propose solutions which may be helpful in choosing a particular LM PCR method for the achievement of the required goal.

Cloning, expression, and biochemical characterization of a cold-active GDSL-esterase of a Pseudomonas sp. S9 isolated from Spitsbergen island soil.

An estS9 gene, encoding an esterase of the psychrotolerant bacterium Pseudomonas sp. S9 was cloned and sequenced. The deduced sequence revealed a protein of 636 amino acid residues with a molecular mass of 69 kDa. Further amino acid sequence analysis revealed that the EstS9 enzyme contained a G-D-S-L motif centered at a catalytic serine, an N-terminal catalytic domain and a C-terminal autotransporter domain. Two recombinant E. coli strains for production of EstS9N (a two domain enzyme) and EstS9Δ (a one domain enzyme) proteins were constructed, respectively. Both recombinant proteins were successfully produced as inclusion bodies and then purified under denaturing conditions. However, because of the low enzymatic activity of the refolded EstS9Δ protein, only the EstS9N protein was further characterized. The purified and refolded EstS9N protein was active towards short-chain p-nitrophenyl esters (C2-C8), with optimal activity for the butyrate (C4) ester. With p-nitrophenyl butyrate as the substrate, the enzyme displayed optimal activity at 35°C and pH 9.0. Additionally, the EstS9N esterase retained ~90% of its activity from 25-40°C and ~40% of its activity at 10°C. Moreover, analysis of its kinetic parameters (Km, kcat, kcat/Km) toward p-nitrophenyl butyrate determined at 15°C and 25°C confirmed that the EstS9 enzyme is cold-adapted. To the best of our knowledge, EstS9 is the third characterized cold-active GDSL-esterase and the first one confirmed to contain an autotransporter domain characteristic for enzymes secreted by the type V secretion system.

First report of seroprevalence of Toxoplasma gondii infection in sheep in Pomerania, northern Poland.

INTRODUCTION AND OBJECTIVE: Toxoplasmosis is parasitic disease which has economic relevance for both veterinary and human medicine. In sheep, toxoplasmosis is a major cause of abortion and can thus cause reproductive problems. The current study aimed to determine the occurrence of anti-Toxoplasma gondii IgG antibodies in sheep from 13 districts of northern Poland and thereby obtain actual data about T. gondii seroprevalence in this population of animals. MATERIALS AND METHODS: Blood samples from 1,646 animals from 99 herds were collected, and an in-house enzyme-linked immunosorbent assay (ELISA) based on native Toxoplasma lysate antigen (TLA) was used for serological testing. The diagnostic sensitivity of diagnostic test used in this study was 98.6%, and specificity 94.9% for the group of 113 sheep sera (74 seropositive and 39 seronegative) previously characterized by using an commercial agglutination test. RESULTS: Antibodies against T. gondii were found in 921 (55.9%) of all tested animals. The percentage of infected sheep was the highest (67.6%) for older animals (>6 years), whereas for younger ones it was significantly lower (50.1% - 57.2% for 1-5-year-old animals, respectively). Furthermore, a higher percentage of seropositive animals was noted among males (63%) than females (55.5%). The results also showed that the size of the herd is not a factor which may affect the seroprevalence of T. gondii infection in the examined population of sheep. CONCLUSION: The results of this study indicate that T. gondii infection in sheep from region of northern Poland is relatively high, and consumption of ovine meat and milk can be regarded as a significant source of infection for humans.

A new Toxoplasma gondii chimeric antigen containing fragments of SAG2, GRA1, and ROP1 proteins-impact of immunodominant sequences size on its diagnostic usefulness.

