Specific Interaction of Novel Friunavirus Phages Encoding Tailspike Depolymerases with Corresponding Acinetobacter baumannii Capsular Types.

Acinetobacter baumannii is one of the most clinically important nosocomial pathogens. The World Health Organisation refers it to its «critical priority¼ category to develop new strategies for effective therapy. This microorganism is capable of producing structurally diverse capsular polysaccharides (CPSs), which serve as primary receptors for A. baumannii bacteriophages carrying polysaccharide-depolymerasing enzymes. In this study, eight novel bacterial viruses that specifically infect A. baumannii strains belonging to K2/K93, K32, K37, K44, K48, K87, K89 and K116 capsular types were isolated and characterized. The overall genomic architecture demonstrated that these viruses are representatives of the Friunavirus genus of the family Autographiviridae The linear double-stranded DNA phage genomes of 41,105-42,402 bp share high nucleotide sequence identity, except for genes encoding structural depolymerases or tailspikes which determine the host specificity. Deletion mutants lacking N-terminal domains of tailspike proteins were cloned, expressed and purified. The structurally defined CPSs of the phage bacterial hosts were cleaved with the specific recombinant depolymerases, and the resultant oligosaccharides that corresponded to monomers or/and dimers of the CPS repeats (K-units) were isolated. Structures of the derived oligosaccharides were established by nuclear magnetic resonance spectroscopy and high-resolution electrospray ionization mass spectrometry. The data obtained showed that all depolymerases studied were glycosidases that cleave specifically the A. baumannii CPSs by the hydrolytic mechanism, in most cases, by the linkage between the K-units.IMPORTANCE Acinetobacter baumannii, a nonfermentative, Gram-negative, aerobic bacterium, is one of the most significant nosocomial pathogens. The pathogenicity of A. baumannii is based on the cooperative action of many factors, one of them being the production of capsular polysaccharides (CPSs) that surround bacterial cells with a thick protective layer. Polymorphism of the chromosomal capsule loci is responsible for the observed high structural diversity of the CPSs. In this study, we describe eight novel lytic phages which have different tailspike depolymerases (TSDs) determining the interaction of the viruses with corresponding A. baumannii capsular types (K-types). Moreover, we elucidate the structures of oligosaccharide products obtained by cleavage of the CPSs by the recombinant depolymerases. We believe that as the TSDs determine phage specificity, the diversity of their structures should be taken into consideration as selection criteria for inclusion of certain phage candidate to the cocktail designed to control A. baumannii with different K-types.

Mechanisms of Acinetobacter baumannii Capsular Polysaccharide Cleavage by Phage Depolymerases.

Aerobic gram-negative bacterium Acinetobacter baumannii has recently become one of the most relevant pathogens associated with hospital-acquired infections worldwide. A. baumannii produces a capsule around the cell, which represents a thick viscous layer of structurally variable capsular polysaccharide (CPS). The capsule protects the bacteria against unfavorable environmental factors and biological systems, including bacteriophages and host immune system. Many A. baumannii phages have structural depolymerases (tailspikes) that specifically recognize and digest bacterial CPS. In this work, we studied the interaction of tailspike proteins of four lytic depolymerase-carrying phages with A. baumannii CPS. Depolymerases of three bacteriophages (Fri1, AS12, and BS46) were identified as specific glycosidases that cleave the CPS of A. baumannii strains 28, 1432, and B05, respectively, by the hydrolytic mechanism. The gp54 depolymerase from bacteriophage AP22 was characterized as a polysaccharide lyase that cleaves the CPS of A. baumannii strain 1053 by ß-elimination at hexuronic acid (ManNAcA) residues.

Elucidation of the K32 Capsular Polysaccharide Structure and Characterization of the KL32 Gene Cluster of Acinetobacter baumannii LUH5549.

Capsular polysaccharide (CPS), isolated from Acinetobacter baumannii LUH5549 carrying the KL32 capsule biosynthesis gene cluster, was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K32 CPS was found to be composed of branched pentasaccharide repeats (K units) containing two residues of ß-D-GalpNAc and one residue of ß-D-GlcpA (ß-D-glucuronic acid) in the main chain and one residue each of ß-D-Glcp and α-D-GlcpNAc in the disaccharide side chain. Consistent with the established CPS structure, the KL32 gene cluster includes genes for a UDP-glucose 6-dehydrogenase (Ugd3) responsible for D-GlcA synthesis and four glycosyltransferases that were assigned to specific linkages. Genes encoding an acetyltransferase and an unknown protein product were not involved in CPS biosynthesis. Whilst the KL32 gene cluster has previously been found in the global clone 2 (GC2) lineage, LUH5549 belongs to the sequence type ST354, thus demonstrating horizontal gene transfer between these lineages.

