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Histophilus somni is one of the predominant bacterial pathogens responsible for bovine respiratory and systemic diseases in cattle. Despite the identification of numerous H. somni virulence factors, little is known about the regulation of such factors. The post-transcriptional regulatory protein Hfq may play a crucial role in regulation of components that affect bacterial virulence. The contribution of Hfq to H. somni phenotype and virulence was investigated following creation of an hfq deletion mutant of H. somni strain 2336 (designated H. somni 2336Δhfq). A comparative analysis of the mutant to the wild-type strain was carried out by examining protein and carbohydrate phenotype, RNA sequence, intracellular survival in bovine monocytes, serum susceptibility, and virulence studies in mouse and calf models. H. somni 2336Δhfq exhibited a truncated lipooligosaccharide (LOS) structure, with loss of sialylation. The mutant demonstrated increased susceptibility to intracellular and serum-mediated killing compared to the wild-type strain. Transcriptomic analysis displayed significant differential expression of 832 upregulated genes and 809 downregulated genes in H. somni 2336Δhfq compared to H. somni strain 2336, including significant downregulation of lsgB and licA, which contribute to LOS oligosaccharide synthesis and sialylation. A substantial number of differentially expressed genes were associated with polysaccharide synthesis and other proteins that could influence virulence. The H. somni 2336Δhfq mutant strain was attenuated in a mouse septicemia model and somewhat attenuated in a calf intrabronchial challenge model. H. somni was recovered less frequently from nasopharyngeal swabs, endotracheal aspirates, and lung tissues of calves challenged with H. somni 2336Δhfq compared to the wild-type strain, and the percentage of abnormal lung tissue in calves challenged with H. somni 2336Δhfq was lower than in calves challenged with the wild-type strain. In conclusion, our results support that Hfq accounts for the regulation of H. somni virulence factors.
Assuntos
Haemophilus somnus , Pasteurellaceae , Animais , Bovinos , Camundongos , Virulência/genética , Haemophilus somnus/genética , Fatores de Virulência/genética , Fatores de Virulência/metabolismo , Proteínas/metabolismo , Monócitos , Pasteurellaceae/genéticaRESUMO
Biofilms are complex, three-dimensional structures that provide a long-established survival mechanism for microorganisms. Biofilms play a substantial role in pathogenesis as they can evade the immune response and be highly resistant to conventional antimicrobials, thus impacting the human health and healthcare system. To address this issue, BMC Microbiology invites submissions to the collection 'Biofilms and its impact on disease'.
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Anti-Infecciosos , Biofilmes , HumanosRESUMO
Antimicrobial resistance is a phenomenon that the present-day world is witnessing that poses a serious threat to global health. The decline in the development of novel therapeutics over the last couple of decades has exacerbated the situation further. In this scenario, the pursuit of new alternative therapeutics to commonly used antibiotics has gained predominance amongst researchers across the world. Antimicrobial peptides (AMPs) from natural sources have drawn significant interest in the recent years as promising pharmacological substitutes over the conventional antibiotics. The most notable advantage of AMPs is that microorganisms cannot develop resistance to them. Insects represent one of the potential sources of AMPs, which are synthesized as part of an innate immune defence against invading pathogens. AMPs from different insects have been extensively studied, and silkworm is one of them. Diverse classes of AMPs (including attacins, cecropins, defensins, enbocins, gloverins, lebocins and moricins) were identified from silkworm that exhibit antimicrobial property against bacteria, fungi and viruses, indicating their potential therapeutic benefits. This review briefs about the immune responses of silkworm to invading pathogens, the isolation of AMPs from silkworms, AMPs reported in silkworms and their activity against various microorganisms.
