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1.
Immunity ; 55(5): 827-846.e10, 2022 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-35483355

RESUMO

Mycobacterium tuberculosis lung infection results in a complex multicellular structure: the granuloma. In some granulomas, immune activity promotes bacterial clearance, but in others, bacteria persist and grow. We identified correlates of bacterial control in cynomolgus macaque lung granulomas by co-registering longitudinal positron emission tomography and computed tomography imaging, single-cell RNA sequencing, and measures of bacterial clearance. Bacterial persistence occurred in granulomas enriched for mast, endothelial, fibroblast, and plasma cells, signaling amongst themselves via type 2 immunity and wound-healing pathways. Granulomas that drove bacterial control were characterized by cellular ecosystems enriched for type 1-type 17, stem-like, and cytotoxic T cells engaged in pro-inflammatory signaling networks involving diverse cell populations. Granulomas that arose later in infection displayed functional characteristics of restrictive granulomas and were more capable of killing Mtb. Our results define the complex multicellular ecosystems underlying (lack of) granuloma resolution and highlight host immune targets that can be leveraged to develop new vaccine and therapeutic strategies for TB.


Assuntos
Mycobacterium tuberculosis , Fibrose Pulmonar , Tuberculose , Animais , Ecossistema , Granuloma , Pulmão , Macaca fascicularis , Fibrose Pulmonar/patologia
2.
BMC Plant Biol ; 24(1): 882, 2024 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-39342076

RESUMO

BACKGROUND: The ANA-grade, encompassing early-diverging angiosperm lineages, Amborellales, Nymphaeales, and Austrobaileyales, represents a fundamental phase in the evolutionary history of flowering plants. Since the completion of key assembly of the Amborella genome, the continuous influx of omics data from the lineage underscores the need for a specialized database. RESULTS: Here, we introduce the ANA-grade Genome Database (ANAgdb, https://anagenome.cn/ ), which integrates multi-omics data including 11 genomes, 167 transcriptomes, and 10 miRNAomes, as well as extensive taxonomic details specific to the ANA-grade. Designed with an array of user-friendly tools, ANAgdb not only facilitates the effective storage, querying, and analysis of data but also enables the integration and dissemination of crucial genomic and taxonomic information. CONCLUSION: By integrating the comprehensive resources and tools, ANAgdb aims to significantly advance research in phylogenomics and taxonomic studies, providing a robust platform for researchers to explore the genetic and morphological diversities of these ancient plant lineages.


Assuntos
Bases de Dados Genéticas , Genoma de Planta , Magnoliopsida , Magnoliopsida/genética , Magnoliopsida/classificação , Filogenia , Genômica , Transcriptoma , MicroRNAs/genética , Multiômica
3.
J Antimicrob Chemother ; 76(8): 2049-2056, 2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-33855344

RESUMO

OBJECTIVES: In the past few decades, multiple-antibiotic-resistant Staphylococcus aureus has emerged and quickly spread in hospitals and communities worldwide. Additionally, the formation of antibiotic-tolerant persisters and biofilms further reduces treatment efficacy. Previously, we identified a sorafenib derivative, SC5005, with bactericidal activity against MRSA in vitro and in vivo. Here, we sought to elucidate the resistance status, mode of action and anti-persister activity of this compound. METHODS: The propensity of S. aureus to develop SC5005 resistance was evaluated by assessment of spontaneous resistance and by multi-passage selection. The mode of action of SC5005 was investigated using macromolecular synthesis, LIVE/DEAD and ATPlite assays and DiOC2(3) staining. The effect of SC5005 on the mammalian cytoplasmic membrane was measured using haemolytic and lactate dehydrogenase (LDH) assays and flow cytometry. RESULTS: SC5005 depolarized and permeabilized the bacterial cytoplasmic membrane, leading to reduced ATP production. Because of this mode of action, no resistance of S. aureus to SC5005 was observed after constant exposure to sub-lethal concentrations for 200 passages. The membrane-perturbing activity of SC5005 was specific to bacteria, as no significant haemolysis or release of LDH from human HT-29 cells was detected. Additionally, compared with other bactericidal antibiotics, SC5005 exhibited superior activity in eradicating both planktonic and biofilm-embedded S. aureus persisters. CONCLUSIONS: Because of its low propensity for resistance development and potent persister-eradicating activity, SC5005 is a promising lead compound for developing new therapies for biofilm-related infections caused by S. aureus.


