RESUMO
Halomonas species are halophilic and alkaliphilic bacteria, which exhibit potential for industrial production of a variety of chemicals, such as polyhydroxyalkanoates and ectoine, by fermentation because of their favorable characteristics, including high-density culturing capacity and low risk of contamination. However, genetic tools to modify the metabolism of Halomonas for suitable fermentation performance are limited. In this study, we developed two independent basic vectors for Halomonas, named pUCpHAw and pHA1AT_32, consisting of ori regions from two plasmids isolated from Halomonas sp. A020, and chloramphenicol- and tetracycline-resistant genes as cloning markers, respectively. These vectors can independently transform and co-transform the Halomonas sp. KM-1 (KM-1). A protein that was highly and constitutively accumulated was identified as a hemolysin coregulated protein (Hcp) based on proteome analysis of KM-1. Using the hcp promoter, various genes, such as phaA and EGFP, were highly expressed. To establish a gene disruption system, the Streptococcus pyogenes cas9 gene and guide RNA for the pyrF gene, a yeast URA3 homologue, were expressed in pUCpHAw and pHA1AT_32, respectively. As a result, gene disruption mutants were isolated based on phenotypes, 5-fluoroorotic acid resistance, and uracil auxotrophy. A combination of KM-1 and these vectors could be a suitable platform for industrial chemical and protein production.
Assuntos
Halomonas , Poli-Hidroxialcanoatos , DNA , Genômica , Halomonas/genética , Halomonas/metabolismo , Plasmídeos/genética , Poli-Hidroxialcanoatos/metabolismoRESUMO
Thermotolerant strains are critical for low-cost high temperature fermentation. In this study, we carried out the thermal adaptation of A. pasteurianus IFO 3283-32 under acetic acid fermentation conditions using an experimental evolution approach from 37ºC to 40ºC. The adapted strain exhibited an increased growth and acetic acid fermentation ability at high temperatures, however, with the trade-off response of the opposite phenotype at low temperatures. Genome analysis followed by PCR sequencing showed that the most adapted strain had 11 mutations, a single 64-kb large deletion, and a single plasmid loss. Comparative phenotypic analysis showed that at least the large deletion (containing many ribosomal RNAs and tRNAs genes) and a mutation of DNA polymerase (one of the 11 mutations) critically contributed to this thermotolerance. The relationship between the phenotypic changes and the gene mutations are discussed, comparing with another thermally adapted A. pasteurianus strains obtained previously.
Assuntos
Acetobacter/fisiologia , Evolução Molecular , Genoma Bacteriano , Termotolerância , Ácido Acético/metabolismo , Acetobacter/genética , Acetobacter/metabolismo , Fermentação , MutaçãoRESUMO
Although pancreatic enzyme replacement therapy (PERT) is effective in the alleviation of pancreatic exocrine insufficiency (PEI)-related symptoms in patients with chronic pancreatitis, its mechanism of action is poorly understood. Recent studies suggest that the intestinal microbiota is associated with the pathogenesis of chronic pancreatitis. Therefore, we hypothesized that PERT exerts its effect by modifying the intestinal microbiota in addition to its presumed role in promoting fat and protein absorption. To explore the mechanism of action of PERT, we analyzed the intestinal microbiotas of two groups of mice treated with either pancrelipase or tap water by using 16S rRNA amplicon sequencing. The results revealed that the bacterial compositions of the pancrelipase-treated mice were significantly different from those of the control samples. Akkermansia muciniphila, a key beneficial bacterium in the intestinal tract, showed a higher relative abundance in the pancrelipase-treated samples than in the control samples. Lactobacillus reuteri, a widely used probiotic bacterium known to relieve intestinal inflammation, also showed a higher relative abundance in the pancrelipase-treated samples. These results suggested that PERT induces the colonization of beneficial bacteria, thereby contributing to the attenuation of PEI-associated symptoms in addition to improvement of the nutritional state.
