RESUMEN
Potential genotoxicity and carcinogenicity of carbon nanotubes (CNT), as well as the underlying mechanisms, remains a pressing topic. The study aimed to evaluate and compare the genotoxic effect and mechanisms of DNA damage under exposure to different types of CNT. Immortalized human cell lines of respiratory origin BEAS-2B, A549, MRC5-SV40 were exposed to three types of CNT: MWCNT Taunit-M, pristine and purified SWCNT TUBALL™ at concentrations in the range of 0.0006-200 µg/ml. Data on the CNT content in the workplace air were used to calculate the lower concentration limit. The genotoxic potential of CNTs was investigated at non-cytotoxic concentrations using a DNA comet assay. We explored reactive oxygen species (ROS) formation, direct genetic material damage, and expression of a profibrotic factor TGFB1 as mechanisms related to genotoxicity upon CNT exposure. An increase in the number of unstable DNA regions was observed at a subtoxic concentration of CNT (20 µg/ml), with no genotoxic effects at concentrations corresponding to industrial exposures being found. While the three test articles of CNTs exhibited comparable genotoxic potential, their mechanisms appeared to differ. MWCNTs were found to penetrate the nucleus of respiratory cells, potentially interacting directly with genetic material, as well as to enhance ROS production and TGFB1 gene expression. For A549 and MRC5-SV40, genotoxicity depended mainly on MWCNT concentration, while for BEAS-2B - on ROS production. Mechanisms of SWCNT genotoxicity were not so obvious. Oxidative stress and increased expression of profibrotic factors could not fully explain DNA damage under SWCNT exposure, and other mechanisms might be involved.
Asunto(s)
Nanotubos de Carbono , Humanos , Nanotubos de Carbono/toxicidad , Especies Reactivas de Oxígeno , Daño del ADN , Línea Celular , ADN , Supervivencia CelularRESUMEN
The ribosomal maturation factor (RimP) is a 17.7 kDa protein and is the assembly factor of the 30S subunit. RimP is essential for efficient processing of 16S rRNA and maturation (assembly) of the 30S ribosome. It was suggested that RimP takes part in stabilization of the central pseudoknot at the early stages of the 30S subunit maturation, and this process may occur before the head domain assembly and later stages of the 30S assembly, but the mechanism of this interaction is still not fully understood. Here we report the assignment of the 1H, 13C and 15N chemical shift in the backbone and side chains of RimP from Staphylococcus aureus. Analysis of chemical shifts of the main chain using TALOS + suggests that the RimP contains eight ß-strands and three α-helices with the topology α1-ß1-ß2-α2- ß3- α3- ß4- ß5- ß6- ß7- ß8. Structural studies of RimP and its complex with the ribosome by integrated structural biology approaches (NMR spectroscopy, X-ray diffraction analysis and cryoelectron microscopy) will allow further screening of highly selective inhibitors of the translation of S. aureus.
Asunto(s)
Ribosomas , Staphylococcus aureus , Microscopía por Crioelectrón , Resonancia Magnética Nuclear Biomolecular , ARN Ribosómico 16S/metabolismo , Proteínas Ribosómicas/química , Ribosomas/metabolismoRESUMEN
Plant-protecting Bacillus sp. strains used as biocontrol agents frequently produce metabolites inhibiting phytopathogenic fungi. Recently, the search for a novel biocontrol agent with a wide spectrum of disease control drew attention to Bacillus subtilis and their related species, including Bacillus mojavensis. In this study, we determined the antifungal properties of the endophytic B. mojavensis PS17 isolated from wheat seeds. Metabolites produced by B. mojavensis PS-17 inhibit the growth of Fusarium graminearum, Fusarium oxysporum, Fusarium chlamydosporum, Ascochyta pisi, Alternaria alternate, Sclerotinia sclerotiorum, Verticillium dahliaee, and Epicoccum nigrum strains. B. mojavensis strain PS17 produces several hydrolytic enzymes, such as chitinase, ß-glucanase, cellulase, lipase, and protease. Additionally, strain B. mojavensis PS17 demonstrates drought tolerance under osmotic pressure of -2.2 MPa and a moderate halotolerance in 5% (w/v) of NaCl. B. mojavensis PS17 on tomato seedlings was able to reduce lesions of Forl ZUM2407 by 48.11% ± 1.07, showing the potentials of B. mojavensis PS17 to be adapted as a biocontrol agent for agricultural use.
