RB49-like Bacteriophages Recognize O Antigens as One of the Alternative Primary Receptors.

The power of most of the enterobacterial O antigen types to provide robust protection against direct recognition of the cell surface by bacteriophage receptor-recognition proteins (RBP) has been recently recognized. The bacteriophages infecting O antigen producing strains of E. coli employ various strategies to tackle this nonspecific protection. T-even related phages, including RB49-like viruses, often have wide host ranges, being considered good candidates for use in phage therapy. However, the mechanisms by which these phages overcome the O antigen barrier remain unknown. We demonstrate here that RB49 and related phages Cognac49 and Whisky49 directly use certain types of O antigen as their primary receptors recognized by the virus long tail fibers (LTF) RBP gp38, so the O antigen becomes an attractant instead of an obstacle. Simultaneously to recognize multiple O antigen types, LTFs of each of these phages can bind to additional receptors, such as OmpA protein, enabling them to infect some rough strains of E. coli. We speculate that the mechanical force of the deployment of the short tail fibers (STF) triggered by the LTF binding to the O antigen or underneath of it, allows the receptor binding domains of STF to break through the O polysaccharide layer.

Escherichia coli bacteriophage Gostya9, representing a new species within the genus T5virus.

Escherichia coli bacteriophage Gostya9 (genus T5virus) was isolated from horse feces collected in Moscow, Russia, in 2013. This phage was associated in a single plaque with the previously reported phage 9g and was subsequently purified. Analysis of the complete genomic sequence of Gostya9 revealed that it is closely related to the T5-like bacteriophage DT57C, which had been isolated at the same location in 2007. These two viruses share 79.5% nucleotide sequence identity, which is below the 95% threshold applied currently to demarcate bacteriophage species. The most significant features distinguishing Gostya9 from DT57C include 1) the presence of one long tail fiber protein gene, 122c (ltf), instead of the two genes, ltfA and ltfB, that are present in DT57C; 2) the absence of the gene for the receptor-blocking lytic conversion lipoprotein precursor llp; and 3) the divergence of the receptor-recognition protein, pb5, which is only distantly related at the amino acid sequence level. The observed features of the Gostya9 adsorption apparatus are suggestive of a possible novel specificity for the final receptor and make this phage interesting for possible direct application in phage therapy of E. coli infections or as a source of receptor-recognition protein for engineering new phage specificities.

Relocalization of Translation Termination and Ribosome Recycling Factors to Stress Granules Coincides with Elevated Stop-Codon Readthrough and Reinitiation Rates upon Oxidative Stress.

Upon oxidative stress, mammalian cells rapidly reprogram their translation. This is accompanied by the formation of stress granules (SGs), cytoplasmic ribonucleoprotein condensates containing untranslated mRNA molecules, RNA-binding proteins, 40S ribosomal subunits, and a set of translation initiation factors. Here we show that arsenite-induced stress causes a dramatic increase in the stop-codon readthrough rate and significantly elevates translation reinitiation levels on uORF-containing and bicistronic mRNAs. We also report the recruitment of translation termination factors eRF1 and eRF3, as well as ribosome recycling and translation reinitiation factors ABCE1, eIF2D, MCT-1, and DENR to SGs upon arsenite treatment. Localization of these factors to SGs may contribute to a rapid resumption of mRNA translation after stress relief and SG disassembly. It may also suggest the presence of post-termination, recycling, or reinitiation complexes in SGs. This new layer of translational control under stress conditions, relying on the altered spatial distribution of translation factors between cellular compartments, is discussed.

Genetic analysis of the cold-sensitive growth phenotype of Burkholderia pseudomallei/thailandensis bacteriophage AMP1.