Assuntos
Bactérias/citologia , Suplementos Nutricionais/microbiologia , Terapia de Reposição de Enzimas/métodos , Microbioma Gastrointestinal/fisiologia , Pâncreas/enzimologia , Pancrelipase/administração & dosagem , Administração Oral , Animais , Bactérias/classificação , Bactérias/efeitos dos fármacos , Fármacos Gastrointestinais , Microbioma Gastrointestinal/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Environmental adaptation is considered as one of the most challenging subjects in biology to understand evolutionary or ecological diversification processes and in biotechnology to obtain useful microbial strains. Temperature is one of the important environmental stresses; however, microbial adaptation to higher temperatures has not been studied extensively. For industrial purposes, the use of thermally adapted strains is important, not only to reduce the cooling expenses of the fermentation system, but also to protect fermentation production from accidental failure of thermal management. Recent progress in next-generation sequencing provides a powerful tool to track the genomic changes of the adapted strains and allows us to compare genomic DNA sequences of conventional strains with those of their closely related thermotolerant strains. In this article, we have attempted to summarize our recent approaches to produce thermotolerant strains by thermal adaptation and comparative genomic analyses of Acetobacter pasteurianus for high-temperature acetic acid fermentations, and Zymomonas mobilis and Kluyveromyces marxianus for high-temperature ethanol fermentations. Genomic analysis of the adapted strains has found a large number of mutations and/or disruptions in highly diversified genes, which could be categorized into groups related to cell surface functions, ion or amino acid transporters, and some transcriptional factors. Furthermore, several phenotypic and genetic analyses revealed that the thermal adaptation could lead to decreased ROS generation in cells that produce higher ROS levels at higher temperatures. Thus, it is suggested that the thermally adapted cells could become robust and resistant to many stressors, and thus could be useful for high-temperature fermentations.
Assuntos
Adaptação Fisiológica , Fermentação , Genoma Bacteriano , Genoma Fúngico , Temperatura Alta , Ácido Acético/metabolismo , Acetobacter/genética , Acetobacter/metabolismo , Acetobacter/fisiologia , Elementos de DNA Transponíveis , Kluyveromyces/genética , Kluyveromyces/metabolismo , Kluyveromyces/fisiologiaRESUMO
Chlamydia is an obligate intracellular bacterial pathogen that replicates solely within a membrane-bound vacuole termed an inclusion. Chlamydia seems to perturb multiple cellular processes of the host, such as, rearrangement of the membrane trafficking system for its intracellular multiplication, and inhibition of host cell apoptosis for persistent infection. In an attempt to clarify host factor involvement in apoptosis regulation, we found that inhibition of Caspase-9 restricted, while Apaf-1 promoted, Chlamydia pneumoniae infection in HEp-2, HeLa, and mouse epithelial fibroblast (MEF) cells. These opposition contributions to the chlamydial infection were confirmed using caspase-9 (-/-) and apaf-1 (-/-) MEFs. Similar phenomena also appeared in the case of infection with Chlamydia trachomatis. Interestingly, caspase-9 in apaf-1 (-/-) MEFs was activated by chlamydial infection but during the infection caspase-3 was not activated. That is, caspase-9 was activated without support for multiplication and activation by Apaf-1, and the activated caspase-9 may be physically disconnected from the caspase cascade. This may be partially explained by the observation of caspase-9 accumulation within chlamydial inclusions. The sequestration of caspase-9 by chlamydia seems to result in apoptosis repression, which is crucial for the chlamydial development cycle. Because Apaf-1 shares domains with intracellular innate immune receptor NOD1, it may play a key role in the strategy to regulate chlamydial infection.