Asunto(s)
Bacillus , Fusarium , Antifúngicos/farmacología , Ascomicetos , Agentes de Control Biológico , Enfermedades de las Plantas , Estrés FisiológicoRESUMEN
Staphylococcus aureus hibernation promoting factor (SaHPF) is responsible for the formation of 100S ribosome dimers, which in turn help this pathogen to reduce energy spent under unfavorable conditions. Ribosome dimer formation strongly depends on the dimerization of the C-terminal domain of SaHPF (CTDSaHPF). In this study, we solved the crystal structure of CTDSaHPF at 1.6â¯Å resolution and obtained a precise arrangement of the dimer interface. Residues Phe160, Val162, Thr171, Ile173, Tyr175, Ile185 andThr187 in the dimer interface of SaHPF protein were mutated and the effects were analyzed for the formation of 100S disomes of ribosomes isolated from S. aureus. It was shown that substitution of any of single residues Phe160, Val162, Ile173, Tyr175 and Ile185 in the SaHPF homodimer interface abolished the ribosome dimerization in vitro.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Ribosómicas/genética , Ribosomas/genética , Infecciones Estafilocócicas/genética , Staphylococcus aureus/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/ultraestructura , Microscopía por Crioelectrón , Dimerización , Hibernación/genética , Humanos , Unión Proteica/genética , Proteínas Ribosómicas/química , Proteínas Ribosómicas/ultraestructura , Ribosomas/ultraestructura , Infecciones Estafilocócicas/microbiología , Staphylococcus aureus/patogenicidadRESUMEN
Staphylococcus aureus hibernation promoting factor (SaHPF) is a 22,2 kDa protein which plays a crucial role in 100S Staphylococcus aureus ribosome formation during stress. SaHPF consists of N-terminal domain (NTD) that prevents proteins synthesis by binding to the 30S subunit at the P- and A-sites, connected through a flexible linker with a C-terminal domain (CTD) that keeps ribosomes in 100S form via homodimerization. Recently obtained 100S ribosome structure of S. aureus by cryo-EM shown that SaHPF-NTD bound to the ribosome active sites, however due to the absence of SaHPF-NTD structure it was modeled by homology with the E. coli hibernation factors HPF and YfiA. In present paper we have determined the solution structure of SaHPF-NTD by high-resolution NMR spectroscopy which allows us to increase structural knowledge about HPF structure from S. aureus.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular/métodos , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas Ribosómicas/química , Proteínas Ribosómicas/metabolismo , Ribosomas/metabolismoRESUMEN
Ribosome binding factor A (RbfA) is a 14.9 kDa adaptive protein of cold shock, which is important for bacterial growth at low temperatures. RbfA can bind to the free 30S ribosomal subunit and interacts with the 5'-terminal helix (helix I) of 16S rRNA. RbfA is important for the efficient processing of 16S rRNA and for the maturation (assembly) of 30S ribosomal subunits. Here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of RbfA from Staphylococcus aureus. Analysis of the backbone chemical shifts by TALOS+ suggests that RbfA contains four α-helixes and three ß-strands with α1-ß1-ß2-α2-α3-ß3-α4 topology. Secondary structure of RbfA have KH-domain fold topology with ßααß subunit which is characterized by a helix-kink-helix motif in which the GxxG sequence is replaced by a conserved AxG sequence, where an Ala residue at position 70 forming an interhelical kink. The solution of the structure of this protein factor and its complex with the ribosome by NMR spectroscopy, X-ray diffraction analysis and cryo-electron microscopy will allow further development of highly selective substances for slowing or completely stopping the translation of the pathogenic bacterium S. aureus, which will interfere with the synthesis and isolation of its pathogenicity factors.
Asunto(s)
Proteínas Bacterianas/química , Resonancia Magnética Nuclear Biomolecular , Staphylococcus aureus/química , Secuencia de Aminoácidos , Isótopos de Nitrógeno , ProtonesRESUMEN
Elongation Factor P (EF-P) is a 20.5 kDa protein that provides specialized translation of special stalling amino acid motifs. Proteins with stalling motifs are often involved in various processes, including stress resistance and virulence. Thus it has been shown that the virulent properties of microorganisms can be significantly reduced if the work of EF-P is disrupted. In order to elucidate the structure, dynamics and function of EF-P from Staphylococcus aureus (S. aureus), here we report backbone and side chains 1H, 13C and 15N chemical shift assignments of EF-P. Analysis of the backbone chemical shifts by TALOS+ suggests that EF-P contains 1 α-helix and 13 ß-strands (ß1-ß2-ß3-ß4-ß5-ß6-ß7-α1-ß8-ß9-ß10-ß11-ß12-ß13). The solution of the structure of this protein by NMR and X-ray diffraction analysis, as well as the structure of the ribosome complex by cryo-electron microscopy, will allow further screening of highly selective inhibitors of the translation of the pathogenic bacterium S. aureus. Here we report the almost complete 1H, 13C, 15N backbone and side chain NMR assignment of a 20.5 kDa EF-P.
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Factores de Elongación de Péptidos/química , Staphylococcus aureus , Secuencia de Aminoácidos , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina betaRESUMEN
Staphylococcus aureus: hibernation-promoting factor (SaHPF) is a 22.2 kDa stationary-phase protein that binds to the ribosome and turns it to the inactive form favoring survival under stress. Sequence analysis has shown that this protein is combination of two homolog proteins obtained in Escherichia coli-ribosome hibernation promoting factor (HPF) (11,000 Da) and ribosome modulation factor RMF (6500 Da). Binding site of E. coli HPF on the ribosome have been shown by X-ray study of Thermus thermophilus ribosome complex. Hence, recent studies reported that the interface is markedly different between 100S from S. aureus and E. coli. Cryo-electron microscopy structure of 100S S. aureus ribosomes reveal that the SaHPF-NTD binds to the 30S subunit as observed for shorter variants of HPF in other species and the C-terminal domain (CTD) protrudes out of each ribosome in order to mediate dimerization. SaHPF-NTD binds to the small subunit similarly to its homologs EcHPF, EcYfiA, and a plastid-specific YfiA. Furthermore, upon binding to the small subunit, the SaHPF-NTD occludes several antibiotic binding sites at the A site (hygromycin B, tetracycline), P site (edeine) and E site (pactamycin, kasugamycin). In order to elucidate the structure, dynamics and function of SaHPF-NTD from S. aureus, here we report the backbone and side chain resonance assignments for SaHPF-NTD. Analysis of the backbone chemical shifts by TALOS+ suggests that SaHPF-NTD contains two α-helices and four ß-strands (ß1-α1-ß2-ß3-ß4-α2 topology). Investigating the long-term survival of S. aureus and other bacteria under antibiotic pressure could lead to advances in antibiotherapy.
Asunto(s)
Proteínas Bacterianas/química , Resonancia Magnética Nuclear Biomolecular , Proteínas Ribosómicas/química , Dominios ProteicosRESUMEN
Monitoring of pathogenic strains of Fusarium oxysporum (Fox), which cause wilt and rots on agricultural and ornamental plants, is important for predicting disease outbreaks. Since both pathogenic and non-pathogenic strains of Fox are ubiquitous and are able to colonize plant roots, detection of Fox DNA in plant material is not the ultimate proof of an ongoing infection which would cause damage to the plant. We followed the colonization of tomato plants by strains Fox f. sp. radicis-lycopersici ZUM2407 (a tomato foot and root rot pathogen), Fox f. sp. radiciscucumerinum V03-2g (a cucumber root rot pathogen) and Fox Fo47 (a well-known non-pathogenic biocontrol strain). We determined fungal DNA concentrations in tomato plantlets by quantitative PCR (qPCR) with primers complementary to the intergenic spacer region (IGS) of these three Fox strains. Two weeks after inoculation of tomato seedlings with these Fox strains, the DNA concentration of Forl ZUM2407 was five times higher than that of the non-compatible pathogen Forc V03-2g and 10 times higher than that of Fo47. In 3-week-old plantlets the concentration of Forl ZUM2407 DNA was at least 10 times higher than those of the other strains. The fungal DNA concentration, as determined by qPCR, appeared to be in good agreement with data of the score of visible symptoms of tomato foot and root rot obtained 3 weeks after inoculation of tomato with Forl ZUM2407. Our results show that targeting of the multicopy ribosomal operon results in a highly sensitive qPCR reaction for the detection of Fox DNA. Since formae speciales of Fox cannot be distinguished by comparison of ribosomal operons, detection of Fox DNA is not evidence of plant infection by a compatible pathogen. Nevertheless, the observed difference in levels of plant colonization between pathogenic and non-pathogenic strains strongly suggests that a concentration of Fox DNA in plant material above the threshold level of 0.005% is due to proliferation of pathogenic Fox.