Bacteriophages related to phage Bp_AMP1 are the most widely spread group of phages infecting Burkholderia pseudomallei-the causative agent of melioidosis. These viruses are also infective against the nonpathogenic host Burkholderia thailandensis, allowing experimental work with them without any special safety precautions. The indirect data as well as the results of the mathematical modelling suggest that the AMP1-like viruses may act as natural biocontrol agents influencing the population levels of B. pseudomallei in soil and water habitats in endemic regions. The cold sensitivity of the lytic growth (CSg) of these phages was suggested to be an important feature modulating the effect of viral infection on host populations in nature. We performed genetic analysis to determine the molecular background of the CSg phenotype of the AMP1 phage. The results indicate that CSg is not due to the lack of any function or product missing at low temperature (25 °C) but results in growth inhibition by a phage-encoded temperature-sensitive genetic switch. We identified phage ORF3 and ORF14 to be involved in the genetic determination of this mechanism.

GTP-independent tRNA delivery to the ribosomal P-site by a novel eukaryotic translation factor.

During translation, aminoacyl-tRNAs are delivered to the ribosome by specialized GTPases called translation factors. Here, we report the tRNA binding to the P-site of 40 S ribosomes by a novel GTP-independent factor eIF2D isolated from mammalian cells. The binding of tRNA(i)(Met) occurs after the AUG codon finds its position in the P-site of 40 S ribosomes, the situation that takes place during initiation complex formation on the hepatitis C virus internal ribosome entry site or on some other specific RNAs (leaderless mRNA and A-rich mRNAs with relaxed scanning dependence). Its activity in tRNA binding with 40 S subunits does not require the presence of the aminoacyl moiety. Moreover, the factor possesses the unique ability to deliver non-Met (elongator) tRNAs into the P-site of the 40 S subunit. The corresponding gene is found in all eukaryotes and includes an SUI1 domain present also in translation initiation factor eIF1. The versatility of translation initiation strategies in eukaryotes is discussed.

Microtubules govern stress granule mobility and dynamics.

Stress granules (SGs) are ribonucleoprotein (RNP)-containing assemblies that are formed in the cytoplasm in response to stress. Previously, we demonstrated that microtubule depolymerization inhibited SG formation. Here, we show that arsenate-induced SGs move throughout the cytoplasm in a microtubule-dependent manner, and microtubules are required for SG disassembly, but not for SG persistence. Analysis of SG movement revealed that SGs exhibited obstructed diffusion on an average, though sometimes SGs demonstrated rapid displacements. Microtubule depolymerization did not influence preformed SG number and size, but significantly reduced the average velocity of SG movement, the frequency of quick movement events, and the apparent diffusion coefficient of SGs. Actin filament disruption had no effect on the SG motility. In cycloheximide-treated cells SGs dissociated into constituent parts that then dissolved within the cytoplasm. Microtubule depolymerization inhibited cycloheximide-induced SG disassembly. However, microtubule depolymerization did not influence the dynamics of poly(A)-binding protein (PABP) in SGs, according to FRAP results. We suggest that the increase of SG size is facilitated by the transport of smaller SGs along microtubules with subsequent fusion of them. At least some protein components of SGs can exchange with the cytoplasmic pool independently of microtubules.

Osteogenic Differentiation Factors of Multipotent Mesenchymal Stromal Cells in the Current Understanding.

BACKGROUND: Molecular genetic mechanisms, signaling pathways, conditions, factors, and markers of the osteogenic differentiation of mesenchymal stem cells (MSCs) are being actively studied and are among the most studied areas in the field of cellular technology. This attention is largely due to the mounting contradictions in the seemingly classical knowledge and the constant updating of results in the analyzed areas. In this regard, we focus on the main classical concepts and some new factors and mechanisms that have a noticeable regulatory effect on the differentiation potential of postnatal MSCs. RESULTS: This review considers the importance of the sources of MSCs for the realization of their differentiation potential, molecular genetic factors and signaling pathways of MSC differentiation, the role of inflammatory cytokines and chemokines in osteogenesis, biomechanical signals, and the effect of conformational changes in the cellular cytoskeleton on MSC differentiation. CONCLUSION: It is concluded that it is necessary to move from studies focused on the effects of local genes to those taking multiple measurements of the gene-regulatory profile and the biomolecules critical for the implementation of numerous, incompletely studied osteogenic factors of endogenous and exogenous origin. Among the cornerstones of future (epi)genetic studies, whether osteomodulatory effects are realized through specific signaling pathways and/or whether cross-signaling with known genes drives the osteogenic differentiation of MSCs remains to be determined.