Assuntos
Apoptose , Fator Apoptótico 1 Ativador de Proteases/genética , Caspase 9/genética , Infecções por Chlamydia/metabolismo , Chlamydia/metabolismo , Epistasia Genética , Animais , Fator Apoptótico 1 Ativador de Proteases/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Suscetibilidade a Doenças/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Interações Hospedeiro-Patógeno , Humanos , CamundongosRESUMO
Gluconacetobacter xylinus (formerly Acetobacter xylinum and presently Komagataeibacter medellinensis) is known to produce cellulose as a stable pellicle. However, it is also well known to lose this ability very easily. We investigated the on and off mechanisms of cellulose producibility in two independent cellulose-producing strains, R1 and R2. Both these strains were isolated through a repetitive static culture of a non-cellulose-producing K. medellinensis NBRC 3288 parental strain. Two cellulose synthase operons, types I and II, of this strain are truncated by the frameshift mutation in the bcsBI gene and transposon insertion in the bcsCII gene, respectively. The draft genome sequencing of R1 and R2 strains revealed that in both strains the bcsBI gene was restored by deletion of a nucleotide in its C-rich region. This result suggests that the mutations in the bcsBI gene are responsible for the on and off mechanism of cellulose producibility. When we looked at the genomic DNA sequences of other Komagataeibacter species, several non-cellulose-producing strains were found to contain similar defects in the type I and/or type II cellulose synthase operons. Furthermore, the phylogenetic relationship among cellulose synthase genes conserved in other bacterial species was analyzed. We observed that the cellulose genes in the Komagataeibacter shared sequence similarities with the γ-proteobacterial species but not with the α-proteobacteria and that the type I and type II operons could be diverged from a same ancestor in Komagataeibacter.
Assuntos
Adaptação Biológica , Vias Biossintéticas/genética , Celulose/biossíntese , Gluconacetobacter xylinus/genética , Gluconacetobacter xylinus/metabolismo , Evolução Molecular , Mutação da Fase de Leitura , Mutagênese Insercional , Óperon , Deleção de Sequência , Homologia de SequênciaRESUMO
Carboxyl group-donated silver (Ag) nanoparticles for coating on medical devices were prepared by a two-phase reduction system in situ. AgNO3 was the Ag ion source, tetraoctylammonium bromide [N(C8H17)4Br] the phase-transfer agent, sodium tetrahydroborate (NaBH4) the reducing agent and 10-carboxy-1-decanthiol (C11H22O2S, CDT) the capping agent. The characterizations of the Ag nanoparticles were conducted by diffuse reflectance Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric differential thermal analysis (TG/DTA) and transmission electron microscope. With CDT capped on Ag nanoparticles, we found that the band around 3,100 cm(-1) was attributed to COO-H stretching vibration, two adsorptions at 2,928 and 2,856 cm(-1) to C-H symmetric/anti-symmetric stretching vibration, and at 1,718 cm(-1) to C=O stretching vibration in the FT-IR spectra. The organic components of the carboxylated Ag nanoparticles were 5.8-25.9 wt%, determined by TG/DTA. The particle sizes of the carboxylated Ag nanoparticles were well controlled by the addition of the capping agent, CDT, into the reaction system. The antimicrobial activity of the Ag nanoparticles covered with different contents of CDT against E. coli was evaluated. Smaller-size Ag nanoparticles showed higher antibacterial activity, which depended on a surface area that attached easily to a microorganism cell membrane.
Assuntos
Antibacterianos/química , Ácidos Carboxílicos/química , Nanopartículas Metálicas/química , Prata/química , Biofilmes , Testes de Sensibilidade MicrobianaRESUMO
Halomonas species, which are aerobic, alkaliphilic, and moderately halophilic bacteria, produce diverse biochemicals. To identify food-related Halomonas strains for bioremediation and the industrial production of biochemicals, 20 strains were isolated from edible seashells, shrimp, and umeboshi (pickled Japanese plum) factory effluents. All isolates were phylogenetically classified into a large clade of Halomonas species. Most isolates, which grew in wide pH (6-13) and salt concentration (0-14%) ranges, exhibited the intracellular accumulation of poly(3-hydroxybutyrate) granules. The characteristics of these isolates varied. A020 isolated from umeboshi factory effluents exhibited enhanced stress tolerance and proliferation and comprised two plasmids. IMZ03 and A020 grew to more than 200 OD600, while IMZ03 produced 3.5% 3-hydroxybutyrate in inorganic medium supplemented with 10% sucrose. The mucus of TK1-1 cultured on agar medium comprised approximately 64| |mM of ectoine. Whole-genome sequencing of A020 was performed to elucidate its origin and genomic characteristics. The genome ana-lysis revealed a region exhibiting synteny with a large virus genome isolated from the ocean, but did not identify any predictable pathogenic genes. Therefore, saline foods and related materials may be suitable resources for isolating Halomonas strains exhibiting unique, useful, and innocuous features.