Assuntos
Staphylococcus aureus Resistente à Meticilina , Infecções Estafilocócicas , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Biofilmes , Humanos , Potenciais da Membrana , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus
4.
Artigo em Inglês | MEDLINE | ID: mdl-30061293

RESUMO

The monobactam scaffold is attractive for the development of new agents to treat infections caused by drug-resistant Gram-negative bacteria because it is stable to metallo-ß-lactamases (MBLs). However, the clinically used monobactam aztreonam lacks stability to serine ß-lactamases (SBLs) that are often coexpressed with MBLs. LYS228 is stable to MBLs and most SBLs. LYS228 bound purified Escherichia coli penicillin binding protein 3 (PBP3) similarly to aztreonam (derived acylation rate/equilibrium dissociation constant [k2/Kd ] of 367,504 s-1 M-1 and 409,229 s-1 M-1, respectively) according to stopped-flow fluorimetry. A gel-based assay showed that LYS228 bound mainly to E. coli PBP3, with weaker binding to PBP1a and PBP1b. Exposing E. coli cells to LYS228 caused filamentation consistent with impaired cell division. No single-step mutants were selected from 12 Enterobacteriaceae strains expressing different classes of ß-lactamases at 8× the MIC of LYS228 (frequency, <2.5 × 10-9). At 4× the MIC, mutants were selected from 2 of 12 strains at frequencies of 1.8 × 10-7 and 4.2 × 10-9 LYS228 MICs were ≤2 µg/ml against all mutants. These frequencies compared favorably to those for meropenem and tigecycline. Mutations decreasing LYS228 susceptibility occurred in ramR and cpxA (Klebsiella pneumoniae) and baeS (E. coli and K. pneumoniae). Susceptibility of E. coli ATCC 25922 to LYS228 decreased 256-fold (MIC, 0.125 to 32 µg/ml) after 20 serial passages. Mutants accumulated mutations in ftsI (encoding the target, PBP3), baeR, acrD, envZ, sucB, and rfaI These results support the continued development of LYS228, which is currently undergoing phase II clinical trials for complicated intraabdominal infection and complicated urinary tract infection (registered at ClinicalTrials.gov under identifiers NCT03377426 and NCT03354754).


Assuntos
Antibacterianos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/genética , Klebsiella pneumoniae/enzimologia , Klebsiella pneumoniae/genética , Monobactamas/farmacologia , Aztreonam/farmacologia , Enterobacteriaceae/efeitos dos fármacos , Enterobacteriaceae/enzimologia , Enterobacteriaceae/genética , Escherichia coli/efeitos dos fármacos , Klebsiella pneumoniae/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Mutação/genética , beta-Lactamases/genética
5.
Artigo em Inglês | MEDLINE | ID: mdl-28096160

RESUMO

Argyrins are natural products with antibacterial activity against Gram-negative pathogens, such as Pseudomonas aeruginosa, Burkholderia multivorans, and Stenotrophomonas maltophilia We previously showed that argyrin B targets elongation factor G (FusA). Here, we show that argyrin B activity against P. aeruginosa PAO1 (MIC = 8 µg/ml) was not affected by deletion of the MexAB-OprM, MexXY-OprM, MexCD-OprJ, or MexEF-OprN efflux pump. However, argyrin B induced expression of MexXY, causing slight but reproducible antagonism with the MexXY substrate antibiotic ciprofloxacin. Argyrin B activity against Escherichia coli increased in a strain with nine tolC efflux pump partner genes deleted. Complementation experiments showed that argyrin was effluxed by AcrAB, AcrEF, and MdtFX. Argyrin B was inactive against Acinetobacter baumannii Differences between A. baumannii and P. aeruginosa FusA proteins at key residues for argyrin B interaction implied that natural target sequence variation impacted antibacterial activity. Consistent with this, expression of the sensitive P. aeruginosa FusA1 protein in A. baumannii conferred argyrin susceptibility, whereas resistant variants did not. Argyrin B was active against S. maltophilia (MIC = 4 µg/ml). Spontaneous resistance occurred at high frequency in the bacterium (circa 10-7), mediated by mutational inactivation of fusA1 rather than by amino acid substitutions in the target binding region. This strongly suggested that resistance occurred at high frequency through loss of the sensitive FusA1, leaving an alternate argyrin-insensitive elongation factor. Supporting this, an additional fusA-like gene (fusA2) is present in S. maltophilia that was strongly upregulated in response to mutational loss of fusA1.


Assuntos
Antibacterianos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Oligopeptídeos/farmacologia , Fator G para Elongação de Peptídeos/antagonistas & inibidores , Acinetobacter/efeitos dos fármacos , Acinetobacter/metabolismo , Proteínas de Bactérias/metabolismo , Burkholderia/efeitos dos fármacos , Burkholderia/metabolismo , Farmacorresistência Bacteriana/genética , Testes de Sensibilidade Microbiana , Fator G para Elongação de Peptídeos/metabolismo , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/metabolismo , Stenotrophomonas maltophilia/efeitos dos fármacos , Stenotrophomonas maltophilia/metabolismo
6.
PLoS Pathog ; 11(3): e1004792, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25815898

RESUMO

The prolonged survival of Mycobacterium tuberculosis (M. tb) in the host fundamentally depends on scavenging essential nutrients from host sources. M. tb scavenges non-heme iron using mycobactin and carboxymycobactin siderophores, synthesized by mycobactin synthases (Mbt). Although a general mechanism for mycobactin biosynthesis has been proposed, the biological functions of individual mbt genes remain largely untested. Through targeted gene deletion and global lipidomic profiling of intact bacteria, we identify the essential biochemical functions of two mycobactin synthases, MbtK and MbtN, in siderophore biosynthesis and their effects on bacterial growth in vitro and in vivo. The deletion mutant, ΔmbtN, produces only saturated mycobactin and carboxymycobactin, demonstrating an essential function of MbtN as the mycobactin dehydrogenase, which affects antigenicity but not iron uptake or M. tb growth. In contrast, deletion of mbtK ablated all known forms of mycobactin and its deoxy precursors, defining MbtK as the essential acyl transferase. The mbtK mutant showed markedly reduced iron scavenging and growth in vitro. Further, ΔmbtK was attenuated for growth in mice, demonstrating a non-redundant role of hydroxamate siderophores in virulence, even when other M. tb iron scavenging mechanisms are operative. The unbiased lipidomic approach also revealed unexpected consequences of perturbing mycobactin biosynthesis, including extreme depletion of mycobacterial phospholipids. Thus, lipidomic profiling highlights connections among iron acquisition, phospholipid homeostasis, and virulence, and identifies MbtK as a lynchpin at the crossroads of these phenotypes.


Assuntos
Proteínas de Bactérias/metabolismo , Ferro/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Oxazóis/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Camundongos , Mycobacterium tuberculosis/genética , Fatores de Virulência/genética
7.
Infect Immun ; 84(8): 2255-2263, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27245412

RESUMO

More people die every year from Mycobacterium tuberculosis infection than from infection by any other bacterial pathogen. Type VII secretion systems (T7SS) are used by both environmental and pathogenic mycobacteria to secrete proteins across their complex cell envelope. In the nonpathogen Mycobacterium smegmatis, the ESX-1 T7SS plays a role in conjugation, and the ESX-3 T7SS is involved in metal homeostasis. In M. tuberculosis, these secretion systems have taken on roles in virulence, and they also are targets of the host immune response. ESX-3 secretes a heterodimer composed of EsxG (TB9.8) and EsxH (TB10.4), which impairs phagosome maturation in macrophages and is essential for virulence in mice. Given the importance of EsxG and EsxH during infection, we examined their regulation. With M. tuberculosis, the secretion of EsxG and EsxH was regulated in response to iron and zinc, in accordance with the previously described transcriptional response of the esx-3 locus to these metals. While iron regulated the esx-3 expression in both M. tuberculosis and M. smegmatis, there is a significant difference in the dynamics of this regulation. In M. smegmatis, the esx-3 locus behaved like other iron-regulated genes such as mbtB In M. tuberculosis, both iron and zinc modestly repressed esx-3 expression. Diminished secretion of EsxG and EsxH in response to these metals altered the interaction of M. tuberculosis with macrophages, leading to impaired intracellular M. tuberculosis survival. Our findings detail the regulatory differences of esx-3 in M. tuberculosis and M. smegmatis and demonstrate the importance of metal-dependent regulation of ESX-3 for virulence in M. tuberculosis.


Assuntos
Proteínas de Bactérias/metabolismo , Metais/metabolismo , Mycobacterium tuberculosis/fisiologia , Tuberculose/microbiologia , Sistemas de Secreção Tipo II , Animais , Regulação Bacteriana da Expressão Gênica , Loci Gênicos , Ferro/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Viabilidade Microbiana , Proteínas Recombinantes , Tuberculose/imunologia , Zinco/metabolismo
8.
PLoS Pathog ; 10(3): e1003994, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24603869

RESUMO

Unlike most bacterial species, Mycobacterium tuberculosis depends on the Clp proteolysis system for survival even in in vitro conditions. We hypothesized that Clp is required for the physiologic turnover of mycobacterial proteins whose accumulation is deleterious to bacterial growth and survival. To identify cellular substrates, we employed quantitative proteomics and transcriptomics to identify the set of proteins that accumulated upon the loss of functional Clp protease. Among the set of potential Clp substrates uncovered, we were able to unambiguously identify WhiB1, an essential transcriptional repressor capable of auto-repression, as a substrate of the mycobacterial Clp protease. Dysregulation of WhiB1 turnover had a toxic effect that was not rescued by repression of whiB1 transcription. Thus, under normal growth conditions, Clp protease is the predominant regulatory check on the levels of potentially toxic cellular proteins. Our findings add to the growing evidence of how post-translational regulation plays a critical role in the regulation of bacterial physiology.


Assuntos
Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Mycobacterium tuberculosis/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Fatores de Transcrição/metabolismo , Reação em Cadeia da Polimerase , Proteólise , Proteômica
9.
Proc Natl Acad Sci U S A ; 109(4): 1257-62, 2012 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-22232695

RESUMO

To measure molecular changes underlying pathogen adaptation, we generated a searchable dataset of more than 12,000 mass spectrometry events, corresponding to lipids and small molecules that constitute a lipidome for Mycobacterium tuberculosis. Iron is essential for M. tuberculosis survival, and the organism imports this metal using mycobactin and carboxymycobactin siderophores. Detection of an unexpected siderophore variant and deletions of genes for iron scavenging has led to a revised mycobactin biosynthesis model. An organism-wide search of the M. tuberculosis database for hypothetical compounds predicted by this model led to the discovery of two families of previously unknown lipids, designated monodeoxymycobactins and monodeoxycarboxymycobactins. These molecules suggest a revised biosynthetic model that alters the substrates and order of action of enzymes through the mycobactin biosynthetic pathway. We tested this model genetically by solving M. tuberculosis lipidomes after deletion of the iron-dependent regulator (ideR), mycobactin synthase B (mbtB), or mycobactin synthase G (mbtG). These studies show that deoxymycobactins are actively regulated during iron starvation, and also define essential roles of MbtG in converting deoxymycobactins to mycobactin and in promoting M. tuberculosis growth. Thus, lipidomics is an efficient discovery tool that informs genetic relationships, leading to a revised general model for the biosynthesis of these virulence-conferring siderophores.


Assuntos
Vias Biossintéticas/fisiologia , Lipídeos/química , Modelos Biológicos , Mycobacterium tuberculosis/metabolismo , Oxazóis/metabolismo , Sideróforos/metabolismo , Cromatografia Líquida de Alta Pressão , Primers do DNA/genética , Bases de Dados Factuais , Ferro/metabolismo , Espectrometria de Massas
10.
Proc Natl Acad Sci U S A ; 108(10): 4176-81, 2011 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-21368134

RESUMO

It is often assumed that antibiotics act on the most vulnerable cellular targets, particularly those that require limited inhibition to block growth. To evaluate this assumption, we developed a genetic method that can inducibly deplete targeted proteins and that mimics their chemical inactivation. We applied this system to current antibiotic targets in mycobacteria. Although depleting some antibiotic targets significantly perturbs bacterial growth, surprisingly, we found that reducing the levels of other targets by more than 97% had little or no effect on growth. For one of these targets, dihydrofolate reductase, metabolic analysis suggested that depletion mimics the use of subinhibitory concentrations of the antibiotic trimethroprim. These observations indicate that some drug targets can exist at levels much higher than are needed to support growth. However, protein depletion can be used to identify promising drug targets that are particularly vulnerable to inhibition.


Assuntos
Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Hidrólise , Dados de Sequência Molecular
11.
Nucleic Acids Res ; 39(6): 2210-20, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21075796

RESUMO

Using a component of the Escherichia coli protein degradation machinery, we have established a system to regulate protein stability in mycobacteria. A protein tag derived from the E. coli SsrA degradation signal did not affect several reporter proteins in wild-type Mycobacterium smegmatis or Mycobacterium tuberculosis. Expression of the adaptor protein SspB, which recognizes this modified tag and helps deliver tagged proteins to the protease ClpXP, strongly decreased the activities and protein levels of different reporters. This inactivation did not occur when the function of ClpX was inhibited. Using this system, we constructed a conditional M. smegmatis knockdown mutant in which addition of anhydrotetracycline (atc) caused depletion of the beta subunit of RNA polymerase, RpoB. The impact of atc on this mutant was dose-dependent. Very low amounts of atc did not prevent growth but increased sensitivity to an antibiotic that inactivates RpoB. Intermediate amounts of RpoB knockdown resulted in bacteriostasis and a more substantial depletion led to a decrease in viability by up to 99%. These studies identify SspB-mediated proteolysis as an efficient approach to conditionally inactivate essential proteins in mycobacteria. They further demonstrate that depletion of RpoB by ∼ 93% is sufficient to cause death of M. smegmatis.


Assuntos
Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Mycobacterium smegmatis/enzimologia , Mycobacterium tuberculosis/enzimologia , Proteínas de Transporte/metabolismo , Estabilidade Enzimática , Proteínas de Escherichia coli/metabolismo , Cinética , Proteínas Luminescentes , Mutação , Peptídeo Hidrolases/genética , Estabilidade Proteica , RNA Bacteriano/química , RNA Bacteriano/metabolismo
12.
Proc Natl Acad Sci U S A ; 107(1): 297-301, 2010 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-20018758

RESUMO

Blood coagulation in humans requires the activity of vitamin K epoxide reductase (VKOR), the target of the anticoagulant warfarin (Coumadin). Bacterial homologs of VKOR were recently found to participate in a pathway leading to disulfide bond formation in secreted proteins of many bacteria. Here we show that the VKOR homolog from the bacterium Mycobacterium tuberculosis, the causative agent of human tuberculosis, is inhibited by warfarin and that warfarin-resistant mutations of mycobacterial VKOR appear in similar locations to mutations found in human patients who require higher doses of warfarin. Deletion of VKOR results in a severe growth defect in mycobacteria, and the growth of M. tuberculosis is inhibited by warfarin. The bacterial VKOR homolog may represent a target for antibiotics and a model for genetic studies of human VKOR. We present a simple assay in Escherichia coli, based on a disulfide-sensitive beta-galactosidase, which can be used to screen for stronger inhibitors of the M. tuberculosis VKOR homolog.


Assuntos
Anticoagulantes/farmacologia , Proteínas de Bactérias/metabolismo , Dissulfetos/química , Oxigenases de Função Mista/antagonistas & inibidores , Mycobacterium tuberculosis/efeitos dos fármacos , Varfarina/farmacologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Estrutura Molecular , Mutação , Mycobacterium tuberculosis/enzimologia , Oxirredução , Alinhamento de Sequência , Vitamina K Epóxido Redutases
13.
Sci Total Environ ; 850: 157939, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-35952878

RESUMO

Electrolytic oxygen aerobic composting (EOAC) is an effective treatment with greater technical superiority and cost advantages for organic solid waste using in situ electrolytic oxygen as a feasible strategy to replace conventional aeration. However, the unclear effects of distribution and variation of in situ electrolytic oxygen on compost maturation in different depth zones of EOAC need further exploration. This study demonstrated that the humification of organic matter was faster at the bottom than in the middle and at the top. The main reason was that the higher oxygen content and lower moisture content in the bottom promoted microbial degradation and heat production, resulting in higher temperatures. The microbial analysis showed that the abundance of typical thermophilic bacteria (such as Cerasibacillus, Lactobacillus, and Pseudogracilibacillus) that could promote compost maturation was higher at the bottom than in the middle and at the top. The finding provided in-depth molecular insights into differentiated humification from bottom to top in EOAC and revealed its further practical engineering applications.


Assuntos
Compostagem , Oxigênio , Solo , Resíduos Sólidos
14.
J Hazard Mater ; 426: 127846, 2022 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-34838365

RESUMO

Aerobic composting is an effective recycling method for the disposal and resource utilization of organic solid waste. However, the inappropriate aeration mode used during conventional aerobic composting (CAC) often results in low oxygen utilization efficiency and loss of temperature, which further leads to a long maturation period and large odorous gas (NH3) pollution. Herein, a novel electrolytic oxygen aerobic composting (EOAC) process was invented first using in-situ oxygen generation for aeration by the electrolysis of water in compost. Our results demonstrated that the germination index (GI) significantly increased during EOAC, and the maturation time of compost was shortened by nearly 50% during EOAC compared to CAC, indicating higher oxygen utilization efficiency during EOAC. Meanwhile, NH3 emissions, N2O emissions, and nitrogen loss during the EOAC process decreased by 61%, 46%, and 21%, respectively, compared to CAC. The total relative abundance of thermophilic and electroactive bacteria during EOAC increased remarkably. EOAC inhibited ammoniation, nitrification, and denitrification, and weakened N-associated functional genes. A techno-economic analysis indicated that EOAC had greater technical superiority and cost advantages compared to CAC. This study represents proof-of-principle for EOAC and suggests that in-situ electrolytic oxygen is a feasible replacement for conventional aeration during aerobic composting.


Assuntos
Compostagem , Eletrólise , Nitrificação , Nitrogênio/análise , Oxigênio , Solo
15.
Sci Total Environ ; 845: 157174, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35809732

RESUMO

Electrolytic oxygen aerobic composting (EOAC) effectively treats organic solid waste by using in-situ electrolytic oxygen for aeration. However, the fundamental mechanism of compost maturity is still unclear. Therefore, we comprehensively characterized dissolved organic matter (DOM) transformation closely related to compost maturity during EOAC. Excitation-emission matrix-parallel factor (EEM-PARAFAC) and Fourier transform infrared (FTIR) analysis confirmed that EOAC quickly decreased organic matter and increased humus substances, accelerating the compost humification process compared with conventional aerobic composting. Electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) analysis reveals that the double bound equivalent and aromaticity index during EOAC are higher than in conventional aerobic composting (CAC), suggesting more aromatic compounds in EOAC. DOM's detailed transformation investigation suggested that low O/C and high H/C compounds were preferentially decomposed during EOAC. Our investigation firstly extends the in-depth molecular mechanisms of humification during EOAC, and reveals its practical engineering applications.


Assuntos
Compostagem , Matéria Orgânica Dissolvida , Compostos Orgânicos , Oxigênio , Solo/química
16.
Ann Otol Rhinol Laryngol ; 120(9): 617-21, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22032077

RESUMO

OBJECTIVES: We sought to identify the prevalence of otitis media with effusion (OME) in urban Chinese children in Xi'an, China. METHODS: Five kindergartens and 3 primary schools were randomly selected in the urban area of Xi'an. Screening otoscopic and tympanometric examinations were performed on 2,902 children (1,491 boys and 1,411 girls) 2 to 8 years of age. Children with an abnormal tympanogram and simultaneous otomicroscopic signs of effusion were given a diagnosis of OME. RESULTS: The overall prevalence of OME was 4.3%. By age group, the prevalence was 14.0% in 2-year-olds, 8.3% in 3-year-olds, 5.0% in 4-year-olds, 4.9% in 5-year-olds, 2.8% in 6-year-olds, 1.7% in 7-year-olds, and 3.2% in 8-year-olds. The prevalence rate for OME was 4.7% for boys versus 3.9% for girls, and 3.0% in the right ear versus 2.7% in the left, showing no statistically significant difference between genders or between ear sides (p > 0.05). CONCLUSIONS: The prevalence of OME in urban areas of Xi'an is not high in comparison with that of the same age group in surrounding areas.


Assuntos
Otite Média com Derrame/epidemiologia , Testes de Impedância Acústica , Criança , Pré-Escolar , China/epidemiologia , Coleta de Dados , Feminino , Humanos , Masculino , Prevalência , Distribuição Aleatória , População Urbana
17.
J Bacteriol ; 190(7): 2496-504, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18223092

RESUMO

Serratia marcescens cells swarm at 30 degrees C but not at 37 degrees C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37 degrees C reduced the expression levels of flhDC(Sm) and hag(Sm) in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37 degrees C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDC(Sm) and synthesis of flagella are significantly increased in the rssA mutant strain at 37 degrees C. Primer extension analysis for determination of the transcriptional start site(s) of flhDC(Sm) revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB approximately P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB approximately P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB approximately P binding site is located between base pair positions -341 and -364 from the translation start codon ATG in the flhDC(Sm) promoter region. The binding site overlaps with the P2 "-35" promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB-P binds to the flhDC(Sm) promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDC(Sm) promoter activity at 37 degrees C. This inhibitory effect was comparatively alleviated at 30 degrees C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37 degrees C.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Serratia marcescens/genética , Transdução de Sinais/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Sequência de Bases , Sítios de Ligação/fisiologia , Imunoprecipitação da Cromatina , Pegada de DNA , Ensaio de Desvio de Mobilidade Eletroforética , Flagelos/genética , Flagelos/metabolismo , Flagelos/fisiologia , Flagelina/metabolismo , Dados de Sequência Molecular , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serratia marcescens/metabolismo , Serratia marcescens/fisiologia , Transdução de Sinais/fisiologia , Sítio de Iniciação de Transcrição
19.
PLoS One ; 13(3): e0193851, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29505586

RESUMO

Acinetobacter baumannii ATCC 19606 can grow without lipooligosaccharide (LOS). Lack of LOS can result from disruption of the early lipid A biosynthetic pathway genes lpxA, lpxC or lpxD. Although LOS itself is not essential for growth of A. baumannii ATCC 19606, it was previously shown that depletion of the lipid A biosynthetic enzyme LpxK in cells inhibited growth due to the toxic accumulation of lipid A pathway intermediates. Growth of LpxK-depleted cells was restored by chemical inhibition of LOS biosynthesis using CHIR-090 (LpxC) and fatty acid biosynthesis using cerulenin (FabB/F) and pyridopyrimidine (acetyl-CoA-carboxylase). Here, we expand on this by showing that inhibition of enoyl-acyl carrier protein reductase (FabI), responsible for converting trans-2-enoyl-ACP into acyl-ACP during the fatty acid elongation cycle also restored growth during LpxK depletion. Inhibition of fatty acid biosynthesis during LpxK depletion rescued growth at 37°C, but not at 30°C, whereas rescue by LpxC inhibition was temperature independent. We exploited these observations to demonstrate proof of concept for a targeted medium-throughput growth restoration screening assay to identify small molecule inhibitors of LOS and fatty acid biosynthesis. The differential temperature dependence of fatty acid and LpxC inhibition provides a simple means by which to separate growth stimulating compounds by pathway. Targeted cell-based screening platforms such as this are important for faster identification of compounds inhibiting pathways of interest in antibacterial discovery for clinically relevant Gram-negative pathogens.


Assuntos
Acinetobacter baumannii/metabolismo , Inibidores da Síntese de Ácidos Graxos/metabolismo , Ácidos Graxos/biossíntese , Lipídeo A/metabolismo , Bioensaio/métodos , Cerulenina/farmacologia , Enoil-(Proteína de Transporte de Acila) Redutase (NADH)/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Treonina/análogos & derivados , Treonina/farmacologia
20.
mSphere ; 3(5)2018 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-30381354

RESUMO

Tight coordination of inner and outer membrane biosynthesis is very important in Gram-negative bacteria. Biosynthesis of the lipid A moiety of lipopolysaccharide, which comprises the outer leaflet of the outer membrane has garnered interest for Gram-negative antibacterial discovery. In particular, several potent inhibitors of LpxC (the first committed step of the lipid A pathway) are described. Here we show that serial passaging of Klebsiella pneumoniae in increasing levels of an LpxC inhibitor yielded mutants that grew only in the presence of the inhibitor. These strains had mutations in fabZ and lpxC occurring together (encoding either FabZR121L/LpxCV37G or FabZF51L/LpxCV37G). K. pneumoniae mutants having only LpxCV37G or LpxCV37A or various FabZ mutations alone were less susceptible to the LpxC inhibitor and did not require LpxC inhibition for growth. Western blotting revealed that LpxCV37G accumulated to high levels, and electron microscopy of cells harboring FabZR121L/LpxCV37G indicated an extreme accumulation of membrane in the periplasm when cells were subcultured without LpxC inhibitor. Significant accumulation of detergent-like lipid A pathway intermediates that occur downstream of LpxC (e.g., lipid X and disaccharide monophosphate [DSMP]) was also seen. Taken together, our results suggest that redirection of lipid A pathway substrate by less active FabZ variants, combined with increased activity from LpxCV37G was overdriving the lipid A pathway, necessitating LpxC chemical inhibition, since native cellular maintenance of membrane homeostasis was no longer functioning.IMPORTANCE Emergence of antibiotic resistance has prompted efforts to identify and optimize novel inhibitors of antibacterial targets such as LpxC. This enzyme catalyzes the first committed step of lipid A synthesis, which is necessary to generate lipopolysaccharide and ultimately the Gram-negative protective outer membrane. Investigation of this pathway and its interrelationship with inner membrane (phospholipid) biosynthesis or other pathways is therefore highly important to the fundamental understanding of Gram-negative bacteria and by extension to antibiotic discovery. Here we exploited the availability of a novel LpxC inhibitor to engender the generation of K. pneumoniae resistant mutants whose growth depends on chemical inhibition of LpxC. Inhibitor dependency resulted from the interaction of different resistance mutations and was based on loss of normal cellular mechanisms required to establish membrane homeostasis. This study provides new insights into the importance of this process in K. pneumoniae and how it may be linked to novel biosynthetic pathway inhibitors.


Assuntos
Proteínas de Bactérias/metabolismo , Klebsiella pneumoniae/crescimento & desenvolvimento , Klebsiella pneumoniae/genética , Lipídeo A/metabolismo , Membranas/metabolismo , Proteínas Mutantes/metabolismo , Mutação de Sentido Incorreto , Proteínas de Bactérias/genética , Homeostase , Proteínas Mutantes/genética
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