This study presents the first evaluation of new Toxoplasma gondii recombinant chimeric antigens containing three immunodominant regions of SAG2, GRA1, and one of two ROP1 fragments differing in length for the serodiagnosis of human toxoplasmosis. The recombinant chimeric antigens SAG2-GRA1-ROP1L (with large fragment of ROP1, 85-396 amino acid residues) and SAG2-GRA1-ROP1S (with a small fragment of ROP1, 85-250 amino acid residues) were obtained as fusion proteins containing His6-tags at both ends using an Escherichia coli expression system. The diagnostic utility of these chimeric antigens was determined using the enzyme-linked immunosorbent assay (ELISA) for the detection of specific anti-T. gondii immunoglobulin G (IgG). The IgG ELISA results obtained for the chimeric antigens were compared to those obtained for the use of Toxoplasma lysate antigen (TLA) and for a mixture of recombinant antigens containing rSAG2, rGRA1, and rROP1. The sensitivity of the IgG ELISA was similar for the SAG2-GRA1-ROP1L chimeric antigen (100 %), the mixture of three proteins (99.4 %) and the TLA (97.1 %), whereas the sensitivity of IgG ELISA with the SAG2-GRA1-ROP1S chimeric antigen was definitely lower, reaching 88.4 %. In conclusion, this study shows that SAG2-GRA1-ROP1L chimeric antigen can be useful for serodiagnosis of human toxoplasmosis with the use of the IgG ELISA assay. Therefore, the importance of proper selection of protein fragments for the construction of chimeric antigen with the highest reactivity in ELISA test is demonstrated.

Characterization of a Single-Stranded DNA-Binding-Like Protein from Nanoarchaeum equitans--A Nucleic Acid Binding Protein with Broad Substrate Specificity.

BACKGROUND: SSB (single-stranded DNA-binding) proteins play an essential role in all living cells and viruses, as they are involved in processes connected with ssDNA metabolism. There has recently been an increasing interest in SSBs, since they can be applied in molecular biology techniques and analytical methods. Nanoarchaeum equitans, the only known representative of Archaea phylum Nanoarchaeota, is a hyperthermophilic, nanosized, obligatory parasite/symbiont of Ignicoccus hospitalis. RESULTS: This paper reports on the ssb-like gene cloning, gene expression and characterization of a novel nucleic acid binding protein from Nanoarchaeum equitans archaeon (NeqSSB-like protein). This protein consists of 243 amino acid residues and one OB fold per monomer. It is biologically active as a monomer like as SSBs from some viruses. The NeqSSB-like protein displays a low sequence similarity to the Escherichia coli SSB, namely 10% identity and 29% similarity, and is the most similar to the Sulfolobus solfataricus SSB (14% identity and 32% similarity). The NeqSSB-like protein binds to ssDNA, although it can also bind mRNA and, surprisingly, various dsDNA forms, with no structure-dependent preferences as evidenced by gel mobility shift assays. The size of the ssDNA binding site, which was estimated using fluorescence spectroscopy, is 7 ± 1 nt. No salt-dependent binding mode transition was observed. NeqSSB-like protein probably utilizes a different model for ssDNA binding than the SSB proteins studied so far. This protein is highly thermostable; the half-life of the ssDNA binding activity is 5 min at 100 °C and melting temperature (T(m)) is 100.2 °C as shown by differential scanning calorimetry (DSC) analysis. CONCLUSION: NeqSSB-like protein is a novel highly thermostable protein which possesses a unique broad substrate specificity and is able to bind all types of nucleic acids.

Prime-boost vaccination with toxoplasma lysate antigen, but not with a mixture of recombinant protein antigens, leads to reduction of brain cyst formation in BALB/c mice.