Assuntos
Halomonas , Biodegradação Ambiental , Alimentos , Halomonas/genética , Filogenia , Cloreto de SódioRESUMO
Four types of zinc (Zn)-doped hydroxyapatite (Zn-HAp) nanoparticles were prepared using calcium nitrate tetrahydrate as an anti-sintering agent during calcination at 600°C for 1 hr, to prevent calcination-induced aggregation. The Zn content of the nanopowders was determined at 0, 4.3, 9.2, and 14.7% [Zn/(Ca + Zn) × 100] using inductively coupled plasma atomic emission spectroscopic analysis. Based on X-ray diffraction analysis, the products were shown to possess an apatite structure without other crystalline impurities. The cell parameters of Zn-HAp nanoparticles decreased with increasing of Zn content in the HAp structures. This tendency implies that Zn ions substituted for Ca sites in the HAp crystal lattices. To investigate the biological effects of Zn-HAp nanoparticles, cell proliferation activity of MC3T3-E1 osteoblasts and antibacterial activity against Escherichia coli were evaluated in vitro. According to the results obtained, Zn-HAp nanoparticles containing of 14.7% Zn ions was noticeable shown shareability of the conflicting activities at 0.1 mg/mL.
Assuntos
Durapatita , Nanopartículas , Antibacterianos/química , Antibacterianos/farmacologia , Proliferação de Células , Durapatita/química , Durapatita/farmacologia , Nanopartículas/química , Difração de Raios X , Zinco/química , Zinco/farmacologiaRESUMO
Percutaneous devices-indwelling catheters-related infections are serious clinical incidents. It is accordingly necessary to develop anti-infective coating materials suitable for the devices for long-term effectiveness. In our research group, highly dispersible and crystalline hydroxyapatite (HAp) nanoparticles doped with metallic or halogen ions possessing antibacterial activities have been developed. In this study, antibacterial, dispersible, and crystalline zinc (Zn)-doped hydroxyapatite [Zn(15)-HAp] nanoparticles substituted with 13.5% Zn content [Zn/(Zn + Ca) × 100] were prepared by a wet chemical method using an anti-sintering agent through calcination. Antibacterial activities of Zn(15)-HAp nanoparticles were evaluated using Escherichia coli (E. coli) and Staphylococcus aureus. The survival rates of the bacteria on Zn(15)-HAp nanoparticles were significantly lower than that on normal HAp (nHAp) coated surfaces, while no influences were observed on proliferation of L929 cells. Even after soaking Zn(15)-HAp nanoparticles in PBS for 2 weeks, the antibacterial activities against E. coli were maintained at a similar level to a 20 min soaking. The bacterial death was related to not only ion-exchange phenomenon between Zn and magnesium ions but also accumulation of reactive oxygen species (ROS) in the cells. Allergic-like reactions-anaphylactoid reactions-might not readily occur with Zn(15)-HAp nanoparticles because the amounts of histamine released from HMC-1 cells co-cultured with nanoparticles were not significantly different to that of nHAp, but were statistically much lower than that of chlorhexidine.