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Small RNAs (sRNA), in association with the global chaperone regulator Hfq, positively or negatively regulate gene expression in bacteria. For this study, Histophilus somni sRNAs that bind to Hfq were identified and then partially characterized. The Hfq-associated sRNAs in H. somni were isolated and identified by co-immunoprecipitation using anti-Hfq antibody, followed by sRNA sequencing. Sequence analysis of the sRNA samples identified 100 putative sRNAs, out of which 16 were present in pathogenic strain 2336, but not in non-pathogenic strain 129Pt. Bioinformatic analyses suggested that the sRNAs HS9, HS79, and HS97 could bind to many genes putatively involved in virulence/biofilm formation. Furthermore, multi-sequence alignment of the sRNA regions in the genome revealed that HS9 and HS97 could interact with sigma 54, which is a transcription factor linked to important bacterial traits, including motility, virulence, and biofilm formation. Northern blotting was used to determine the approximate size, abundance and any processing events attributed to the sRNAs. Selected sRNA candidates were confirmed to bind Hfq, as determined by electrophoretic mobility shift assays using sRNAs synthesized by in vitro transcription and recombinant Hfq. The exact transcriptional start site of the sRNA candidates was determined by RNA ligase-mediated rapid amplification of cDNA ends, followed by cloning and sequencing. This is the first investigation of H. somni sRNAs that show they may have important regulatory roles in virulence and biofilm formation.
Assuntos
Pasteurellaceae , Pequeno RNA não Traduzido , Northern Blotting , Agregação Celular , Biologia ComputacionalRESUMO
UspE is a global regulator in Escherichia coli. To study the function of Histophilus somni uspE, strain 2336::TnuspE was identified from a bank of mutants generated with EZ::Tn5™
Assuntos
Biofilmes , Infecções por Haemophilus , Haemophilus somnus , Virulência , Animais , Infecções por Haemophilus/microbiologia , Haemophilus somnus/genética , Haemophilus somnus/patogenicidade , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Camundongos , Mutação , Virulência/genéticaRESUMO
Adaptation to changing environmental conditions is crucial for the survival of microorganisms. Bacteria have evolved various mechanisms to cope with osmotic stress. Here, we report the identification and functional characterization of the osmotic stress response operon, betIBA, in Acinetobacter nosocomialis. The betIBA operon encodes enzymes that are important for the conversion of choline to the osmoprotectant, glycine betaine. The betIBA operon is polycistronic and is under the regulation of the first gene, betI, of the same operon. A bioinformatics analysis revealed the presence of a BetI-binding motif upstream of the betIBA operon, and electrophoretic mobility shift assays confirmed the specific binding of BetI. An mRNA expression analysis revealed that expression of betI, betB, and betA genes is elevated in a betI-eletion mutant compared with the wild type, confirming that the autorepressor BetI represses the betIBA operon in A. nosocomialis. We further found that the betIBA operon is under the transcriptional control of the quorum-sensing (QS) regulator, AnoR in, A. nosocomialis. A subsequent analysis of the impact of BetI on expression of the QS genes, anoR and anoI, demonstrated that BetI acts as a repressor of anoR and anoI. In addition, it was noticed that the osmotic stress response regulator, OmpR might play an important role in controlling the expression of betIBA operon in A. nosocomialis. Collectively, these data demonstrate that QS and osmotic stress-response systems are correlated in A. nosocomialis and that the expression of genes in both systems is finely tuned by various feedback loops depending on osmolarity conditions.
Assuntos
Acinetobacter/metabolismo , Proteínas de Bactérias/metabolismo , Óperon , Percepção de Quorum , Proteínas Repressoras/metabolismo , Acinetobacter/genética , Regulação Bacteriana da Expressão Gênica , OsmorregulaçãoRESUMO
Multidrug efflux pumps play an important role in antimicrobial resistance and pathogenicity in bacteria. Here, we report the functional characterization of the RND (resistance-nodulation- division) efflux pump, AcrAB, in Acinetobacter nosocomialis. An in silico analysis revealed that homologues of the AcrAB efflux pump, comprising AcrA and AcrB, are widely distributed among different bacterial species. Deletion of acrA and/or acrB genes led to decreased biofilm/pellicle formation and reduced antimicrobial resistance in A. nosocomialis. RNA sequencing and mRNA expression analyses showed that expression of acrA/B was downregulated in a quorum sensing (QS) regulator (anoR)-deletion mutant, indicating transcriptional activation of the acrAB operon by AnoR in A. nosocomialis. Bioassays showed that secretion of N-acyl homoserine lactones (AHLs) was unaffected in acrA and acrB deletion mutants; however, AHL secretion was limited in a deletion mutant of acrR, encoding the acrAB regulator, AcrR. An in silico analysis indicated the presence of AcrR-binding motifs in promoter regions of anoI (encoding AHL synthase) and anoR. Specific binding of AcrR was confirmed by electrophoretic mobility shift assays, which revealed that AcrR binds to positions -214 and -217 bp upstream of the translational start sites of anoI and anoR, respectively, demonstrating transcriptional regulation of these QS genes by AcrR. The current study further addresses the possibility that AcrAB is controlled by the osmotic stress regulator, OmpR, in A. nosocomialis. Our data demonstrate that the AcrAB efflux pump plays a crucial role in biofilm/pellicle formation and antimicrobial resistance in A. nosocomialis, and is under the transcriptional control of a number of regulators. In addition, the study emphasizes the interrelationship of QS and AcrAB efflux systems in A. nosocomialis.