Calcium Phosphate Coating Prepared by Microarc Oxidation Affects hTERT Expression, Molecular Presentation, and Cytokine Secretion in Tumor-Derived Jurkat T Cells.

Calcium phosphate (CaP) materials are among the best bone graft substitutes, but their use in the repair of damaged bone in tumor patients is still unclear. The human Jurkat T lymphoblast leukemia-derived cell line (Jurkat T cells) was exposed in vitro to a titanium (Ti) substrate (10 × 10 × 1 mm3) with a bilateral rough (average roughness index (Ra) = 2-5 µm) CaP coating applied via the microarc oxidation (MAO) technique, and the morphofunctional response of the cells was studied. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectroscope (EDX) analyses showed voltage-dependent (150-300 V) growth of structural (Ra index, mass, and thickness) and morphological surface and volume elements, a low Ca/PaT ratio (0.3-0.6), and the appearance of crystalline phases of CaHPO4 (monetite) and ß-Ca2P2O7 (calcium pyrophosphate). Cell and molecular reactions in 2-day and 14-day cultures differed strongly and correlated with the Ra values. There was significant upregulation of hTERT expression (1.7-fold), IL-17 secretion, the presentation of the activation antigens CD25 (by 2.7%) and CD95 (by 5.15%) on CD4+ cells, and 1.5-2-fold increased cell apoptosis and necrosis after two days of culture. Hyperactivation-dependent death of CD4+ cells triggered by the surface roughness of the CaP coating was proposed. Conversely, a 3.2-fold downregulation in hTERT expression increased the percentages of CD4+ cells and their CD95+ subset (by 15.5% and 22.9%, respectively) and inhibited the secretion of 17 of 27 test cytokines/chemokines without a reduction in Jurkat T cell survival after 14 days of coculture. Thereafter, cell hypoergy and the selection of an hTERT-independent viable CD4+ subset of tumor cells were proposed. The possible role of negative zeta potentials and Ca2+ as effectors of CaP roughness was discussed. The continuous (2-14 days) 1.5-6-fold reductions in the secretion of vascular endothelial growth factor (VEGF) by tumor cells correlated with the Ra values of microarc CaP-coated Ti substrates seems to limit surgical stress-induced metastasis of lymphoid malignancies.

High-throughput LPS profiling as a tool for revealing of bacteriophage infection strategies.

O-antigens of Gram-negative bacteria modulate the interactions of bacterial cells with diverse external factors, including the components of the immune system and bacteriophages. Some phages need to acquire specific adhesins to overcome the O-antigen layer. For other phages, O-antigen is required for phage infection. In this case, interaction of phage receptor binding proteins coupled with enzymatic degradation or modification of the O-antigen is followed by phage infection. Identification of the strategies used by newly isolated phages may be of importance in their consideration for various applications. Here we describe an approach based on screening for host LPS alterations caused by selection by bacteriophages. We describe an optimized LPS profiling procedure that is simple, rapid and suitable for mass screening of mutants. We demonstrate that the phage infection strategies identified using a set of engineered E. coli 4 s mutants with impaired or altered LPS synthesis are in good agreement with the results of simpler tests based on LPS profiling of phage-resistant spontaneous mutants.

Structure and gene cluster of the O antigen of Escherichia coli F17, a candidate for a new O-serogroup.