Assuntos
Durapatita , Nanopartículas , Antibacterianos/química , Antibacterianos/farmacologia , Clorexidina , Materiais Revestidos Biocompatíveis/química , Durapatita/química , Escherichia coli , Halogênios , Histamina , Íons , Magnésio/química , Nanopartículas/química , Pirenos , Espécies Reativas de Oxigênio , Zinco/químicaRESUMO
Aim: To clarify whether there is any association between the extent of Chlamydia pneumoniae (C. pneumoniae) infection and plaque instability or post-directional coronary atherectomy (DCA) restenosis, we determined the frequency of C. pneumoniae infection and its localization in symptomatic coronary atherosclerotic plaques using specimens obtained from DCA. Methods and results: Immunohistochemistry (IHC) and real-time polymerase chain reaction (RT-PCR) revealed the existence of C. pneumoniae in all 50 specimens of coronary atherosclerotic plaques obtained by DCA. C. pneumoniae-positive cell ratio determined with IHC or copy numbers of C. pneumoniae DNA detected by RT-PCR did not differ significantly between patients with stable angina pectoris and those with acute coronary syndrome (IHC: 16.4 ± 7.6% vs 18.0 ± 7.1%, P = .42; RT-PCR: no. of cases with high copy numbers 12/25 vs 10/25, P = .78), or between patients with subsequent post-DCA restenosis and those without (IHC: 17.1 ± 8.0% vs 18.0 ± 7.4%, P = .74; RT-PCR: 5/12 vs 10/21, P = 1.00). Conclusions: C. pneumoniae was highly prevalent in coronary atherosclerotic plaques of patients who underwent DCA. However, the extent of C. pneumoniae infection in coronary atherosclerotic plaques was not associated with plaque instability or post-DCA restenosis.
RESUMO
Gluconacetobacter xylinus is involved in the industrial production of cellulose. We have determined the genome sequence of G. xylinus NBRC 3288, a cellulose-nonproducing strain. Comparative analysis of genomes of G. xylinus NBRC 3288 with those of the cellulose-producing strains clarified the genes important for cellulose production in Gluconacetobacter.
Assuntos
Ácido Acético/análise , Celulose/biossíntese , Condimentos/microbiologia , Genoma Bacteriano , Gluconacetobacter xylinus/genética , Sequência de Bases , Gluconacetobacter xylinus/isolamento & purificação , Gluconacetobacter xylinus/metabolismo , Dados de Sequência MolecularRESUMO
RhoH is an atypical small G protein with defective GTPase activity that is specifically expressed in hematopoietic lineage cells. RhoH has been implicated in regulation of several physiological processes including hematopoiesis, integrin activation, and T cell differentiation and activation. In the present study, we investigated the role of RhoH in mast cells by generating RhoH knockout mice. Despite observing normal development of mast cells in vivo, passive systemic anaphylaxis and histamine release were impaired in these mice. We also observed defective degranulation and cytokine production upon FcepsilonRI ligation in RhoH-deficient bone marrow-derived mast cells. Furthermore, FcepsilonRI-dependent activation of Syk and phosphorylation of its downstream targets, including LAT, SLP76, PLCgamma1, and PLCgamma2 were impaired, however phosphorylation of the gamma-subunit of FcepsilonRI remained intact. We also found RhoH-Syk association that was greatly enhanced by active Fyn. Our results indicate that RhoH regulates FcepsilonRI signaling in mast cells by facilitating Syk activation, possibly as an adaptor molecule for Syk.
Assuntos
Mastócitos/enzimologia , Mastócitos/imunologia , Receptores de IgE/fisiologia , Transdução de Sinais/imunologia , Fatores de Transcrição/fisiologia , Proteínas rho de Ligação ao GTP/fisiologia , Animais , Degranulação Celular/genética , Degranulação Celular/imunologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Ativação Enzimática/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mastócitos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Anafilaxia Cutânea Passiva/genética , Anafilaxia Cutânea Passiva/imunologia , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Proteínas rho de Ligação ao GTP/biossíntese , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Acetobacter species have been used for brewing traditional vinegar and are known to have genetic instability. To clarify the mutability, Acetobacter pasteurianus NBRC 3283, which forms a multi-phenotype cell complex, was subjected to genome DNA sequencing. The genome analysis revealed that there are more than 280 transposons and five genes with hyper-mutable tandem repeats as common features in the genome consisting of a 2.9-Mb chromosome and six plasmids. There were three single nucleotide mutations and five transposon insertions in 32 isolates from the cell complex. The A. pasteurianus hyper-mutability was applied for breeding a temperature-resistant strain grown at an unviable high-temperature (42 degrees C). The genomic DNA sequence of a heritable mutant showing temperature resistance was analyzed by mutation mapping, illustrating that a 92-kb deletion and three single nucleotide mutations occurred in the genome during the adaptation. Alpha-proteobacteria including A. pasteurianus consists of many intracellular symbionts and parasites, and their genomes show increased evolution rates and intensive genome reduction. However, A. pasteurianus is assumed to be a free-living bacterium, it may have the potentiality to evolve to fit in natural niches of seasonal fruits and flowers with other organisms, such as yeasts and lactic acid bacteria.