Assuntos
Acinetobacter/fisiologia , Proteínas de Bactérias/fisiologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Percepção de QuorumRESUMO
Mycobacterial cell walls comprise thick and diverse lipids and glycolipids that act as a permeability barrier to antibiotics or other chemical agents. The use of OH radicals from a non-thermal plasma jet (NTPJ) for the inactivation of mycobacteria in aqueous solution was adopted as a novel approach. Addition of water vapor in a nitrogen plasma jet generated OH radicals, which converted to hydrogen peroxide (H2O2) that inactivated non-pathogenic Mycobacterium smegmatis and pathogenic Mycobacterium tuberculosis H37Rv. A stable plasma plume was obtained from a nitrogen plasma jet with 1.91 W of power, killing Escherichia coli and mycobacteria effectively, whereas addition of catalase decreased the effects of the former. Mycobacteria were more resistant than E. coli to NTPJ treatment. Plasma treatment enhanced intracellular ROS production and upregulation of genes related to ROS stress responses (thiolrelated oxidoreductases, such as SseA and DoxX, and ferric uptake regulator furA). Morphological changes of M. smegmatis and M. tuberculosis H37Rv were observed after 5 min treatment with N2+H2O plasma, but not of pre-incubated sample with catalase. This finding indicates that the bactericidal efficacy of NTPJ is related to the toxicity of OH and H2O2 radicals in cells. Therefore, our study suggests that NTPJ treatment may effectively control pulmonary infections caused by M. tuberculosis and nontuberculous mycobacteria (NTM) such as M. avium or M. abscessus in water.
Assuntos
Antibacterianos/farmacologia , Radical Hidroxila/farmacologia , Mycobacterium/efeitos dos fármacos , Gases em Plasma/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Meios de Cultura/química , Escherichia coli/efeitos dos fármacos , Escherichia coli/fisiologia , Peróxido de Hidrogênio/análise , Peróxido de Hidrogênio/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Mycobacterium/fisiologia , Nitrogênio/análise , Nitrogênio/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Gases em Plasma/química , Espécies Reativas de Oxigênio/metabolismo , Água/análiseRESUMO
BACKGROUND: Acinetobacter has emerged recently as one of the most challenging nosocomial pathogens because of its increased rate of antimicrobial resistance. The genetic complexity and genome diversity, as well as the lack of adequate knowledge on the pathogenic determinants of Acinetobacter strains often hinder with pathogenesis studies for the development of better therapeutics to tackle this nosocomial pathogen. OBJECTIVES: In this study, we comparatively analyzed the whole genome sequence of a virulent Acinetobacternosocomialis strain NCTC 8102. METHODS: The genomic DNA of A. nosocomialis NCTC 8102 was isolated and sequenced using PacBio RS II platform. The sequenced genome was functionally annotated and gene prediction was carried out using the program, Glimmer 3. The phylogenetic analysis of the genome was performed using Mega 6 program and the comparative genome analysis was carried out by BLAST (Basic Local Alignment Search Tool). RESULTS: The complete genome analysis depicted that the genome consists of a circular chromosome with an average G + C content of 38.7%. The genome comprises 3700 protein-coding genes, 96 RNA genes (18 rRNA, 74 tRNA and 4 ncRNA genes), and 91 pseudogenes. In addition, 6 prophage regions comprising 2 intact, 1 incomplete and 3 questionable ones and 18 genomic islands were identified in the genome, suggesting the possible occurrence of horizontal gene transfer in this strain. Comparative genome analysis of A. nosocomialis NCTC 8102 genome with the already sequenced A. nosocomialis strain SSA3 showed an average nucleotide identity of 99.0%. In addition, the number of prophages and genomic islands were higher in the A. nosocomialis NCTC 8102 genome compared to that of the strain SSA3. 14 of the genomic islands were unique to A. nosocomialis NCTC 8102 compared to strain SSA3 and they harbored genes which are involved in virulence, multidrug resistance, biofilm formation and bacterial pathogenesis. CONCLUSION: We sequenced the whole genome of A. nosocomialis strain NCTC 8102 followed by comparatively genome analysis. The study provides valuable information on the genetic features of A. nosocomialis strain and the data from this study would assist in further studies for the development of control measures for this nosocomial pathogen.