Escherichia coli F17 isolated from horse feces was studied in respect to the O antigen (O polysaccharide) structure and genetics. The lipopolysaccharide was isolated by phenol-water extraction of bacterial cells and cleaved by mild acid hydrolysis to yield the O polysaccharide, which was studied by sugar analysis and selective solvolysis with CF3CO2H along with one- and two-dimensional 1H and 13C NMR spectroscopy. The O polysaccharide was found to have a branched pentasaccharide repeat (O-unit) containing one residue each of d-galactose, d-mannose, l-rhamnose, d-glucuronic acid, and N-acetyl-d-glucosamine; about 2/3 units bear a side-chain glucose residue. To our knowledge, the F17 O-polysaccharide structure established is unique among known bacterial polysaccharide structures. The O-antigen gene cluster of E. coli F17 between the conserved genes galF and gnd was sequenced and found to be 99% identical to that of E. coli 102,755 assigned to a novel OgN8 genotype (A. Iguchi, S. Iyoda, K. Seto, H. Nishii, M. Ohnishi, H. Mekata, Y. Ogura, T. Hayashi, Front. Microbiol. 7 (2016) 765). Genes in the cluster were annotated taking into account the F17 O-polysaccharide structure. The data obtained confirm that E. coli F17 and E. coli strains belonging to the OgN8 genotype can be considered as a candidate to a new E. coli O-serogroup. The O antigen of this novel type was demonstrated to make for an effective shield protecting the intimate outer membrane surface of bacteria from direct interaction with bacteriophages.

Cellular and Molecular Basis of Osteoblastic and Vascular Niches in the Processes of Hematopoiesis and Bone Remodeling (A Short Review of Modern Views).

In evolutionary processes, human bone marrow has formed as an organ depot of various types of cells that arise from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs). Vital HSC activity is controlled through molecular interactions with the niche microenvironment. The review describes current views on the formation of key molecular and cellular components of the HSC niche, which ensure maintenance of home ostasis in stem cell niches, obtained from studies of their role in regulating the proliferation and differentiation of HSCs, including the physiological, reparative and pathological remodeling of bone tissue. Due to rapid developments in biotechnology, tissue bioengineering, and regenerative medicine, information can be useful for developing biomimetic and bioinspired materials and implants that provide an effective bone/bone marrow recovery process after injuries and, to a greater extent, diseases of various etiologies.

Protein kinase A, which regulates intracellular transport, forms complexes with molecular motors on organelles.

Major signaling cascades have been shown to play a role in the regulation of intracellular organelle transport . Aggregation and dispersion of pigment granules in melanophores are regulated by the second messenger cAMP through the protein kinase A (PKA) signaling pathway ; however, the exact mechanisms of this regulation are poorly understood. To study the role of signaling molecules in the regulation of pigment transport in melanophores, we have asked the question whether the components of the cAMP-signaling pathway are bound to pigment granules and whether they interact with molecular motors to regulate the granule movement throughout the cytoplasm. We found that purified pigment granules contain PKA and scaffolding proteins and that PKA associates with pigment granules in cells. Furthermore, we found that the PKA regulatory subunit forms two separate complexes, one with cytoplasmic dynein ("aggregation complex") and one with kinesin II and myosin V ("dispersion complex"), and that the removal of PKA from granules causes dissociation of dynein and disruption of dynein-dependent pigment aggregation. We conclude that cytoplasmic organelles contain protein complexes that include motor proteins and signaling molecules involved in different components of intracellular transport. We propose to call such complexes 'regulated motor units' (RMU).

Disruption of microtubules inhibits cytoplasmic ribonucleoprotein stress granule formation.

Stress granules are RNP-containing particles arising in the cytoplasm in response to environmental stress. They are dynamic structures assembling and disassembling in the cytoplasm very rapidly. We have studied whether the cytoskeleton is involved in the formation of stress granules. Stress granules were induced in CV-1 cells by sodium arsenate treatment and visualized by immunofluorescent staining with antibodies either to the p170 subunit of eIF3 or to poly(A)-binding protein. Treatment with sodium arsenate for 30-120 min led to assembling of stress granules in a majority of CV-1 cells. Disruption of MT array with nocodazole treatment abolished arsenate-induced formation of stress granules. A similar effect was induced by the microtubule-depolymerizing drug vinblastine, though the influence of the microtubule-stabilizing drug paclitaxel was opposite. Nocodazole treatment did not prevent arsenate-induced phosphorylation of the eIF-2alpha factor, essential for stress granule formation, suggesting that the presence of intact MT array is required for granule assembly. Unexpectedly, treatment of cells with the actin filament-disrupting drug latrunculin B slightly enhanced stress granule formation. We propose that stress granule formation is microtubule-dependent process and likely is facilitated by the motor protein-driven movement of individual stress granule components (e.g., mRNP) along microtubules.