INTRODUCTION: Infection with the ubiquitous parasite Toxoplasma gondii is a threat for immunocompromised patients and pregnant women and effective immune-prophylaxis is still lacking. METHODS: Here we tested a mixture of recombinant T. gondii antigens expressed in different developmental stages, i.e., SAG1, MAG1 and GRA7 (SMG), and a lysate derived from T. gondii tachyzoites (TLA) for prophylactic vaccination against cyst formation. Both vaccine formulations were applied systemically followed by an oral TLA-booster in BALB/c mice. RESULTS: Systemic priming with SMG and oral TLA-booster did not show significant induction of protective immune responses. In contrast, systemic priming and oral booster with TLA induced higher levels of Toxoplasma-specific IgG, IgG1 and IgG2a in sera as well as high levels of Toxoplasma-specific IgG1 in small intestines. Furthermore, high levels of Toxoplasma-specific Th1-, Th17- and Th2-associated cytokines were only detected in restimulated splenocytes of TLA-vaccinated mice. Importantly, in mice orally infected with T. gondii oocysts, only TLA-vaccination and booster reduced brain cysts. Furthermore, sera from these mice reduced tachyzoites invasion of Vero cells in vitro, indicating that antibodies may play a critical role for protection against Toxoplasma infection. Additionally, supernatants from splenocyte cultures of TLA-vaccinated mice containing high levels of IFN-γ lead to substantial production of nitric oxide (NO) after incubation with macrophages in vitro. Since NO is involved in the control of parasite growth, the high levels of IFN-γ induced by vaccination with TLA may contribute to the protection against T. gondii. CONCLUSION: In conclusion, our data indicate that prime-boost approach with TLA, but not with the mixture of recombinant antigens SMG, induces effective humoral and cellular Toxoplasma-specific responses and leads to significant reduction of cerebral cysts, thereby presenting a viable strategy for further vaccine development against T. gondii infection.

Serodiagnosis of Toxoplasma gondii infection in farm animals (horses, swine, and sheep) by enzyme-linked immunosorbent assay using chimeric antigens.

Toxoplasma gondii infects all warm-blooded animals including humans, causing serious public health problems and great economic loss in the animal husbandry. Commonly used serological tests for diagnosis of toxoplasmosis involve preparation of whole Toxoplasma lysate antigen (TLA) from tachyzoites. The production of this antigen is associated with high costs and lengthy preparation and the possibility of staff infection. There are also some difficulties in the standardization of such tests. One approach in order to improve the diagnosis of T. gondii infection is to use recombinant chimeric antigens in place of the TLA, which was confirmed by studies in the serodiagnosis of toxoplasmosis in humans. In this paper, we assess, for the first time, the diagnostic utility of five T. gondii recombinant chimeric antigens (MIC1-MAG1-SAG1S, SAG1L-MIC1-MAG1, SAG2-GRA1-ROP1S, SAG2-GRA1-ROP1L, and GRA1-GRA2-GRA6) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs) with sera from three different groups of livestock animals (horses, pigs, and sheep). The reactivity of individual chimeric antigens was analyzed in relation to the results obtained in IgG ELISAs based on a mixture of three antigens (M1: rSAG1+rMIC1+rMAG1, M2: rSAG2+rGRA1+rROP1, and M3: rGRA1+rGRA2+rGRA6) and referenced to TLA. All chimeric antigens were characterized by high specificity (100%), and the sensitivity of the IgG ELISAs based on chimeric antigens was variable (between 28.4% and 100%) and mainly dependent on the animal species. The chimeric antigens were generally more reactive than mixtures of three antigens. The most effective for the diagnosis of toxoplasmosis was SAG2-GRA1-ROP1L, which can detect specific anti-T. gondii antibodies in 100%, 93.8%, and 100% of positive serum samples from horses, pigs, and sheep, respectively. The present study shows that recombinant chimeric antigens can be successfully used to diagnose T. gondii infection in farm animals, and can replace the commonly used TLA.

Identification of antigen Ag43 in uropathogenic Escherichia coli Dr+ strains and defining its role in the pathogenesis of urinary tract infections.

Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are amongst the most common bacterial infectious diseases in the developed world. The urovirulence of UPEC is mainly associated with the surface-exposed fimbrial adhesins and adhesins of the autotransporter (AT) family. The best studied of these proteins is antigen Ag43 mediating cell aggregation, adhesion and biofilm development as the causes of chronic UTIs. The E. coli IH11128 Dr(+) (dra (+)) strain of the Dr/Afa(+) family of adhesins possesses two major surface-exposed virulence factors: Dr fimbrial polyadhesin and DraD protein (fimbrial tip subunit or protein component of the adhesive sheath). Here, we identified for the first time, to our knowledge, the agn43 gene encoding Ag43 in the WT clinical isolate of UPEC Dr(+) as a new virulence factor not yet tested. We also found that Dr fimbrial expression, which like Ag43 is under the control of a phase-variable mechanism, did not exclude Ag43 surface presentation. However, the presence of Dr fimbriae supported by other structures on the cell surface caused a physical neutralization of Ag43-mediated autoaggregation during in vitro growth. The fimbrial bundling further increased the distance between the adjacent Ag43(+) cells, thus preventing head-to-tail association between surface-exposed Ag43 subunits and their interactions with the host cells. The investigations showed that Ag43 did not act as a specific adhesin and invasin, conversely to the major virulence factors of E. coli Dr(+), but played significant roles in the viability and metabolic activity of bacterial cells forming biofilm, and in the survival of bacteria within invaded epithelial cells.

New recombinant chimeric antigens, P35-MAG1, MIC1-ROP1, and MAG1-ROP1, for the serodiagnosis of human toxoplasmosis.

The aim of the study was to evaluate the usefulness of 3 chimeric Toxoplasma gondii antigens, P35-MAG1, MIC1-ROP1 and MAG1-ROP1, in the serodiagnosis of an acute toxoplasmosis in humans. Proteins were produced as fusion proteins containing His tags ends and then further purified by metal affinity chromatography. Their application for the diagnosis of recently acquired T. gondii infection was tested in IgG and IgM enzyme-linked immunosorbent assays (ELISAs). At 100%, 77.3%, and 86.4%, respectively, the reactivity of the IgG ELISA using P35-MAG1, MIC1-ROP1, and MAG1-ROP1 for sera from patients where acute toxoplasmosis was suspected was significantly higher than for the samples from people with a chronic infection, at 26.2%, 36.1%, and 32.8%, respectively. Moreover, P35-MAG1, MIC1-ROP1, and MAG1-ROP1 detected IgM antibodies with a reactivity at 81.8%, 72.7%, and 59.1%, respectively. The results presented in the article show that, particularly, P35-MAG1 may be useful in the preliminary detection of recent T. gondii infection.

The optimal mixture of Toxoplasma gondii recombinant antigens (GRA1, P22, ROP1) for diagnosis of ovine toxoplasmosis.

Toxoplasmosis, caused by Toxoplasma gondii, is the major parasitic disease affecting sheep. Infection not only results in significant reproductive losses in these animals, but has public health implications since consumption of infected meat can facilitate zoonotic transmission. Although several serological tests are currently used for diagnosis of ovine toxoplasmosis, production of reliable reagents is a constraint and therefore there is a need to develop new diagnostic tools. In this paper, we assess for the first time, the preliminary diagnostic utility of 19 T. gondii recombinant antigens (GRA1, GRA2ex2, GRA4, GRA5, GRA6, GRA9, SAG1, SAG4, BSR4, P22, ROP1, P36, MIC1ex2, MIC1ex34, MIC3, MAG1, BAG1, LDH1, and LDH2) in immunoglobulin G (IgG) enzyme-linked immunosorbent assays (IgG ELISAs). Following an initial evaluation, eight recombinant antigens (GRA1, GRA9, SAG1, SAG4, P22, MIC1ex2, MIC3, ROP1) were chosen for subsequent testing and comparison against the native Toxoplasma lysate antigen (TLA) in IgG ELISAs using 88 sera from naturally infected sheep and 20 sera from healthy animals. The reactivity of these antigens was variable with the best results for GRA1, P22, ROP1 and TLA. High sensitivity and specificity (100%) was noted for GRA1, ROP1 and TLA; P22 showed a slightly lower sensitivity (98.9%) but the same high specificity (100%). Four different combinations of these antigens (M1: GRA1+ROP1; M2: GRA1+P22; M3: P22+ROP1; M4: GRA1+P22+ROP1) were tested against the same pool of ovine sera; all IgG-positive serum samples were detected by all of the mixtures. However, the most effective for diagnosis of toxoplasmosis in sheep, based on the highest absorbance values, was the mixture M4 containing three proteins. High sensitivity and specificity (100%) was observed from tests containing either M4 or TLA antigens with a new pool of sera (93 seropositive and 35 seronegative). Thus, the present study shows that a cocktail of GRA1+P22+ROP1 recombinant proteins can be used to diagnose T. gondii infection in sheep, and consequently will assist in epidemiological studies.

Characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila, Flavobacterium psychrophilum, Psychrobacter arcticus, Psychrobacter cryohalolentis, Psychromonas ingrahamii, Psychroflexus torquis, and Photobacterium profundum.

BACKGROUND: Single-stranded DNA-binding proteins (SSBs) play essential roles in DNA replication, recombination and repair in Bacteria, Archaea and Eukarya. In recent years, there has been an increasing interest in SSBs, since they find numerous applications in diverse molecular biology and analytical methods. RESULTS: We report the characterization of single-stranded DNA-binding proteins from the psychrophilic bacteria Desulfotalea psychrophila (DpsSSB), Flavobacterium psychrophilum (FpsSSB), Psychrobacter arcticus (ParSSB), Psychrobacter cryohalolentis (PcrSSB), Psychromonas ingrahamii (PinSSB), Photobacterium profundum (PprSSB), and Psychroflexus torquis (PtoSSB). The proteins show a high differential within the molecular mass of their monomers and the length of their amino acid sequences. The high level of identity and similarity in respect to the EcoSSB is related to the OB-fold and some of the last amino acid residues. They are functional as homotetramers, with each monomer encoding one single stranded DNA binding domain (OB-fold). The fluorescence titrations indicated that the length of the ssDNA-binding site size is approximately 30 ± 2 nucleotides for the PinSSB, 31 ± 2 nucleotides for the DpsSSB, and 32 ± 2 nucleotides for the ParSSB, PcrSSB, PprSSB and PtoSSB. They also demonstrated that it is salt independent. However, when the ionic strength was changed from low salt to high, binding-mode transition was observed for the FpsSSB, at 31 ± 2 nucleotides and 45 ± 2 nucleotides, respectively. As expected, the SSB proteins under study cause duplex DNA destabilization. The greatest decrease in duplex DNA melting temperature was observed in the presence of the PtoSSB 17 °C. The SSBs in question possess relatively high thermostability for proteins derived from cold-adapted bacteria. CONCLUSION: The results showed that SSB proteins from psychrophilic microorganisms are typical bacterial SSBs and possess relatively high thermostability, offering an attractive alternative to other thermostable SSBs in molecular biology applications.

A new human IgG avidity test, using mixtures of recombinant antigens (rROP1, rSAG2, rGRA6), for the diagnosis of difficult-to-identify phases of toxoplasmosis.

The preliminary diagnostic utility of two mixtures of Toxoplasma gondii recombinant antigens (rROP1+rSAG2 and rROP1+rGRA6) in IgG ELISA and IgG avidity test has been evaluated. A total of 173 serum samples from patients with toxoplasmosis and seronegative people were examined. The sensitivity of IgG ELISA for rROP1+rSAG2 and rROP1+rGRA6 was 91.1% and 76.7%, respectively, while the reactivity for sera from patients where acute toxoplasmosis was suspected was higher, at 100% and 95.4%, respectively, than for people with chronic infection, at 88.2% and 70.6%. In this study a different trend in avidity maturation of IgG antibodies for two mixtures of proteins in comparison with native antigen was observed. The results suggest that a new IgG avidity test using the mixtures of recombinant antigens may be useful for the diagnosis of difficult-to-identify phases of toxoplasmosis. For this reason, selected mixtures after the additional tests on groups of sera with well-defined dates of infection could be used as a better alternative to the native antigens of the parasite in the serodiagnosis of human T. gondii infection.

Identification and characterization of single-stranded DNA-binding protein from the facultative psychrophilic bacteria Pseudoalteromonas haloplanktis.

Single-stranded DNA-binding protein (SSB) plays an important role in DNA metabolism such as DNA replication, repair, and recombination, and is essential for cell survival. This study reports on the ssb-like gene cloning, gene expression and characterization of a single-stranded DNA-binding protein of Pseudoalteromonas haloplanktis (PhaSSB) and is the first report of such a protein from psychrophilic microorganism. PhaSSB possesses a high sequence similarity to Escherichia coli SSB (48% identity and 57% similarity) and has the longest amino acid sequence (244 amino acid residues) of all the known bacterial SSBs with one OB-fold per monomer. An analysis of purified PhaSSB by means of chemical cross-linking experiments, sedimentation analysis and size exclusion chromatography revealed a stable tetramer in solution. Using EMSA, we characterized the stoichiometry of PhaSSB complexed with a series of ssDNA homopolymers, and the size of the binding site was determined as being approximately 35 nucleotides long. In fluorescence titrations, the occluded site size of PhaSSB on poly(dT) is 34 nucleotides per tetramer under low-salt conditions (2mM NaCl), but increases to 54-64 nucleotides at higher-salt conditions (100-300mM NaCl). This suggests that PhaSSB undergoes a transition between ssDNA binding modes, which is observed for EcoSSB. The binding properties of PhaSSB investigated using SPR technology revealed that the affinity of PhaSSB to ssDNA is typical of SSB proteins. The only difference in the binding mode of PhaSSB to ssDNA is a faster association phase, when compared to EcoSSB, though compensated by faster dissociation rate. When analyzed by differential scanning calorimetry (DSC), the melting temperature (Tm) was determined as 63 °C, which is only a few degrees lower than for EcoSSB.

Epidemiological study of Toxoplasma gondii infection among cattle in Northern Poland.

Toxoplasmosis, caused by Toxoplasma gondii, is a significant disease in livestock and humans. Because of medical and veterinary importance it is essential to study the prevalence of T. gondii infection among human and animals in various parts of the word. In this study, 4033 cattle from eight provinces of Northern Poland (belonging to 190 herds) were tested for IgG antibodies against T. gondii by an in-house ELISA technique based on native Toxoplasma lysate antigen. The diagnostic sensitivity of test used in this study was 96.3%, and specificity was 98% for the group of 77 cattle sera (27 seropositive and 50 seronegative) previously characterized with the use of agglutination and immunofluorescence methods. A 127 (3.15%) out of all tested animals belonging to 72 (37.9%) out of 190 herds were founded as positive. Furthermore, our results showed that the way of feeding and farming, the size of the herd and the age of animals have the influence on the prevalence of toxoplasmosis among cattle. The percentage of infected cattle was the highest for old animals which belongs to the small herds with the traditional way of farming. These results indicate that T. gondii infection in cattle from Northern Poland is relatively low and consumption of beef and milk can be regarded as a poor source of infection for humans.

Cloning and characterization of a novel cold-active glycoside hydrolase family 1 enzyme with ß-glucosidase, ß-fucosidase and ß-galactosidase activities.

BACKGROUND: Cold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds. RESULTS: A bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and ß-linked oligosaccharides, and had remarkably high ß-galactosidase, ß-glucosidase and ß-fucosidase activities. Because of the lack of information about the usefulness of ß-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for ß-glucosidase or ß-galactosidase. The BglMKg had maximal ß-galactosidase and ß-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for ß-galactosidase activity was 6.5, whereas the optimum pH for ß-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed ß-glucosidase substrates than ß-galactosidase ones. CONCLUSIONS: BglMKg is a small, monomeric, cold-active ß-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.