Assuntos
Acetobacter/genética , Genoma Bacteriano , Instabilidade Genômica , Acetobacter/metabolismo , Adaptação Fisiológica/genética , Sequência de Bases , Elementos de DNA Transponíveis , DNA Bacteriano/química , Variação Genética , Genômica , Genótipo , Temperatura Alta , Dados de Sequência Molecular , Mutação , Fenótipo , Plasmídeos/genética , Sequências de Repetição em TandemRESUMO
We developed a specific and rapid detection system for Rickettsia japonica and R. heilongjiangensis, the causative agents of spotted fever, using a TaqMan minor groove binder probe for a particular open reading frame (ORF) identified by the R. japonica genome project. The target ORF was present only in R. japonica-related strains.
Assuntos
Rickettsia/isolamento & purificação , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Rickettsia/genética , Infecções por Rickettsia/diagnósticoRESUMO
Rac1, one of the Rho family small guanosine triphosphatases, has been shown to work as a "molecular switch" in various signal transduction pathways. To assess the function of Rac1 in the differentiation process of CD4 single-positive (CD4-SP) T cells from CD4CD8 double-positive (DP) cells, we used a DP cell line DPK, which can differentiate into CD4-SP cells upon TCR stimulation in vitro. DPK expressing dominant-negative (dn)Rac1 underwent massive apoptosis upon TCR stimulation and resulted in defective differentiation of CD4-SP cells. Conversely, overexpression of dnRac2 did not affect differentiation. TCR-dependent actin polymerization was inhibited, whereas early ERK activation was unaltered in dnRac1-expressing DPK. We found that TCR-dependent induction of Bcl-2 was suppressed greatly in dnRac1-expressing DPK, and this suppression was independent of actin rearrangement. Furthermore, introduction of exogenous Bcl-2 inhibited TCR-dependent induction of apoptosis and restored CD4-SP generation in dnRac1-expressing DPK without restoring TCR-induced actin polymerization. Collectively, these data indicate that Rac1 is critical in differentiation of CD4-SP from the DP cell line by preventing TCR-induced apoptosis via Bcl-2 up-regulation.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Timo/imunologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Apoptose/imunologia , Diferenciação Celular/imunologia , Linhagem Celular , Humanos , Timo/citologiaRESUMO
Chlamydia pneumoniae is an obligate intracellular pathogen responsible for respiratory diseases, including pneumonia and bronchitis, and is highly involved in chronic diseases, including atherosclerosis, asthma, and Alzheimer's disease. We previously showed that the host apoptotic factor caspase-9 played a crucial role for chlamydial multiplication and host apoptosis inhibition by chlamydial infection. To identify chlamydial genes interacting with human caspase-9, yeast two-hybrid screening was performed and 5 chlamydial genes, including Cpj0838 and pmpG were isolated from the C. pneumoniae genomic library. Pull-down experiments showed that caspase-9 physically bound to the Cpj0838 product and chlamydial cells, which contain PmpG proteins. This study could provide a clue to understanding host-Chlamydia interactions, especially the apoptosis repression by Chlamydia infection.