Structure and Gene Cluster of the K93 Capsular Polysaccharide of Acinetobacter baumannii B11911 Containing 5-N-Acetyl-7-N-[(R)-3-hydroxybutanoyl]pseudaminic Acid.

Capsular polysaccharide (CPS) assigned to the K93 type was isolated from the bacterium Acinetobacter baumannii B11911 and studied by sugar analysis along with one- and two-dimensional 1H and 13C NMR spectroscopy. The CPS was found to contain a derivative of pseudaminic acid, and the structure of the branched tetrasaccharide repeating unit was established. Genes in the KL93 capsule biosynthesis locus were annotated and found to be consistent with the CPS structure established. The K93 CPS has the α-d-Galp-(1→6)-ß-d-Galp-(1→3)-d-GalpNAc trisaccharide fragment in common with the K14 CPS of Acinetobacter nosocomialis LUH 5541 and A. baumannii D46. It also shares the ß-d-Galp-(1→3)-d-GalpNAc disaccharide fragment and the corresponding predicted Gal transferase Gtr5, as well as the initiating GalNAc-1-P transferase ItrA2, with a number of A. baumannii strains.

[Viral sorption on polyaniline, carbon nanotubes and their based nanocomposites].

The paper gives data on the sorption of influenza virus pandemic strain A/IIV-Moscow/01/2009 (H1N1)swl, avian influenza viruses with A/H5 and A/H7 hemagglutinin, poliomyelitis virus, and T4-D bacteriophage on polyaniline sorbents, carbon nanotubes, and their based nanocomposites. The sorption of viruses occurred in different solutions at 4-37 degrees C during 15 min or more. The rate of viral sorption depended on the structure of sorbents.

[The properties of the Pseudomonas aeruginosa bacteriophage phiPMG1].

The properties of the isolated Pseudomonas aeruginosa bacteriophage phiPMG1 include the lytic infection cycle, and the formation of a broad halo (semi-transparent zone) around the plaques. We consider phiPMG1 as a potential member of therapeutic cocktails of live phages, and as a source of peptidoglycan and lipopolysaccharide degrading enzymes. Partial sequencing of phiPMG1 genome has revealed high similarity with known temperate P. aeruginosa phage D3. An open reading frame encoding lytic transglycosilase was identified in the genome. This enzyme PMG MUR was obtained in recombinant form, and its activity and substrate specificity has been studied.

[Results of hematopoietic cell transplantation in the first complete remission in children with acute myeloid leukemia from an intermediate risk group].

AIM: To analyze the results of allogeneic and autologous hemopoietic cell transplantations (allo- and auto-HCT) in children with acute myeloid leukemia (AML) from an intermediate risk group, most of which were performed using lower-intensity conditioning modes. SUBJECTS AND METHODS. The study enrolled 36 children from an intermediate risk group, who had undergone auto-HCT (n = 22) or allo-HCT (n = 14) in December 1994 to December 2008. The patients' age was 0.7 to 16.6 years (median 12.8 years). Chemotherapeutic conditioning regimens were applied to all the patients. Melphalan was a basic myeloablative agent in 83.3% of cases. RESULTS: With a median follow-up of 4.6 years (1.1-13.8 years), three-year relapse-free survival (RFS) was 80.4%; overall survival (OS) was 65.6%. Recurrences were documented only in 6 (16.6%) patients from the auto-HCT. Transplantation-associated mortality (TAM) was 13.8% (five patients died). After allo-HCT versus auto-HCT, RFS, OS, and TAM were 100 and 68.7% (p = 0.03), 93.2 and 55.5% (p = 0.02), and 7.1 and 18.2%, respectively. Acute and chronic graft-versus-host reactions developed in 57.1 and 23.1%, respectively. CONCLUSION: Transplantation of allogeneic hemopoietic cells from a compatible related donor in the intermediate risk group children with AML, by using melphalan-based conditioning regimen, demonstrates a high survival rate with the minimum toxicity.

[Chromosomal translocation t(8,21) in acute myeloid leukemia of children: prognostic value of additional karyotype abnormalities].

Prognostic significance of additional karyotype abnormalities was studied in 73 children with t(8,21) acute myeloid leukemia (AML). Additional chromosomal aberrations were documented in 61 cases (83.6%). The loss of sex chromosomes and/or deletion of the long arm of chromosome 9 (9q-) were predominant abnormalities, in agreement with the literature data. Other additional abnormalities detected in 14 cases were tentatively designated as "atypical". Comparison of pretreatment cytogenetic data and those obtained during relapses revealed the previously unknown rise in the frequency of atypical abnormalities in AML relapses (to 63.6% vs 19.2% at the first presentation, p < 0.005). It is supposed that atypical additional abnormalities reflect relatively late stages of leukemia, and their presence before therapy predicts poor prognosis. In fact, general, relapse-free, and uneventful survival rates in patients with atypical abnormalities were significantly lower that in the remaining patients with t(8;21) AML. Poor survival was associated not only with early relapses but also with high mortality from fatal infections soon after onset of treatment. The incidence of fatal infections in this group was significantly higher than in patients without atypical abnormalities (p = 0.027). Atypical additional abnormalities are rather variable and each variant should to be specifically characterized to estimate its prognostic significance. Our results need to be verified in a larger-scale multicentre study.

[Complex karyotype abnormalities in pediatric acute myeloid leukemia].

A majority of the data on the prognostic significance of distinct chromosome changes and combinations of them in pediatric acute myeloid leukemia (AML) has been derived from adult studies, with not numerous published data in pediatric patients. One of points needed to be clarified is prognostic significance of complex karyotype (at least 3 unrelated abnormalities). We investigated characteristic features of complex karyotype in newly diagnosed pediatric AML de novo. Cell clones with complex karyotype were found in 35 of 254 (13.8%) patients at the age from 0 to 15 years studied prior to therapy. The group was divided into 2 subgroups depending on presence of favorable chromosome abnormalities, i.e. (see symbols)(8;21), t(15;17) and inv(16). The abnormalities were absent in 20 cases (1st subgroup), in 15 remaining patients they were identified (2nd subgroup). In 2nd subgroup karyotypes were not so considerably changed and no adverse risk markers were detected as distinct from 1st subgroup. New data were obtained for complex karyotype differences of adult and pediatric AML. In the great majority (76%) of complex karyotypes in our adult patients chromosome abnormalities associated with adverse risk were found but in pediatric patients their frequency was significantly less (30%). The highest rate of complex karyotype we observed in children at the age from 0 to 3 years. Similar data were not published earlier. Complex karyotype is considered to be characteristic of older AML patients and in the majority of the patients the karyotype contains markers of adverse risk. Possibly, worse outcome in older AML patients is connected with the markers but not with multiple chromosome changes. New data of frequency and the peculiarities of complex karyotype in pediatric AML are important for understanding of AML pathogenesis and for development a more effective AML treatment.

[Clinico-laboratory variants of the course and results of therapy of hepatitis-associated aplastic anemias in children].

AIM: To study clinical and laboratory characteristics of hepatitides and evaluate efficacy of immunosuppressive therapy and transplantation of the bone marrow in hepatitis-associated aplastic anemia (HAAA). MATERIAL AND METHODS: A retrospective analysis of case histories of children with HAAA was made. For all the patients standard tests for detection of aquired aplastic anemia and hepatitis were conducted. Transplantation of hemopoietic stem cells (THSC) from HLA-identical donors was made in 4 patients, 25 patients were treated with combined immunosuppressive therapy (antithymocytic globulin--ATG plus cyclosporin A -CsA), one patients received monotherapy with CsA, two--prednisolone and a short course of CsA, one child was untreated. RESULTS: Of 260 children admitted to hospital from April 1989 to July 2005 for aquired aplastic anemia, 33 (12.7%) met diagnostic criteria of HAAA. Boys to girls ratio was 267. Hepatitides were severe: median of alaninaminotransferase concentration was 1215 IU/l, aspartataminotransferase--789 IU/l, bilirubin--152.5 mcmol/l. Median of the interval from hepatitis symptoms to documentation of pancytopenia was 66 days (0-204 days). All four patients after THSC are alive for 30-72 months. Probability of complete remission after the first course of ATG+CsA is 0.72 +/- 0.09, probability of survival 0.81 +/- 0.07, median of the interval to transfusion independence--50 days. CONCLUSION: HAAA prognosis is good only in administration of up-to-date therapy. After seronegative hepatitis it is necessary to control hemogram parameters and in the presence of minimal cytopenia patients should be directed to hematological hospital.

Functional role of the N-terminal domain of bacteriophage T4 gene product 11.

Bacteriophage T4 late gene product 11 (gp11), the three-dimensional structure of which has been solved by us to 2.0 A resolution, is a part of the virus' baseplate. The gp11 polypeptide chain consists of 219 amino acid residues and the functionally active protein is a three-domain homotrimer. In this work, we have studied the role of gp11 N-terminal domain in the formation of a functionally active trimer. Deletion variants of gp11 and monoclonal antibodies recognizing the native conformation of gp11 trimer have been selected. Long deletions up to a complete removal of the N-terminal domain, containing 64 residues, do not affect the gp11 trimerization, but considerably change the protein structure and lead to the loss of its ability to incorporate into the baseplate. However, the deletion of the first 17 N-terminal residues results in functionally active protein that can complete the 11(-)-defective phage particles in in vitro complementation assay. This region of the polypeptide chain is probably essential for gp11-gp10 stable complex formation at the early stages of phage baseplate assembly in vivo. A study of the gp10 deletion variants suggests that the central domain of gp10 trimer is responsible for the interaction with gp11.

Molecular architecture of bacteriophage T4.

In studying bacteriophage T4--one of the basic models of molecular biology for several decades--there has come a Renaissance, and this virus is now actively used as object of structural biology. The structures of six proteins of the phage particle have recently been determined at atomic resolution by X-ray crystallography. Three-dimensional reconstruction of the infection device--one of the most complex multiprotein components--has been developed on the basis of cryo-electron microscopy images. The further study of bacteriophage T4 structure will allow a better understanding of the regulation of protein folding, assembly of biological structures, and also mechanisms of functioning of the complex biological molecular machines.

[Time course changes in the count of peripheral granulocytes in children with acquired aplastic anemias treated with antithymocyte globulin, cyclosporin A and granulocytic colony stimulating factor]. / Dinamika kolichestva granulotsitov v perifericheskoi krovi u detei s priobretennymi aplasticheskimi anemiiami, poluchivshikh lechenie antitimotsitarnym globulinom, tsiklosporinom A i granulotsitarnym koloniestimuliruiushchim faktorom.

AIM: To examine the pattern of changes in the count of peripheral granulocytes in children with aplastic anemias (AA), receiving a combined immunosuppressive therapy with antithymocytic globulin (ATG) and cyclosporin A in combination with granulocytic colony-stimulating factor (G-CSF). MATERIALS AND METHODS: 31 children (17 boys and 14 girls) aged 2-15 years (median 9 years) with newly diagnosed severe and very severe acquired AA took a combined immunosuppressive therapy with ATG and cyclosporin A in combination with G-CSF in an initial dose of 10 micrograms/kg a day. RESULTS: A three-linear and response was recorded in 19 (61%) children, an isolated granulocytic response was in 26 (84%). The interval median before the recovery of granulocytes to 1.5 x 10(9)/l and 5 x 10(9)/l was 19 and 38 days, respectively. CONCLUSION: Use of G-CSF may increase the count of granulocytes in the vast majority of patients with AA, without dramatic influence on the frequency of a three-linear response. Intermittent use of G-CSF may maintain the count of granulocytes long at the safe level and reduce the cost of treatment.

Properties of bacteriophage T4 baseplate protein encoded by gene 8.

Gene product 8 (gp8, 344 amino acids per monomer) of bacteriophage T4 is one of the baseplate structural proteins. We constructed an expression vector of gp8 and developed a method for purification of recombinant protein. CD spectroscopy showed that gp8 is an alpha/beta type structural protein. Its polypeptide chain consists of nearly 40% beta-structure and 15% alpha-helix. These data agree with results of prediction of secondary structure based on the amino acid sequence of the protein. The sedimentation coefficient under standard conditions (S20,w) is 4.6S. Analytical ultracentrifugation results demonstrated that gp8 in solution has two types of oligomers--dimer and tetramer. The tetramer of gp8 may be included in the wedge (1/6 of the baseplate), and the dimer may be an intermediate product of association.

Structure of bacteriophage T4 gene product 11, the interface between the baseplate and short tail fibers.

Bacteriophage T4, like all other viruses, is required to be stable while being transmitted from host to host, but also is poised to eject efficiently and rapidly its double-stranded DNA genome to initiate infection. The latter is coordinated by the recognition of receptors on Escherichia coli cells by the long tail fibers and subsequent irreversible attachment by the short tail fibers. These fibers are attached to the baseplate, a multi-subunit assembly at the distal end of the tail. Recognition and attachment induce a conformational transition of the baseplate from a hexagonal to a star-shaped structure. The crystal structure of gene product 11 (gp11), a protein that connects the short tail fibers to the baseplate, has been determined to 2.0 A resolution using multiple wavelength anomalous dispersion with Se. This structure is compared to the trimeric structure of gp9, which connects the baseplate with the long tail fibers. The structure of gp11 is a trimer with each monomer consisting of 218 residues folded into three domains. The N-terminal domains form a central, trimeric, parallel coiled coil surrounded by the middle "finger" domains. The fingers emanate from the carboxy-terminal beta-annulus domain, which, by comparison with the T4 whisker "fibritin" protein, is probably responsible for trimerization. The events leading from recognition of the host to the ejection of viral DNA must be communicated along the assembled trimeric (gp9)(3) attached to the long tail fibers via the trimeric baseplate protein (gp10)(3) to the trimeric (gp11)(3) and the trimeric short tail fibers.

Transformation of a fragment of beta-structural bacteriophage T4 adhesin to stable alpha-helical trimer.

Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a beta-helix, is supposed to be a major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile beta-structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant alpha-helical structure. This structure is probably similar to that of fibritin, which has a triple alpha-helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.

Structure of bacteriophage T4 fibritin M: a troublesome packing arrangement.

Fibritin, a 52 kDa product of bacteriophage T4 gene wac, forms 530 A long fibers, named whiskers, that attach to the phage neck and perform a helper function during phage assembly. Fibritin is a homotrimer, with its predominant central domain consisting of 12 consecutive alpha-helical coiled-coil segments linked together by loops. The central domain is flanked by small globular domains at both ends. Fibritin M is a genetically engineered fragment of the wild type and contains 74 amino-acid residues corresponding to the last coiled-coil segment and the complete carboxy-terminal domain. The crystals of fibritin M belong to the rare space group P3 with three crystallographically independent trimers in the unit cell. The structure has been established at 1.85 A resolution by combining molecular and isomorphous replacement techniques. One of the two heavy-atom derivatives used was gaseous xenon. A substantial fraction of residues in each independent trimer is disordered to various extents in proportion to the lack of restraints on the molecules provided by the lattice contacts. Accurate modeling of the solvent present in the crystals was crucial for achieving good agreement with experimental data.

Preliminary crystallographic studies of bacteriophage T4 fibritin confirm a trimeric coiled-coil structure.

Fibritin, a 52-kDa product of gene wac of bacteriophage T4, forms fibrous "whiskers" that connect to the phage tail and facilitate the later stages of phage assembly. Preliminary experiments suggest that fibritin is a trimer, and its predominant central part has a parallel alpha-helical coiled-coil structure. To investigate the oligomerization and function of fibritin, we have designed and studied two related deletion mutants, denoted M and E, that consist of its last 75 and 120 amino acids, respectively. Both proteins contain part of the coiled-coil region and the 29 amino acid carboxy-terminal domain essential for the trimerization of fibritin. The proteins are expressed as a soluble product in an Escherichia coli system. We have obtained crystals of fibritins M and E. Complete native X-ray diffraction data sets have been collected to 1.85 and 2.7 A resolution, respectively. The crystals have space group P3 with a=44.3 A, c=91.3 A (fibritin M) and R32 with a=41.2 A, b=358.7 A (fibritin E) in the hexagonal setting. Symmetry and packing considerations show that fibritin is a triple coiled coil.

["Cyclosporin dependence" in the treatment of severe aplastic anemia in children]. / "Tsikloporinovaia zavisimost'" pri lechenii tiazhelykh aplasticheskikh anemii u detei.

Cyclosporin A (CA) was used in the treatment of 2 patients with acquired aplastic anemia. Upon reaching hematological remission these children had recurrence consequent to dose lowering or discontinuation of CA. The remission occurred again when full-dose CA treatment was resumed. Slow 2-year decrease in CA dose led to uneventful end of CA treatment without deterioration of blood picture.