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1.
Infect Immun ; : IAI0031521, 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34543119

RESUMO

Mycobacterium tuberculosis is a chronic infectious disease pathogen. To date, tuberculosis is a major infectious disease that endangers human health. To better prevent and treat tuberculosis, it is important to study the pathogenesis of M. tb. Based on early-stage laboratory research results, in this study, we verified the upregulation of sod2 in Bacillus Calmette-Guérin (BCG) and H37Rv infection. By detecting BCG/H37Rv intracellular survival in sod2-silenced and sod2- overexpressing macrophages, sod2 was found to promote the intracellular survival of BCG/H37Rv. Then, miR-495 was determined to be downregulated by BCG/H37Rv. BCG/H37Rv can upregulate sod2 expression by miR-495 to promote the intracellular survival of BCG/H37Rv through a decline in ROS levels. This study provides a theoretical basis for developing new drug targets and treating tuberculosis.

2.
J Microbiol ; 59(9): 854-860, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34382147

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) is an important zoonotic pathogen that places severe burdens on public health and animal husbandry. There are many pathogenic factors in E. coli. The type VI secretion system (T6SS) is a nano-microbial weapon that can assemble quickly and inject toxic effectors into recipient cells when danger is encountered. T6SSs are encoded in the genomes of approximately 25% of sequenced Gram-negative bacteria. When these bacteria come into contact with eukaryotic cells or prokaryotic microbes, the T6SS assembles and secretes associated effectors. In the porcine ExPEC strain PCN033, we identified four classic rearrangement hotspot (Rhs) genes. We determined the functions of the four Rhs proteins through mutant construction and protein expression. Animal infection experiments showed that the Δrhs-1CT, Δrhs-2CT, Δrhs-3CT, and Δrhs-4CT caused a significant decrease in the multiplication ability of PCN033 in vivo. Cell infection experiments showed that the Rhs protein is involved in anti-phagocytosis activities and bacterial adhesion and invasion abilities. The results of this study demonstrated that rhs1, rhs3, and rh4 plays an important role in the interaction between PCN033 and host cell. Rhs2 has contribution to cell and mice infection. This study helps to elucidate the pathogenic mechanism governing PCN033 and may help to establish a foundation for further research seeking to identify potential T6SS effectors.


Assuntos
Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli/metabolismo , Doenças dos Suínos/microbiologia , Animais , Aderência Bacteriana , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/metabolismo , Feminino , Intestinos/microbiologia , Camundongos , Família Multigênica , Suínos
3.
Int J Mol Sci ; 22(12)2021 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-34198513

RESUMO

BACKGROUND: Pulmonary disease caused by Mycobacterium abscessus (M. abscessus) spreads around the world, and this disease is extremely difficult to treat due to intrinsic and acquired resistance of the pathogen to many approved antibiotics. M. abscessus is regarded as one of the most drug-resistant mycobacteria, with very limited therapeutic options. METHODS: Whole-cell growth inhibition assays was performed to screen and identify novel inhibitors. The IC50 of the target compounds were tested against THP-1 cells was determined to calculate the selectivity index, and then time-kill kinetics assay was performed against M. abscessus. Subsequently, the synergy of oritavancin with other antibiotics was evaluated by using checkerboard method. Finally, in vivo efficacy was determined in an immunosuppressive murine model simulating M. abscessus infection. RESULTS: We have identified oritavancin as a potential agent against M. abscessus. Oritavancin exhibited time-concentration dependent bactericidal activity against M. abscessus and it also displayed synergy with clarithromycin, tigecycline, cefoxitin, moxifloxacin, and meropenem in vitro. Additionally, oritavancin had bactericidal effect on intracellular M. abscessus. Oritavancin significantly reduced bacterial load in lung when it was used alone or in combination with cefoxitin and meropenem. CONCLUSIONS: Our in vitro and in vivo assay results indicated that oritavancin may be a viable treatment option against M. abscessus infection.


Assuntos
Antibacterianos/uso terapêutico , Lipoglicopeptídeos/uso terapêutico , Infecções por Mycobacterium não Tuberculosas/tratamento farmacológico , Infecções por Mycobacterium não Tuberculosas/microbiologia , Mycobacterium abscessus/fisiologia , Animais , Antibacterianos/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Humanos , Imunossupressão , Espaço Intracelular/microbiologia , Lipoglicopeptídeos/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium abscessus/efeitos dos fármacos , Mycobacterium tuberculosis/efeitos dos fármacos , Células THP-1
4.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072443

RESUMO

As an important zoonotic pathogen, Streptococcus suis (S. suis) infection has been reported to be a causative agent for variety of diseases in humans and animals, especially Streptococcal toxic shock-like syndrome (STSLS), which is commonly seen in cases of severe S. suis infection. STSLS is often accompanied by excessive production of inflammatory cytokines, which is the main cause of death. This calls for development of new strategies to avert the damage caused by STSLS. In this study, we found for the first time that Baicalein, combined with ampicillin, effectively improved severe S. suis infection. Further experiments demonstrated that baicalein significantly inhibited the hemolytic activity of SLY by directly binding to SLY and destroying its secondary structure. Cell-based assays revealed that Baicalein did not exert toxic effects and conferred protection in S. suis-infected cells. Interestingly, compared with ampicillin alone, Baicalein combined with ampicillin resulted in a higher survival rate in mice severely infected with S. suis. At the same time, we found that baicalein can be combined with meropenem against MRSA. In conclusion, these results indicate that baicalein has a good application prospect.


Assuntos
Antibacterianos/farmacologia , Flavanonas/farmacologia , Infecções Estreptocócicas/microbiologia , Streptococcus suis/efeitos dos fármacos , Animais , Antibacterianos/química , Citocinas/biossíntese , Modelos Animais de Doenças , Farmacorresistência Bacteriana/efeitos dos fármacos , Quimioterapia Combinada , Flavanonas/química , Hemólise/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/metabolismo , Infecções Estreptocócicas/patologia , Relação Estrutura-Atividade
5.
Molecules ; 26(7)2021 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-33915741

RESUMO

As an important zoonotic pathogen, Streptococcus suis (S. suis) can cause a variety of diseases both in human and animals, especially Streptococcal toxic shock-like syndrome (STSLS), which commonly appears in severe S. suis infection. STSLS is often accompanied by excessive production of inflammatory cytokines, which is the main cause of host death. Therefore, it is urgent to find a new strategy to relieve the damage caused by STSLS. In this study, we found, for the first time, that apigenin, as a flavonoid compound, could combine with ampicillin to treat severe S. suis infection. Studies found that apigenin did not affect the growth of S. suis and the secretion of suilysin (SLY), but it could significantly inhibit the hemolytic activity of SLY by directly binding to SLY and destroying its secondary structure. In cell assays, apigenin was found to have no significant toxic effects on effective concentrations, and have a good protective effect on S. suis-infected cells. More importantly, compared with the survival rate of S. suis-infected mice treated with only ampicillin, the survival rate of apigenin combined with an ampicillin-treated group significantly increased to 80%. In conclusion, all results indicate that apigenin in combination with conventional antibiotics can be a potential strategy for treating severe S. suis infection.


Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Apigenina/farmacologia , Infecções Estreptocócicas/tratamento farmacológico , Infecções Estreptocócicas/microbiologia , Streptococcus suis/efeitos dos fármacos , Ampicilina/química , Ampicilina/uso terapêutico , Animais , Antibacterianos/química , Apigenina/química , Apigenina/uso terapêutico , Sítios de Ligação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Quimioterapia Combinada , Eritrócitos/efeitos dos fármacos , Proteínas Hemolisinas/antagonistas & inibidores , Proteínas Hemolisinas/química , Interações Hospedeiro-Patógeno , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Testes de Sensibilidade Microbiana , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ligação Proteica , Infecções Estreptocócicas/diagnóstico , Infecções Estreptocócicas/metabolismo , Relação Estrutura-Atividade , Resultado do Tratamento
6.
Appl Environ Microbiol ; 87(10)2021 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-33674433

RESUMO

Streptococcal toxic shock-like syndrome (STSLS) caused by the epidemic strain of Streptococcus suis leads to severe inflammation and high mortality. The life and health of humans and animals are also threatened by the increasingly severe antimicrobial resistance in Streptococcus suis There is an urgent need to discover novel strategies for the treatment of S. suis infection. Suilysin (SLY) is considered to be an important virulence factor in the pathogenesis of S. suis In this study, ellipticine hydrochloride (EH) was reported as a compound that antagonizes the hemolytic activity of SLY. In vitro, EH was found to effectively inhibit SLY-mediated hemolytic activity. Furthermore, EH had a strong affinity for SLY, thereby directly binding to SLY to interfere with the hemolytic activity. Meanwhile, it was worth noting that EH was also found to have a significant antibacterial activity. In vivo, compared with traditional ampicillin, EH not only significantly improved the survival rate of mice infected with S. suis 2 strain Sc19 but also relieved lung pathological damage. Furthermore, EH effectively decreased the levels of inflammatory cytokines (interleukin-6 [IL-6], tumor necrosis factor alpha [TNF-α]) and blood biochemistry enzymes (alanine transaminase [ALT], aspartate transaminase [AST], creatine kinase [CK]) in Sc19-infected mice. Additionally, EH markedly reduced the bacterial load of tissues in Sc19-infected mice. In conclusion, our findings suggest that EH can be a potential compound for treating S. suis infection in view of its antibacterial and antihemolysin activity.IMPORTANCE In recent years, the inappropriate use of antibiotics has unnecessarily caused the continuous emergence of resistant bacteria. The antimicrobial resistance of Streptococcus suis has also become an increasingly serious problem. Targeting virulence can reduce the selective pressure of bacteria on antibiotics, thereby alleviating the development of bacterial resistance to a certain extent. Meanwhile, the excessive inflammatory response caused by S. suis infection is considered the primary cause of acute death. Here, we found that ellipticine hydrochloride (EH) exhibited effective antibacterial and antihemolysin activities against S. suis in vitro In vivo, compared with ampicillin, EH had a significant protective effect on S. suis serotype 2 strain Sc19-infected mice. Our results indicated that EH, with dual antibacterial and antivirulence effects, will contribute to treating S. suis infections and alleviating the antimicrobial resistance of S. suis to a certain extent. More importantly, EH may develop into a promising drug for the prevention of acute death caused by excessive inflammation.


Assuntos
Antibacterianos/uso terapêutico , Proteínas de Bactérias/metabolismo , Elipticinas/uso terapêutico , Proteínas Hemolisinas/metabolismo , Infecções Estreptocócicas/tratamento farmacológico , Streptococcus suis , Fatores de Virulência/metabolismo , Animais , Antibacterianos/farmacologia , Citocinas/sangue , Modelos Animais de Doenças , Elipticinas/farmacologia , Feminino , Hemólise/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Infecções Estreptocócicas/sangue , Streptococcus suis/efeitos dos fármacos , Streptococcus suis/crescimento & desenvolvimento , Streptococcus suis/metabolismo
7.
ACS Synth Biol ; 10(4): 756-765, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33755417

RESUMO

Menaquinone-7 (MK-7) possesses wide health and medical value, and the market demand for MK-7 has increased. Metabolic engineering for MK-7 production in Escherichia coli still remains challenging due to the characteristics of the competing quinone synthesis, and cells mainly synthesized menaquinones under anaerobic conditions. To increase the production of MK-7 in engineered E. coli strains under aerobic conditions, we divided the whole MK-7 biosynthetic pathway into three modules (MVA pathway, DHNA pathway, and MK-7 pathway) and systematically optimized each of them. First, by screening and enhancing Idi expression, the amounts of MK-7/DMK-7 increased significantly. Then, in the MK-7 pathway, by combinatorial overexpression of endogenous MenA and exogenous UbiE, and fine-tuning the expression of HepPPS, MenA, and UbiE, 70 µM MK-7 was achieved. Third, the DHNA synthetic pathway was enhanced, and 157 µM MK-7 was achieved. By the combinational metabolic engineering strategies and membrane engineering, an efficient metabolic engineered E. coli strain for MK-7 synthesis was developed, and 200 µM (129 mg/L) MK-7 was obtained in shake flask experiment, representing a 306-fold increase compared to the starting strain. In the scale-up fermentation, 2074 µM (1350 mg/L) MK-7 was achieved after 52 h fermentation with a productivity of 26 mg/L/h. This is the highest titer of MK-7 ever reported. This study offers an alternative method for MK-7 production from biorenewable feedstock (glucose) by engineered E. coli. The high titer of our process should make it a promising cost-effective resource for MK-7.

8.
Mol Carcinog ; 59(12): 1392-1408, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33079409

RESUMO

Countless evidence suggests that long noncoding RNAs (lncRNAs) are involved in human malignant cancers, including esophageal squamous cell carcinoma (ESCC), although their exact function remains unclear. In the present study, we aimed to investigate the roles and molecular mechanisms of the lncRNA LOC440173 in ESCC progression. Microarray analysis and quantitative real-time polymerase chain reaction were conducted to measure the expression levels of LOC440173 and miR-30d-5p. The biological function of this lncRNA was investigated using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, clone formation, and transwell assays, as well as flow cytometry and Western blot analysis. The function of LOC440173 was validated in vivo using tumor xenografts. The regulatory network of LOC440173/miR-30d-5p/HDAC9 was established using bioinformatic analysis and verified with dual-luciferase reporter assays, RNA immunoprecipitation assay, and rescue experiments. The expression level of LOC440173 was significantly increased in ESCC tissues and esophageal carcinoma cells. High LOC440173 expression was correlated with histological grade, tumor invasion depth, lymph node metastasis, and TNM stage. Overexpression of LOC440173 promoted esophageal cancer cell proliferation, migration, and invasion, as well as the epithelial-mesenchymal transition (EMT) process in vitro, and facilitated tumor growth in vivo. MicroRNA-30d-5p (miR-30d-5p) was downregulated in ESCC tissues and acted as a direct binding target of LOC440173 during the regulation of HDAC9 expression in esophageal carcinoma cells. In conclusion, LOC440173 exerts a promotive role in ESCC tumorigenesis by targeting the miR-30d-5p/HDAC9 axis and regulating the EMT process. LOC440173 might be a new therapeutic target for the treatment of ESCC.


Assuntos
Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Histona Desacetilases/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Idoso , Animais , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Progressão da Doença , Transição Epitelial-Mesenquimal , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Regulação para Cima
9.
Arch Biochem Biophys ; 692: 108522, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32781051

RESUMO

About one quarter of people worldwide are infected with tuberculosis, and multi-drug resistant tuberculosis (MDR-TB) remains a health threat. It is known that two-Component Signal Transduction Systems (TCSs) of Mycobacterium tuberculosis are closely related to tuberculosis resistance, but the mechanism by which orphan response protein Rv3143 regulates strain sensitivity to drug is still unclear. This study found that Rv3143 overexpression resulted in approximately two-fold increase in Mycobacterium smegmatis antibiotic sensitivity. Transcriptome sequencing indicated that 198 potential genes were regulated by Rv3143, affecting the sensitivity of the strain to rifampicin (RIF). MSMEG_4740 promoter binding with Rv3143, was screened out by surface plasmon resonance (SPR). Rv1524, the homologous gene of MSMEG_4740, belonging to the glycosyltransferase (Gtf) family, was related to cell wall modification. By measuring ethidium bromide (EB) accumulation, we found when Rv3143 or MSMEG_4740, or Rv1524 was overexpressed, the cell wall permeability of Mycobacterium smegmatis was increased. In addition, a combination of Rv3143 and RIF was observed. Our findings provide a new strategy for treating drug-resistant tuberculosis by increasing the expression of Rv3143 to enhance the strain sensitivity to antibiotics.


Assuntos
Proteínas de Bactérias/metabolismo , Parede Celular/metabolismo , Farmacorresistência Bacteriana , Mycobacterium smegmatis/metabolismo , Proteínas de Bactérias/genética , Parede Celular/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Mycobacterium smegmatis/genética , Permeabilidade/efeitos dos fármacos , Rifampina/farmacologia , Transcriptoma/efeitos dos fármacos
10.
ACS Synth Biol ; 9(9): 2610-2615, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786359

RESUMO

Developing sustainable agricultural practices will require increasing our understanding of plant-microbe interactions. To study these interactions, new genetic tools for manipulating nonmodel microbes will be needed. To help meet this need, we recently reported development of chassis-independent recombinase-assisted genome engineering (CRAGE). CRAGE relies on cassette exchange between two pairs of mutually exclusive lox sites and allows direct, single-step chromosomal integration of large, complex gene constructs into diverse bacterial species. We then extended CRAGE by introducing a third mutually exclusive lox site, creating CRAGE-Duet, which allows modular integration of two constructs. CRAGE-Duet offers advantages over CRAGE, especially when a cumbersome recloning step is required to build single-integration constructs. To demonstrate the utility of CRAGE-Duet, we created a set of strains from the plant-growth-promoting rhizobacterium Pseudomonas simiae WCS417r that expressed various fluorescence marker genes. We visualized these strains simultaneously under a confocal microscope, demonstrating the usefulness of CRAGE-Duet for creating biological systems to study plant-microbe interactions.

11.
J Cell Mol Med ; 24(16): 8962-8976, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32583631

RESUMO

Long non-coding RNAs (lncRNAs) have been well demonstrated to emerge as crucial regulators in cancer progression, and they can function as regulatory network based on their interactions. Although the biological functions of FAM83H-AS1 have been confirmed in various tumour progressions, the underlying molecular mechanisms of FAM83H-AS1 in oesophageal squamous cell carcinoma (ESCC) remained poorly understood. To address this, we treated human oesophageal cancer cell line Eca109 cells with TGF-ß and found FAM83H-AS1 was notably overexpressed. In the present study, FAM83H-AS1 was observed to be significantly up-regulated in ESCC tissues and was associated with TNM stage, pathological differentiation and lymph node metastasis. FAM83H-AS1 reinforced oesophageal cancer cell proliferation, migration and invasion, and participated in epithelial-to-mesenchymal transition (EMT) process at mRNA and protein levels. In addition, a concordant regulation between FAM83H-AS1 and its sense strand FAM83H was detected at the transcriptional and translational levels. Furthermore, FAM83H-AS1 could act as competing endogenous RNA to affect the expression of Girdin by sponging miR-10a-5p verified by RIP and luciferase reporter assays. Consequently, the study provided a unique perspective of FAM83H-AS1 in ESCC progression, which may be considered as potential biomarker and therapeutic target for ESCC therapy.


Assuntos
Carcinoma de Células Escamosas do Esôfago/genética , MicroRNAs/genética , Proteínas dos Microfilamentos/genética , Proteínas/genética , Proteínas de Transporte Vesicular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metástase Linfática/genética , Metástase Linfática/patologia , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , RNA Mensageiro/genética , Regulação para Cima/genética
12.
BMC Pediatr ; 20(1): 288, 2020 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-32517812

RESUMO

BACKGROUND: Secondary hemophagocytic lymphohistiocytosis (HLH) is a rare hyperinflammatory syndrome that requires prompt diagnosis and appropriate treatment. A risk-stratification model that could be used to identify high-risk pediatric patients with HLH who should be considered for second-line therapies, including salvage regimens and allogeneic hematopoietic cell transplantation (HCT), was developed. METHODS: The medical records of 88 pediatric patients (median age 1.4 years, range 0.2-15 years) with non-malignancy associated secondary HLH were retrospectively reviewed. Treatment strategies included dexamethasone, etoposide, and cyclosporine. RESULTS: Survival analysis showed HLH patients with infections other than Epstein-Barr virus (EBV) and unknown causes experienced better 5-year overall survival (OS) than patients with HLH due to autoimmune disease, EBV or immunodeficiency (76% vs. 65, 33.3, 11%, p < 0.001). On multivariate analysis, among all patients, non-response at 8 weeks was the most powerful predictor of poor OS. When treatment response was excluded, hemoglobin < 60 g/L and albumin < 25 g/L at diagnosis were associated with poor OS. In patients with EBV-HLH, hemoglobin < 60 g/L at diagnosis was associated with poor OS. A prognostic risk score was established and weighted based on hazard ratios calculated for three parameters measured at diagnosis: hemoglobin < 60 g/L (2 points), platelets < 30 × 109/L (1 point), albumin < 25 g/L (2 points). Five-year OS of low-risk (score 0-1), intermediate-risk (score 2), and poor-risk (score ≥ 3) patients were 88, 38, and 22%, respectively (p < 0.001). CONCLUSIONS: These findings indicate that clinicians should be aware of predictive factors at diagnosis and consider 8-week treatment response to identify patients with high-risk of disease progression and the need for second-line therapy and allogeneic HCT.


Assuntos
COVID-19 , Infecções por Vírus Epstein-Barr , Linfo-Histiocitose Hemofagocítica , Neoplasias , Adolescente , Criança , Pré-Escolar , Herpesvirus Humano 4 , Humanos , Lactente , Linfo-Histiocitose Hemofagocítica/diagnóstico , Linfo-Histiocitose Hemofagocítica/etiologia , Linfo-Histiocitose Hemofagocítica/terapia , Prognóstico , Estudos Retrospectivos , SARS-CoV-2 , Síndrome de Resposta Inflamatória Sistêmica , Resultado do Tratamento
13.
Front Microbiol ; 11: 806, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32528422

RESUMO

Extraintestinal pathogenic Escherichia coli (ExPEC) strains are the cause of a majority of human extraintestinal infections globally, resulting in enormous direct economic and medical costs. The plasmid-mediated, colistin-resistant gene mcr-1 has broken through the ultimate defense line against MDR Gram-negative pathogens. There is an urgent need to discover the new compound intended for colistin-resistant E. coli. In this study, antibacterial targets of ellipticine hydrochloride (EH) were confirmed by localized surface plasmon resonance (LSPR) and decatenation assay. The LSPR analysis exhibited good binding between EH and E. coli topoisomerase IV. In this study, a synergistic effect is obvious in the combination of EH and colistin, to which eight of ten strains showed synergy, while two isolates (20%) showed no difference. The bacteria enumeration analysis of EH treatment group suggested that the decreased bacterial titer can be observed in various tissues of infected mice. EH treatment significantly decreased the levels of a variety of pro-inflammatory factors, such as TNF-α and IL-6. Moreover, other related lesions, such as inflammatory cell infiltration, alveolar interstitial congestion, and edema were observed to be relieved to different extents. This study reveals the anti-E. coli potential activities and molecular mechanism of EH and the therapeutical effectiveness of EH application to animals. It provides us with a new option for fighting against multidrug-resistant ExPEC infections in the future.

14.
Molecules ; 25(1)2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31861925

RESUMO

Background: Tuberculosis remains a global disease that poses a serious threat to human health, but there is lack of new and available anti-tuberculosis agents to prevent the emergence of drug-resistant strains. To address this problem natural products are still potential sources for the development of novel drugs. Methods: A whole-cell screening approach was utilized to obtain a natural compound enniatin A1 from a natural products library. The target compound's antibacterial activity against Mycobacterium tuberculosis (M. tuberculosis) was evaluated by using the resazurin reduction micro-plate assay (REMA) method. The cytotoxicity of the compound against Vero cells was measured to calculate the selectivity index. The intracellular inhibition activity of enniatin A1 was determined. We performed its time-kill kinetic assay against M. tuberculosis. We first tested its synergistic effect in combination with the first and second-line anti-tuberculosis drugs. Finally, we measured the membrane potential and intracellular ATP levels of M. tuberculosis after exposure to enniatin A1. Results: We identified enniatinA1 as a potential antibacterial agent against M. tuberculosis, against which it showed strong selectivity. Enniatin A1 exhibited a time-concentration-dependent bactericidal effect against M. tuberculosis, and it displayed synergy with rifamycin, amikacin, and ethambutol. After exposure to enniatinA1, the membrane potential and intracellular ATP levels of M. tuberculosis was significantly decreased. Conclusions: Enniatin A1 exhibits the positive potential anti-tuberculosis agent characteristics.


Assuntos
Trifosfato de Adenosina/metabolismo , Antituberculosos/farmacologia , Depsipeptídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Mycobacterium tuberculosis/metabolismo , Tuberculose/tratamento farmacológico , Animais , Antituberculosos/agonistas , Chlorocebus aethiops , Depsipeptídeos/agonistas , Avaliação de Medicamentos , Sinergismo Farmacológico , Humanos , Células THP-1 , Células Vero
15.
Leuk Res ; 87: 106265, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31698307

RESUMO

Acute myeloid leukemia (AML) is a malignant myeloid hematopoietic stem and progenitor cell disease. Studies have shown that the long noncoding RNA (lncRNA) myocardial infarction associated transcript (MIAT) is abundantly expressed in multiple human solid tumors. However, the expression and role of MIAT in AML has not been explored previously. In this study, we find that MIAT is overexpressed in AML patient specimens and AML cell lines. Importantly, upregulation of MIAT is closely related with poor clinical outcome. Further investigations reveal that knockdown of MIAT inhibits the colony formation and proliferation, meanwhile, accelerates the apoptosis of AML cells in vitro. Consistently, MIAT knockdown slows AML progression in immunodeficient mice. Mechanistically, we confirm that MIAT can function as a sponge to inhibit microRNA-495 (miR-495), a tumor suppressor, in AML cells. Collectively, our results demonstrate that MIAT is involved in promoting the progression of AML, at least partly, through negative regulation of miR-495, and therefore provide a promising target for treatment of AML.


Assuntos
Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , MicroRNAs/genética , RNA Longo não Codificante/fisiologia , Animais , Estudos de Casos e Controles , Linhagem Celular Tumoral , Progressão da Doença , Regulação para Baixo/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Células HL-60 , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , RNA Longo não Codificante/genética
16.
Nat Microbiol ; 4(12): 2498-2510, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31611640

RESUMO

It is generally believed that exchange of secondary metabolite biosynthetic gene clusters (BGCs) among closely related bacteria is an important driver of BGC evolution and diversification. Applying this idea may help researchers efficiently connect many BGCs to their products and characterize the products' roles in various environments. However, existing genetic tools support only a small fraction of these efforts. Here, we present the development of chassis-independent recombinase-assisted genome engineering (CRAGE), which enables single-step integration of large, complex BGC constructs directly into the chromosomes of diverse bacteria with high accuracy and efficiency. To demonstrate the efficacy of CRAGE, we expressed three known and six previously identified but experimentally elusive non-ribosomal peptide synthetase (NRPS) and NRPS-polyketide synthase (PKS) hybrid BGCs from Photorhabdus luminescens in 25 diverse γ-Proteobacteria species. Successful activation of six BGCs identified 22 products for which diversity and yield were greater when the BGCs were expressed in strains closely related to the native strain than when they were expressed in either native or more distantly related strains. Activation of these BGCs demonstrates the feasibility of exploiting their underlying catalytic activity and plasticity, and provides evidence that systematic approaches based on CRAGE will be useful for discovering and identifying previously uncharacterized metabolites.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Vias Biossintéticas/genética , Engenharia Genética/métodos , Família Multigênica , Recombinases/metabolismo , Metabolismo Secundário/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Genoma Bacteriano , Peptídeo Sintases , Photorhabdus/genética , Policetídeo Sintases/genética
17.
Appl Environ Microbiol ; 85(18)2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31285192

RESUMO

Microorganisms that release plant-available phosphate from natural soil phosphate stores may serve as biological alternatives to costly and environmentally damaging phosphate fertilizers. To explore this possibility, we engineered a collection of root bacteria to release plant-available orthophosphate from phytate, an abundant phosphate source in many soils. We identified 82 phylogenetically diverse phytase genes, refactored their sequences for optimal expression in Proteobacteria, and then synthesized and engineered them into the genomes of three root-colonizing bacteria. Liquid culture assays revealed 41 engineered strains with high levels of phytate hydrolysis. Among these, we identified 12 strains across three bacterial hosts that confer a growth advantage on the model plant Arabidopsis thaliana when phytate is the sole phosphate source. These data demonstrate that DNA synthesis approaches can be used to generate plant-associated strains with novel phosphate-solubilizing capabilities.IMPORTANCE Phosphate fertilizers are essential for high-yield agriculture yet are costly and environmentally damaging. Microbes that release soluble phosphate from naturally occurring sources in the soil are appealing, as they may reduce the need for such fertilizers. In this study, we used synthetic biology approaches to create a collection of engineered root-associated microbes with the ability to release phosphate from phytate. We demonstrate that these strains improve plant growth under phosphorus-limited conditions. This represents a first step in the development of phosphate-mining bacteria for future use in crop systems.


Assuntos
Arabidopsis/microbiologia , Fosfatos/metabolismo , Ácido Fítico/metabolismo , Raízes de Plantas/metabolismo , Proteobactérias/metabolismo , Microrganismos Geneticamente Modificados/metabolismo , Raízes de Plantas/microbiologia , Proteobactérias/genética
18.
Technol Cancer Res Treat ; 18: 1533033819852990, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31138034

RESUMO

Alterations in microRNAs expression can accelerate the development of human cancers. However, the role of miR-153-3p in acute lymphoblastic leukemia remains unknown. The expression of miR-153-3p in acute lymphoblastic leukemia cell lines was measured by quantitative real-time polymerase chain reaction. Effects of miR-153-3p expression on acute lymphoblastic leukemia cell proliferation, migration, and invasion were examined by Cell Counting Kit-8 assay, wound healing assay, and Transwell invasion assay, respectively. We then validated inhibitor of growth protein 2 as a direct target of miR-153-3p through bioinformatics analysis, luciferase activity reporter assay, and Western blot assay. The miR-153-3p expression was decreased in acute lymphoblastic leukemia cell lines. Cell proliferation, migration, and invasion of acute lymphoblastic leukemia were obviously decreased by miR-153-3p overexpression. Moreover, inhibitor of growth protein 2 was validated as a direct target of miR-153-3p and the overexpression of inhibitor of growth protein 2 reversed the suppressive effects of miR-153-3p on acute lymphoblastic leukemia cell behaviors. Based on these results, we provided evidence that miR-153-3p might be a target for the treatment of acute lymphoblastic leukemia.


Assuntos
Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Proteínas de Homeodomínio/genética , MicroRNAs/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Interferência de RNA , Receptores Citoplasmáticos e Nucleares/genética , Proteínas Supressoras de Tumor/genética , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Humanos
19.
Curr Med Sci ; 38(4): 640-647, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30128873

RESUMO

This study aimed to assess the relationship of OAS2 rs739901 5,-flanking C/A polymorphisms with the susceptibility to Enterovirus-71 (EV71) infection. We investigated 294 hand-foot-mouth disease (HFMD) Chinese children with EV71 infection (165 mild cases and 129 encephalitis cases). The improved multiplex ligation detection reaction (iMLDR) technique was used to test the genotypes. In EV71-infected patients, the CA genotype distribution (P=0.007), A allele frequency (OR 1.32,95% CI 1.0-1.7, P=0.034) and CA+AA carriage frequency (P=0.003) of OAS2 rs739901 5'-flanking were obviously elevated as compared with controls, but there were no statistically significant differences between mild cases and encephalitis cases. In EV71-infected patients, the counts of white blood cells (P=0.034) and blood glucose concentrations (P=0.042) were raised in A carriers (CA+AA). Among different genotypes of encephalitis cases, the contents of cerebrospinal fluid (CSF) showed no significant differences. IFN-γ levels in EV71-infected patients were higher than those in controls (mild group vs. control group, P<0.01; encephalitis group vs. control group, P<0.01;). In encephalitis cases, IFN-γ levels were reduced (P<0.05) in A carriers compared to CC genotype, however, there were no significant differences between genotypes CA and AA (P=0.226). These findings suggest that OAS2 rs739901 5'-flanking C/A genetic polymorphisms involve the susceptibility to EV71 infection, and A allele might be a risk factor of the susceptibility to EV-71 infection.


Assuntos
2',5'-Oligoadenilato Sintetase/genética , Doença de Mão, Pé e Boca/genética , Polimorfismo de Nucleotídeo Único , Glicemia/metabolismo , Estudos de Casos e Controles , Criança , Pré-Escolar , China , Feminino , Frequência do Gene , Doença de Mão, Pé e Boca/sangue , Heterozigoto , Humanos , Interferon gama/sangue , Masculino
20.
J Ind Microbiol Biotechnol ; 45(7): 567-577, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29546662

RESUMO

Increasing availability of new genomes and putative biosynthetic gene clusters (BGCs) has extended the opportunity to access novel chemical diversity for agriculture, medicine, environmental and industrial purposes. However, functional characterization of BGCs through heterologous expression is limited because expression may require complex regulatory mechanisms, specific folding or activation. We developed an integrated workflow for BGC characterization that integrates pathway identification, modular design, DNA synthesis, assembly and characterization. This workflow was applied to characterize multiple phenazine-modifying enzymes. Phenazine pathways are useful for this workflow because all phenazines are derived from a core scaffold for modification by diverse modifying enzymes (PhzM, PhzS, PhzH, and PhzO) that produce characterized compounds. We expressed refactored synthetic modules of previously uncharacterized phenazine BGCs heterologously in Escherichia coli and were able to identify metabolic intermediates they produced, including a previously unidentified metabolite. These results demonstrate how this approach can accelerate functional characterization of BGCs.


Assuntos
Proteínas de Bactérias/genética , Família Multigênica , Fenazinas/metabolismo , Vias Biossintéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo
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