Assuntos
Apoptose , Proteínas da Membrana Bacteriana Externa/metabolismo , Caspase 9/metabolismo , Infecções por Chlamydophila/microbiologia , Chlamydophila pneumoniae/genética , Proteínas da Membrana Bacteriana Externa/genética , Chlamydophila pneumoniae/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Técnicas do Sistema de Duplo-HíbridoRESUMO
A group of genes thought to encode members of the unique chlamydial polymorphic membrane protein (pmp) family were recently described in the Chlamydophila felis genome. This study aimed to commence characterisation of a subset of 12 of these putative pmp genes by developing and using gene-specific real-time (Q)PCR assays to confirm their presence in a wide range of C. felis field isolates and laboratory strains, and to look for pmp mRNA expression during in vitro infection. Sequencing of 525-698 base pair regions of pmp genes 7, 9-11, 13-20 for two laboratory strains of C. felis and alignment with the published Fe/C-56 sequence found only a single nucleotide polymorphism present in pmp9. Following the development of gene-specific (Q)PCR assays, analysis of genomic DNA extracted from 40 C. felis field isolates and 4 laboratory strains found that all 12 pmp genes were represented in all cases. Reverse transcription (RT)-QPCR analysis of RNA extracted from cell cultures at 24 and 48 h post inoculation with 1 of 5 different strains of C. felis detected transcripts for all 12 pmp genes at both time points. Analysis of the relative levels of pmp gene transcription suggested that down-regulation of the expression of multiple C. felis pmp genes occurs between 24 and 48 h post inoculation. This study provides the first evidence that 12 of the putative pmp C. felis genes are transcribed during in vitro infection, and shows that these genes are present in a large range of C. felis field isolates and multiple passage laboratory-grown strains.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Doenças do Gato/microbiologia , Infecções por Chlamydophila/veterinária , Chlamydophila/genética , Polimorfismo de Nucleotídeo Único , Transcrição Gênica , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Gatos , Linhagem Celular , Infecções por Chlamydophila/microbiologia , DNA Bacteriano/química , Regulação para Baixo , Gliceraldeído-3-Fosfato Desidrogenases/genética , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , RNA Bacteriano/química , RNA Bacteriano/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Alinhamento de SequênciaRESUMO
BACKGROUND: Thirteen patients with chlorhexidine-silver sulfadiazine-impregnated catheters have experienced serious anaphylactic shock in Japan. These adverse reactions highlight the lack of commercially available catheters impregnated with strong antibacterial chemical agents. A system should be developed that can control both biocompatibility and antibacterial activity. SUMMARY: Hydroxyapatite (HAp) is biocompatible with bone and skin tissues. To provide antibacterial activity by using an external physical stimulus, titanium (Ti) ions were doped into the HAp structure. Highly dispersible, Ti-doped HAp (Ti-HAp) nanoparticles suitable as a coating material were developed. In 3 kinds of Ti-HAp [Ti/(Ca + Ti) = 0.05, 0.1, 0.2], the Ti content in the HAp was approximately 70% of that used in the Ti-HAp preparation, as determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES). ICP-AES and X-ray diffraction showed Ti ions were well substituted into the HAp lattice. The nanoparticles were almost uniformly coated on a polyethylene (PE) sheet in a near-monolayer with a surface coverage ratio >95%. The antibacterial activity of the Ti-HAp nanoparticles containing 7.3% Ti ions and coating the sheet was evaluated by calculating the survival ratio of Pseudomonas aeruginosa on the coated sheet after ultraviolet (UV) irradiation. The Ti-HAp-coated sheet showed a 50% decrease in the number of P. aeruginosa compared with that on an uncoated control PE sheet after UV irradiation for 30 s. Key Messages: A system of biocompatibility and antibacterial activity with an on/off switch controlled by external UV stimulation was developed. The system is expected to be applicable in long-term implanted intravascular catheters.
Assuntos
Antibacterianos/farmacologia , Catéteres , Materiais Revestidos Biocompatíveis/química , Nanopartículas/química , Pseudomonas aeruginosa/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/efeitos da radiação , Durapatita/química , Humanos , Teste de Materiais , Nanopartículas/efeitos da radiação , Polietileno/química , Pseudomonas aeruginosa/citologia , Titânio/química , Raios UltravioletaRESUMO
Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 µg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides.