A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice

Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.     

Yersiniosis in Czechoslovakia. Results of a long-term study involving microbiological laboratories of the hygienic service

The paper reports on 17 years' experience of yersiniosis surveillance. Despite the fact that the microbiological identification of Y. enterocolitica 03 was successfully carried out by virtually all the microbiological laboratories of the Hygienic Service, reported cases of yersiniosis account for only one sixtieth part of alimentary infections. Enteric disease is the prevailing form of yersiniosis, extraintestinal ones being very rare. Most of the implicated patients were children, namely boys. An analysis of symptoms was performed based on the results of two epidemiological studies focussed around 215 and 37 cases of yersiniosis, indicating the involvement of the upper respiratory tract at the very beginning of the disease. The role of not sufficiently cooked pork as a vehicle of infection was proved only once (presswurst), however interhuman transmission of yersiniosis was confirmed in 4 cases with an incubation period of approximately 7 days. There appear to be many obscure areas in the epidemiology of yersiniosis, which may be illuminated using cultivation procedures to detect even small amounts of yersinia.     

Use of an in vitro allergy test for the differential diagnosis of human brucellosis and yersiniosis

The degree of the allergic reorganization in the body of brucellosis and yersiniosis patients was determined by the study of their blood and serum samples in the in vitro allergy test (the direct and indirect leukocytolysis test). The allergy test is highly specific and recommended as a differential diagnostic method in brucellosis and yersiniosis infections.     

The outer membrane porin from Yersinia enterocolitica in yersiniosis diagnostics

The diagnostic test system based on a species-specific antigen, pore-forming protein from the outer membrane of Yersinia enterocolitica, for yersiniosis verification by the method of ELISA has been developed and approved. The proposed ELISA test system is characterized by high sensitivity (95%) and specificity (89%) and provides a differential diagnostics of yersiniosis from other acute enteric infections with similar clinical manifestations. In comparison with the commercial diagnostics based on indirect hemagglutination reaction, which is conventionally used in clinical practice, the porin-based ELISA provides the high level (90–95%) of yersiniosis identification at early (1st week) and late (2nd–4th week) stages of infection process. It has been found that the ELISA test system reveals antibodies to the Y. enterocolitica porin in patient’s serum irrespective of the serological variant of causative agent.     

DNA cleavage by CgII and NgoAVII requires interaction between N- and R-proteins and extensive nucleotide hydrolysis

The stress-sensitive restriction-modification (RM) system CglI from Corynebacterium glutamicum and the homologous NgoAVII RM system from Neisseria gonorrhoeae FA1090 are composed of three genes: a DNA methyltransferase (M.CglI and M.NgoAVII), a putative restriction endonuclease (R.CglI and R.NgoAVII, or R-proteins) and a predicted DEAD-family helicase/ATPase (N.CglI and N.NgoAVII or N-proteins). Here we report a biochemical characterization of the R- and N-proteins. Size-exclusion chromatography and SAXS experiments reveal that the isolated R.CglI, R.NgoAVII and N.CglI proteins form homodimers, while N.NgoAVII is a monomer in solution. Moreover, the R.CglI and N.CglI proteins assemble in a complex with R₂N₂ stoichiometry. Next, we show that N-proteins have ATPase activity that is dependent on double-stranded DNA and is stimulated by the R-proteins. Functional ATPase activity and extensive ATP hydrolysis (∼170 ATP/s/monomer) are required for site-specific DNA cleavage by R-proteins. We show that ATP-dependent DNA cleavage by R-proteins occurs at fixed positions (6–7 nucleotides) downstream of the asymmetric recognition sequence 5′-GCCGC-3′. Despite similarities to both Type I and II restriction endonucleases, the CglI and NgoAVII enzymes may employ a unique catalytic mechanism for DNA cleavage.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia lipopolisacharydes and Yop proteins by ELISA

The antibodies against the somatic antigens of Y. enterocolitica O3, O8, O9, O5,27,Y. pseudotuberculosis I, and released proteins Yop were detected using the ELISA in 1634 serum samples and 84 synovial fluids collected from 1290 persons suspected for yersiniosis, as well as 200 serum samples from healthy individuals (blood donors). The presence of antibody in diagnostically significant titres for somatic antigens of Yersinia were detected by ELISA in 20.5% and 50.6%, antibodies for released proteins Yop in 11.5% and 28.4% respectively of blood donors and patients suspected for yersiniosis. The antibody against the O3 antigen of Y. enterocolitica was the most frequently detected antibody while the most infrequent was the antibody for the antigen from the 08 serologic group. The results of the study showed that the humoral response picture to Yersinia antigens in the course of yersiniosis in humans is dependent on the age and sex of the patient, duration of the infection, and clinical manifestations. Most frequently the elevated antibody levels were detected among patients with erythema nodosum and patients with gastrointestinal symptoms. The frequency of occurrence of antibodies for most antigens of Yersinia, together with age increased reaching its peak, on the average, among individuals aged 21 - 40 years. Analysis of individual cases showed that by the end of the first week of infection, elevated levels of antibodies for somatic antigens of Yersinia are evident. On the other hand, antibodies for released proteins Yop as a matter of rule appear in the second week from the onset of clinical symptoms. Within this early phase of infection immunoglobulins of the A and M classes dominate reaching their highest level in the second to third week of the infection. In majority of the individuals studied antibodies of the IgG class reached their highest level much later in relation to those of the IgA and IgM classes. Significant differences were found in IgA antibody detection among individuals with clinical manifestations of stomachaches and arthritis. Nevertheless, among individuals with clinical symptoms of stomachaches, these immunoglobulins as a matter of principle disappear with a period of 2-3 months from the onset of clinical symptoms. In individuals with arthritis however the aforementioned immunoglobulins maintained at considerable levels even after a year. In joint-fluid samples obtained from patients with arthritis antibodies for Yersinia antigens were detected in similar levels just as obtained simultaneously serum from those individuals.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to enterobacterial common antigen (ECA)

The antibodies against the Enterobacterial Common Antigen (ECA) were detected using the ELISA in 293 serum samples collected from 185 persons suspected for yersiniosis, as well as 115 serum samples from healthy individuals (blood donors). The presence of IgA antibody in diagnostically significant titres for ECA were detected by ELISA in 3.5%, IgG in 13.0%, and IgM in 5.2% of blood donors. Statistical analysis showed that the frequency of detecting antibodies for ECA among the patients with yersiniosis was significantly higher (p < 0.05) in relation to the blood donors. Most frequently the elevated antibody levels were detected among patients with reactive arthritis (IgA 29.2%, IgG 35.4%, IgM 16.7%) while the most infrequent among patients with abdominal pain in acute phase of yersiniosis (IgA 14.9%, IgG 25.3%, IgM 19.5%). The level of antibodies for ECA, together with age increased reaching its peak, on the average, among individuals aged 41 - 60 years. In majority of the individuals studied antibodies of the IgG class reached the level much higher in relation to those of the IgA and IgM classes. The obtained results showed that the detection of antibodies to ECA may be useful in serodiagnosis of Yersinia infections.     

Utilization of released proteins (RPs) of Yersinia enterocolitica for serologic diagnosis of yersiniosis. III. Western-blot

The usefulness of the immunoblotting using released proteins (Yersinia outer proteins-Yop) as the antigen for the serological diagnosis of yersiniosis was estimated. The IgA, IgG, and IgM antibody responses of patients with yersiniosis and healthy blood donors were studied by western-blot prepared in our laboratory, and two commercial assays. The results indicate that antibodies of all three classes are most consistently directed against the proteins of YopD, YopM and YopE. Good correlations between the three western-blots were obtained for all proteins except the protein V-AG. Patients with yersinia-triggered reactive arthritis have IgA class antibodies against the YopD more often and for longer period than the non-arthritic patients with yersiniosis.     

Possibility of the serological differential diagnosis of human brucellosis and yersiniasis

The results of the examination of patients with brucellosis and yersiniosis (serotype 0-9) are presented. The possibility of making, in principle, the differential serological diagnosis of brucellosis and yersiniosis in the agglutination test with the use of Yersinia OH-antigen has been established.     

Serum immunoglobulin IgG subclass distribution of antibody responses to Yop proteins and lipopolysaccharide of Yersinia enterocolitica in patients with yersiniosis.

To assess the humoral immunological responses at the IgG subclass level in yersiniosis specific antibody responses against lipopolysaccharide of Yersinia enterocolitica 03 (LPS) and Yersinia Yop proteins were analyzed by ELISA. Thirty five patients with arthritis and forty nine patients with uncomplicated yersiniosis were included in the study. Analysis of the IgG subclass responses to the LPS revealed that the subclass distribution for both groups of patients was IgG2>IgG1>IgG3. The concentration of IgG4 was below detection level. The predominant antibody responses to Yop proteins were IgG1>IgG3>IgG2>IgG4 but the frequency of detection of particular IgG subclass antibodies were dependent on the age of patients. Generally, the frequency of occurrence of IgG2 antibodies for Yop proteins of Yersinia together increased with age reaching its peak among individuals aged above 40 years. On the other hand, IgG1 for Yop proteins and IgG3 for Y. enterocolitica LPS were diagnosed more often in serum samples obtained from children than from adults. We also found significantly higher frequency of IgG4 to Yop proteins of Y. enterocolitica in men than in women.     

MAMPs and MIMPs: proposed classifications for inducers of innate immunity

Plants encode a sophisticated innate immune system. Resistance against potential pathogens often relies on active responses. Prerequisite to the induction of defences is recognition of the pathogenic threat. Significant advances have been made in our understanding of the non-self molecules that are recognized by plants and the means by which plants perceive them. Established terms describing these recognition events, including microbe-associated molecular pattern (MAMP), MAMP-receptor, effector, and resistance (R) protein, need clarification to represent our current knowledge adequately. In this review we propose criteria to classify inducers of plant defence as either MAMPs or microbe-induced molecular patterns (MIMPs). We refine the definition of MAMP to mean a molecular sequence or structure in ANY pathogen-derived molecule that is perceived via direct interaction with a host defence receptor. MIMPs are modifications of host-derived molecules that are induced by an intrinsic activity of a pathogen-derived effector and are perceived by a host defence receptor. MAMP-receptors have previously been classified separately from R-proteins as a discrete class of surveillance molecules. However, MAMP-receptors and R-proteins cannot be distinguished on the basis of their protein structures or their induced responses. We propose that MAMP-receptors and MIMP-receptors are each a subset of R-proteins. Although our review is based on examples from plant pathogens and plants, the principles discussed might prove applicable to other organisms.     

Epizootiological evaluation of intestinal yersiniasis in Sakhalin Province

The work presents the data obtained as the result of the study of intestinal yersiniosis in the synanthropic and natural foci of the Sakhalin region. The circle of Yersinia carriers spreading this infection in the household and natural biocenoses has been established. For the first time the data on the water-type natural focus of yersiniosis with its specific spatial structure, detected on the Tyuleny Island, are published.     

Clinicoimmunological monitoring of therapy in patients with associated forms of yersiniosis

Characteristics of the clinical process and immunological profile in children with yersiniosis as a monoinfection or in association with acute intenstinal infections and virus hepatitis A are presented. The efficacy of the immunotropic therapy with cycloferon, an interferon inductor, and recombinant interferon in the patients with the viral and bacterial association of the disease (yersiniosis + hepatitis A) and initial disbalance of the serum cytokines was estimated. Dependence of the interferon clinicolaboratory efficacy on the initial levels of serum y-interferon, IL2 and IIA, promoting shorter terms of hyperthermia, diarrhea syndrome and cytolysis syndrome was shown. It allowed to optimize the scheme of the pathogenetic therapy of Yersinia mixed infection.     

Application of released proteins (RPs) of Yersinia enterocolitica for serological diagnosis of yersiniosis. II. Gene recombination for production of the YopD protein

The aim of this study was to evaluate the usefulness of gene recombination technique using the pET-30 Ek/LIC expression vector for production a 36 kDa released protein called YopD and evaluate of this purified protein as antigen in serodiagnosis of yersiniosis. Protein YopD of Y. enterocolitica was expressing in Escherichia coli BL21 (DE3) using the pET-30 Ek/LIC expression vector. Purification of the expressed enzyme from suspensions of E. coli cells treated with Bug Buster Protein/Extraction Reagent was accomplished by immobilised metal (Ni2+) affinity column chromatography (His-trap). The IgM, IgG and IgA class antibodies to YopD were measured in 100 serum samples collected from patients suspected for yersiniosis and 100 blood donors. The obtained results were compared to the results of ELISA with released proteins isolated from the culture of Y. enterocolitica supernatant under calcium deficient conditions and commercial ELISA with recombinant released proteins. A very high (94.0-100.0%) specificity and good sensitivity (55.2-80.4%) were displayed by the ELISA with YopD in relation to other two ELISA. The results of our study showed that recombinant YopD protein purified by chromatography of bio-affinity may be used in serodiagnosis of yersiniosis as a high specific antigen free of Yersinia lipopolysaccharides.     

Yersinia lipopolysaccharide and its biological activity

The data on the structure and biological activity of the lipopolysaccharide (LPS) of Yersinia as an important virulence factor are analyzed. The biological effects of LPS are characterized by dose dependence: small doses stimulate the intensity of phagocytosis, while large doses decrease phagocytic activity and produce cytotoxic effect. Yersinia LPS plays an important role in the development of such consequences of yersiniosis as reactive arthritis, erythema nodosum, Reiter's syndrome. Yersinia LPS is a widespread component for the diagnostics of yersiniosis and pseudotuberculosis.     

Effect of cryosubstance Chagi on deposition or isolation of 90Sr and on the effect of prolonged external exposure to gamma-radiation

Positive effect on localisation and bringing out of 90Sr from organism were gained in the series of experiments on the rats Wistar after using per orum alcohol extract, water suspension and water extract of Inonotus obliquus (Chaga). Everyday per orum infusion of Inonotus water extract into the BALB-line mice under conditions of a prolonged (during two months) external total gamma-irradiation with power dose 0.025 sGr/min has a positive effect on increase an average life duration, are slow down the development of leycopenia, hold lipid peroxide oxidation in the blood and in critic tissues and the R-proteins in blood serum on the level, close to the intact control; appearance, activity and behaviour of the animals were the same.     

Effects of dietary probiotic supplementations on prevention/treatment of yersiniosis disease

Aims:  To evaluate Enterobacter cloacae and Bacillus mojavensis , isolated from rainbow trout gut in the present study, as a probiotic to control yersiniosis disease.
Methods and Results:  A strain of Ent. cloacae and B. mojavensis , isolated from the digestive tract of rainbow trout had an antagonistic effect to Yersinia ruckeri , which causes yersiniosis. After feeding fish with 1 × 10⁸ cells g⁻¹ probiotic containing feed for 60 days, the fish survival rate increased to 99·2% following challenge with Y. ruckeri compared with controls that had 35% survival rate. Effects of Ent. cloacae and B. mojavensis on weight gains and stimulation of red blood cells, white blood cells, platelets and hemoglobin were also evaluated in separate groups of fish fed probiotic containing feed for 2 months. Probiotic significantly affected white blood cells, hemoglobin and weight gains of the experimental fish.
Conclusions:  Enterobacter cloacae and B. mojavensis , can be used to prevent and control yersiniosis disease.
Significance and Impact of the Study:  In conclusion, concomitant use of Ent. cloacae and B. mojavensis as a feed supplement is beneficial to rainbow trout. Use of these organisms can protect fish from yersiniosis and enhance digestibility and utilization of feed. Use of such probiotics may also limit the use of antibiotics and other chemicals in control and treatment of diseases, and thus contribute to the effort to reduce environmental contamination by residual antibiotics and chemicals .     

Identification and characterization of pathogenic Yersinia enterocolitica isolates by PCR and pulsed-field gel electrophoresis

Approximately 550 to 600 yersiniosis patients are reported annually in Sweden. Although pigs are thought to be the main reservoir of food-borne pathogenic Yersinia enterocolitica, the role of pork meat as a vehicle for transmission to humans is still unclear. Pork meat collected from refrigerators and local shops frequented by yersiniosis patients (n=48) were examined for the presence of pathogenic Yersinia spp. A combined culture and PCR method was used for detection, and a multiplex PCR was developed and evaluated as a tool for efficient identification of pathogenic food and patient isolates. The results obtained with the multiplex PCR were compared to phenotypic test results and confirmed by pulsed-field gel electrophoresis (PFGE). In all, 118 pork products (91 raw and 27 ready-to-eat) were collected. Pathogenic Yersinia spp. were detected by PCR in 10% (9 of 91) of the raw pork samples (loin of pork, fillet of pork, pork chop, ham, and minced meat) but in none of the ready-to-eat products. Isolates of Y. enterocolitica bioserotype 4/O:3 were recovered from six of the PCR-positive raw pork samples; all harbored the virulence plasmid. All isolates were recovered from food collected in shops and, thus, none were from the patients' home. When subjected to PFGE, the six isolates displayed four different NotI profiles. The same four NotI profiles were also present among isolates recovered from the yersiniosis patients. The application of a multiplex PCR was shown to be an efficient tool for identification of pathogenic Y. enterocolitica isolates in naturally contaminated raw pork.     

Long-term yersiniosis surveillance project in Czechoslovakia

The paper presents an epidemiological analysis of 8,232 cases of yersiniosis caused by Y. enterocolitica 03 over 1972-1988 as reported by Hygienic Stations. The steady epidemiological characteristics of yersiniosis were the prevalence of children and boys and a typical seasonal pattern with differences between the Czech and Slovak Republics. In the Czech Republic, there were several incidence peaks following, when summarized, the incidence curve of other alimentary infections, whereas in the Slovak Republic morbidity reached its peak during winter months. The numbers of isolated strains oscillated significantly between districts and by years. It appears that the number of positive findings in different districts is not only an objective value reflecting a mosaic-like pattern of incidence but also depends on the quality of microbiological diagnostics.