Assuntos
Acinetobacter/genética , Genoma Bacteriano , Filogenia , Acinetobacter/classificação , Acinetobacter/patogenicidade , Biofilmes , Ilhas Genômicas , Anotação de Sequência Molecular , Prófagos/genética , Virulência/genéticaRESUMO
NemR is an electrophile-sensing regulator which controls two enzymes required for the detoxification of reactive electrophiles: N-ethylmaleimide (NEM) reductase and glyoxalase I in Escherichia coli. Both enzymes are essential for bacterial survival in the presence of toxic reactive electrophiles, such as N-ethylmaleimide and methyl glyoxal. Here, we report the identification and characterization of NemR from Acinetobacter nosocomialis, a nosocomial pathogen. We confirmed that nemR and the nemA gene which encodes N-ethylmaleimide reductase form a single operon, which is in accordance with the reports from E. coli. Bioinformatic analysis revealed the presence of an NemR binding motif in the promoter regions of nemRA operon and gloA (encoding glyoxalase I) and the binding was confirmed by gel mobility shift assay. The deletion of nemR resulted in increased biofilm/pellicle formation in A. nosocomialis. mRNA expression analysis revealed that NemR acts as a repressor of the nemRA operon and gloA, and that the repressor function is inactivated by the addition of toxic Cys modification agents, contributing to bacterial survival. In addition, it was demonstrated that the nemRA operon is positively regulated by the quorum sensing regulator, AnoR and the operon plays a role in biofilm/pellicle formation in A. nosocomialis.
Assuntos
Acinetobacter/efeitos dos fármacos , Acinetobacter/enzimologia , Antioxidantes/metabolismo , Proteínas de Bactérias/metabolismo , Etilmaleimida/toxicidade , Glioxal/toxicidade , Proteínas Repressoras/metabolismo , Acinetobacter/genética , DNA Bacteriano/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Ordem dos Genes , Desintoxicação Metabólica Fase I , Óperon , Ligação Proteica , Proteínas Repressoras/genéticaRESUMO
Biofilm formation in healthcare is an issue of considerable concern, as it results in increased morbidity and mortality, imposing a significant financial burden on the healthcare system. Biofilms are highly resistant to conventional antimicrobial therapies and lead to persistent infections. Hence, there is a high demand for novel strategies other than conventional antibiotic therapies to control biofilm-based infections. There are two approaches which have been employed so far to control biofilm formation in healthcare settings: one is the development of biofilm inhibitors based on the understanding of the molecular mechanism of biofilm formation, and the other is to modify the biomaterials which are used in medical devices to prevent biofilm formation. This review will focus on the recent advances in anti-biofilm approaches by interrupting the quorum-sensing cellular communication system and the multidrug efflux pumps which play an important role in biofilm formation. Research efforts directed towards these promising strategies could eventually lead to the development of better anti-biofilm therapies than the conventional treatments.
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Isoprene is a valuable precursor for synthetic rubber and a signature product of terpenoid pathways. Here, we developed an isoprene biosensor by employing a TbuT transcriptional regulator of Ralstonia pickettii to express a fluorescent reporter gene in response to intracellular isoprene in engineered Escherichia coli. The TbuT regulator recognizes isoprene as its less-preferred effector molecule; thus, we amplified the reporter gene expression using a T7 RNA polymerase-mediated transcriptional cascade and iteratively tuned the promoter transcribing tbuT to improve the sensitivity for detecting isoprene. When the engineered E. coli cells expressed heterologous genes for isoprene biosynthesis, the intracellular isoprene was expelled and the tbuT transcription factor was subsequently activated, leading to gfp expression. The chromosomal isoprene biosensor showed a linear correlation between GFP fluorescence and intracellular isoprene concentration. Using this chromosomal isoprene biosensor, we successfully identified the highest isoprene producer among four different E. coli strains producing different amounts of isoprene. The isoprene biosensor presented here can enable high-throughput screening of isoprene synthases and metabolic pathways for efficient and sustainable production of bioisoprene in engineered microbes.
Assuntos
Técnicas Biossensoriais/métodos , Butadienos/análise , Escherichia coli/metabolismo , Hemiterpenos/análise , Engenharia Metabólica/métodos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hemiterpenos/biossíntese , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , Ralstonia pickettii/genética , Espectrometria de Fluorescência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Virais/genéticaRESUMO
Multidrug efflux systems contribute to antimicrobial resistance and pathogenicity in bacteria. Here, we report the identification and characterization of a transcriptional regulator AcrR controlling the yet uncharacterized multidrug efflux pump, AcrAB in Acinetobacter nosocomialis. In silico analysis revealed that the homologs of AcrR and AcrAB are reported in the genomes of many other bacterial species. We confirmed that the genes encoding the AcrAB efflux pump, acrA and acrB forms a polycistronic operon which is under the control of acrR gene upstream of acrA. Bioinformatic analysis indicated the presence of AcrR binding motif in the promoter region of acrAB operon and the specific binding of AcrR was confirmed by electrophoretic mobility shift assay (EMSA). The EMSA data showed that AcrR binds to -89 bp upstream of the start codon of acrA. The mRNA expression analysis depicted that the expression of acrA and acrB genes are elevated in the deletion mutant compared to that in the wild type confirming that AcrR acts as a repressor of acrAB operon in A. nosocomialis. The deletion of acrR resulted in increased motility, biofilm/pellicle formation and invasion in A. nosocomialis. We further analyzed the role of AcrR in A. nosocomialis pathogenesis in vivo using murine model and it was shown that acrR mutant is highly virulent inducing severe infection in mouse leading to host death. In addition, the intracellular survival rate of acrR mutant was higher compared to that of wild type. Our data demonstrates that AcrR functions as an important regulator of AcrAB efflux pump and is associated with several phenotypes such as motility, biofilm/pellicle formation and pathogenesis in A. nosocomialis.
Assuntos
Acinetobacter/crescimento & desenvolvimento , Acinetobacter/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Repressoras/metabolismo , Infecções por Acinetobacter/microbiologia , Infecções por Acinetobacter/patologia , Animais , Sítios de Ligação , Biologia Computacional , DNA Bacteriano/metabolismo , Modelos Animais de Doenças , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Membrana Transportadoras/genética , Camundongos , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Repressoras/genética , Análise de Sobrevida , VirulênciaRESUMO
Urinary tract infections (UTIs) are one of the most common types of bacterial infection in humans in various parts of the world and are caused mainly by uropathogenic Escherichia coli (UPEC). A total of 58 UPEC isolates from urine were characterized by serotyping and pulsed-field gel electrophoresis (PFGE). The majority of the UPEC strains belonged to serogroups O2 and O6. The UPEC strains were grouped under different pulsotypes and majority of them belonged to serogroups O2 and O6. Among the 14 virulence factors considered, 13 were present in various serogroups. The virulence genes fimH and sfa were present in all the isolates while none of the isolates carried lt-1. The strains exhibited 36 different virulence patterns, of which 11, referred to as UP (UPEC pattern) 1 to UP 11 were most common. Antibiotic resistance profiling of the UPEC isolates revealed that the serogroups O2 and O6 contain the highest number of resistant strains. The data from the current study depicting the distribution of UPEC strains among various serogroups and pulsotypes, and the occurrence of virulence genes and antibiotics resistance offer useful information on the epidemiological features of UPEC in Korea for the enhanced surveillance of potential emergence of UPEC.
Assuntos
Infecções Urinárias/epidemiologia , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Adolescente , Criança , Pré-Escolar , Escherichia coli/genética , Escherichia coli/patogenicidade , Infecções por Escherichia coli/epidemiologia , Feminino , Humanos , Masculino , República da Coreia/epidemiologia , Sorogrupo , Sorotipagem , Sistema Urinário/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Virulência , Fatores de Virulência/análiseRESUMO
Urinary tract infections (UTIs) are among the most common infections in humans, predominantly caused by uropathogenic Escherichia coli (UPEC). The diverse genomes of UPEC strains mostly impede disease prevention and control measures. In this study, we comparatively analyzed the whole genome sequence of a highly virulent UPEC strain, namely UPEC 26-1, which was isolated from urine sample of a patient suffering from UTI in Korea. Whole genome analysis showed that the genome consists of one circular chromosome of 5,329,753 bp, comprising 5064 protein-coding genes, 122 RNA genes (94 tRNA, 22 rRNA and 6 ncRNA genes), and 100 pseudogenes, with an average G+C content of 50.56%. In addition, we identified 8 prophage regions comprising 5 intact, 2 incomplete and 1 questionable ones and 63 genomic islands, suggesting the possibility of horizontal gene transfer in this strain. Comparative genome analysis of UPEC 26-1 with the UPEC strain CFT073 revealed an average nucleotide identity of 99.7%. The genome comparison with CFT073 provides major differences in the genome of UPEC 26-1 that would explain its increased virulence and biofilm formation. Nineteen of the total GIs were unique to UPEC 26-1 compared to CFT073 and nine of them harbored unique genes that are involved in virulence, multidrug resistance, biofilm formation and bacterial pathogenesis. The data from this study will assist in future studies of UPEC strains to develop effective control measures.
Assuntos
Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Composição de Bases/genética , Sequência de Bases/genética , Mapeamento Cromossômico , DNA Bacteriano/genética , Escherichia coli/genética , Infecções por Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genoma Bacteriano/genética , Ilhas Genômicas , Filogenia , Infecções Urinárias/genética , Virulência , Fatores de Virulência/genética , Sequenciamento Completo do Genoma/métodosRESUMO
Genetic circuit-based biosensors are useful in detecting target metabolites or in vivo enzymes using transcription factors (Tx) as a molecular switch to express reporter signals, such as cellular fluorescence and antibiotic resistance. Herein, a phenol-detecting Tx (DmpR) was employed as a critical tool for enzyme engineering, specifically for the rapid analysis of numerous mutants with multiple mutations at the active site of tryptophan-indole lyase (TIL, EC 4.1.99.1). Cellular fluorescence was monitored cell-by-cell using flow cytometry to detect the creation of phenolic compounds by a new tyrosine-phenol-lyase (TPL, EC 4.1.99.2). In the TIL scaffold, target amino acids near the indole ring (Asp137, Phe304, Val394, Ile396 and His463) were mutated randomly to construct a large diversity of specificity variations. Collection of candidate positives by cell sorting using flow cytometry and subsequent shuffling of beneficial mutations identified a critical hit with four mutations (D137P, F304D, V394L, and I396R) in the TIL sequence. The variant displayed one-thirteenth the level of TPL activity, compared with native TPLs, and completely lost the original TIL activity. The findings demonstrate that hypersensitive, Tx-based biosensors could be useful critically to generate new activity from a related template, which would alleviate the current burden to high-throughput screening.
Assuntos
Evolução Molecular Direcionada/métodos , Redes Reguladoras de Genes/fisiologia , Tirosina Fenol-Liase/metabolismo , Proteínas de Bactérias/genética , Domínio Catalítico , Citrobacter freundii/enzimologia , Escherichia coli/enzimologia , Citometria de Fluxo/métodos , Corantes Fluorescentes , Ensaios de Triagem em Larga Escala/métodos , Modelos Moleculares , Fenol/análise , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Tirosina Fenol-Liase/análiseRESUMO
Autophagy is known to be a vital homeostatic defense process that controls mycobacterial infection. However, the relationship between autophagy response and the virulence of Mycobacterium abscessus strain UC22 has not been reported. Here, we demonstrate that M. abscessus induces autophagy and inhibits autophagy flux in murine macrophages. Further, the rough variant of M. abscessus, UC22 that is a highly virulent clinical isolate, significantly inhibited autophagic flux than the smooth variant of M. abscessus ATCC 19977. In addition, it was noticed that the intracellular survival of UC22 is significantly enhanced by blocking the autophagosome-lysosome fusion in macrophages compared to the smooth variant. However, Mycobacterium smegmatis did not block autophagy flux in murine macrophages. Besides, we confirmed that the lipid components of M. abscessus UC22 play a role in autophagosome formation. These data suggest that the virulent M. abscessus might be able to survive and grow within autophagosomes by preventing the autophagosome-lysosome fusion and their clearance from the cells.
Assuntos
Autofagia , Macrófagos/microbiologia , Mycobacterium abscessus/fisiologia , Animais , Feminino , Lipídeos , Macrófagos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Células RAW 264.7RESUMO
GlxR is considered as a global transcriptional regulator controlling a large number of genes having broad physiological aspects in Corynebacterium glutamicum. However, the expression profile revealing the transcriptional control of glxR has not yet been studied in detail. DNA affinity chromatography experiments revealed the binding of transcriptional regulators SucR, RamB, GlxR, and a GntR-type protein (hereafter denoted as GntR3) to the upstream region of glxR. The binding of different regulators to the glxR promoter was confirmed by EMSA experiments. The expression of glxR was analyzed in detail under various carbon sources in the wild-type and different mutant strains. The sucR and gntR3 deletion mutants showed decreased glxR promoter activities, when compared with the wild type, irrespective of the carbon sources. The promoter activity of glxR was derepressed in the ramB deletion mutant under all the tested carbon sources. These results indicate that SucR and GntR3 are acting as activators of GlxR, while RamB plays a repressor. As expected, the expression of glxR in the cyaB and glxR deletion mutants was derepressed under different media conditions, indicating that GlxR is autoregulated.
Assuntos
Corynebacterium glutamicum/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/isolamento & purificação , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , Fatores de Transcrição/isolamento & purificação , Carbono/metabolismo , Cromatografia de Afinidade , Proteínas de Ligação a DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Deleção de Genes , Perfilação da Expressão Gênica , Ligação Proteica , Fatores de Transcrição/genéticaRESUMO
The cyclic AMP receptor protein (CRP) homolog, GlxR, controls the expression of several genes involved in the regulation of diverse physiological processes in Corynebacterium glutamicum. In silico analysis has revealed the presence of glxR binding sites upstream of genes adhA, ald, and ptsG, encoding glucose-specific phosphotransferase system protein, alcohol dehydrogenase (ADH), and acetaldehyde dehydrogenase (ALDH), respectively. However, the involvement of the GlxR-cAMP complex on the expression of these genes has been explored only in vitro. In this study, the expressions of ptsG, adhA, and ald were analyzed in detail using an adenylate cyclase gene (cyaB) deletion mutant and glxR deletion mutant. The specific activities of ADH and ALDH were increased in both the mutants in glucose and glucose plus ethanol media, in contrast to the wild type. In accordance, the promoter activities of adhA and ald were derepressed in the cyaB mutant, indicating that glxR acts as a repressor of adhA. Similarly, both the mutants exhibited derepression of ptsG regardless of the carbon source. These results confirm the involvement of GlxR on the expression of important carbon metabolic genes; adhA, ald, and ptsG.