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1.
Food Microbiol ; 120: 104476, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38431322

RESUMO

Globally, the spread of multidrug-resistant Pseudomonas aeruginosa, Escherichia coli, and Klebsiella pneumoniae from food to humans poses a severe threat to public health. The aim of this study was to assess the co-occurrence of colistin and ß-lactamase resistance genes in E. coli, K. pneumoniae, and P. aeruginosa strains isolated from faeces of abattoir broiler chickens. The E. coli, P. aeruginosa and K. pneumoniae isolates were successfully detected from faecal samples by polymerase chain reaction (PCR) at infection rates of 60.7%, 22.5% and 16.7% respectively. The isolates displayed the highest levels of antibiotic resistance (AR) against ampicillin (82.3%) and amoxicillin-clavulanic acid (74.2%) for E. coli, followed by cefoxitin (70.6%) for K. pneumoniae, whilst P. aeruginosa displayed 26.1% antibiotic resistance (AR) against both ampicillin and colistin sulphate. The colistin mcr-1 gene was harboured by 46.8%, 47.1% and 21.7%, E. coli, K. pneumonia and P. aeruginosa isolates respectively. Ten out of 62 (16.1%), 6/17 (35.3%), 4/23 (17.4%) isolates were phenotypically classified as ESBL E. coli, K. pneumoniae, and P. aeruginosa respectively. The ESBL-E. coli isolates respectively possessed blaCTX-M (60%), blaTEM (20%) and blaCTX-M-9 (10%) genes. The ESBL-K. pneumoniae harboured, blaCTX-M (50%), blaOXA (33%), blaCARB (17%), and blaCTX-M-9 (17%) genes respectively, whilst, P. aeruginosa isolates respectively carried blaTEM (75%), blaCTX-M (50%), blaOXA (25%) and blaCARB (25%) genes. Molecular analysis identified the blaCTX-Mß-lactamase-encoding genes collectively from E. coli, P. aeruginosa, K. pneumoniae isolates. Colistin and ß-lactamase genes were present in only 16.7%, 6.9%, and 2.9% of E. coli, K. pneumoniae, and P. aeruginosa isolates, respectively. A total of 17, 7 and 3 isolates for E. coli, K. pneumoniae and P. aeruginosa respectively carried both colistin and ß-lactamase antibiotics resistant genes. This is a public health threat that points to a challenge in the treatment of infections caused by these zoonotic bacteria. Data generated from this study will contribute to formulation of new strategies for combating spread of E. coli, K. pneumoniae, and P. aeruginosa isolates as well as prevention of their AR development.


Assuntos
Infecções por Escherichia coli , Escherichia coli , Humanos , Animais , Klebsiella pneumoniae/genética , Antibacterianos/farmacologia , Pseudomonas aeruginosa/genética , Galinhas , Colistina/farmacologia , Farmacorresistência Bacteriana/genética , beta-Lactamases/genética , Infecções por Escherichia coli/microbiologia , Ampicilina , Testes de Sensibilidade Microbiana
2.
Can Vet J ; 65(3): 259-266, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38434158

RESUMO

Objectives: To evaluate the effects of a cell-free supernatant from Lactococcus lactis (CFSM) on performance and diarrhearelated parameters and the presence of F4+ enterotoxigenic E. coli (ETEC) in piglets during post-weaning, and to evaluate the in vitro effect of the CFSM on faeG gene expression in an E. coli F4+. Animals and procedure: In 3 trials with 90 piglets per trial, pigs were assigned to receive a placebo or 1 of 2 CFSM treatments and observed for diarrhea and performance. Fecal swabs were taken to determine the presence of ETEC. Quantitative RT-PCR was used to assess faeG gene expression in E. coli 21259 after treatment with CFSM at 50 mg/mL. Results: The CFSM administered for 14 d at a dose of 24 mg/kg BW (2X) reduced diarrhea-related parameters compared to the placebo. Quantitative RT-PCR showed that, in E. coli 21259 treated with CFSM at 50 mg/mL, expression of the faeG gene was significantly repressed (P < 0.0001) relative to that in the untreated control. Conclusion: The evaluated CFSM reduced the frequency and prevalence of diarrhea in a field situation. The in vitro treatment had an inhibitory effect on the expression of the faeG gene in F4+ E. coli 21259.


Effet d'un surnageant de culture de Lactococcus lactis sur la diarrhée et les paramètres de performance des porcelets en période post-sevrage et sur l'expression du gène faeG in vitro. Objectifs: Évaluer les effets d'un surnageant acellulaire de Lactococcus lactis (CFSM) sur les paramètres de performance et de diarrhée et la présence d'E. coli entérotoxinogène F4+ (ETEC) chez les porcelets en post-sevrage, et évaluer l'effet in vitro du CFSM sur l'expression du gène faeG dans un E. coli F4+. Animaux et procédure: Dans 3 essais portant sur 90 porcelets par essai, les porcs ont reçu un placebo ou 1 des 2 traitements CFSM et ont été observés pour détecter la diarrhée et leurs performances. Des prélèvements fécaux ont été effectués pour déterminer la présence d'ETEC. La RT-PCR quantitative a été utilisée pour évaluer l'expression du gène faeG dans E. coli 21259 après traitement avec CFSM à 50 mg/mL. Résultats: Le CFSM administré pendant 14 jours à une dose de 24 mg/kg de poids corporel (2X) a réduit les paramètres liés à la diarrhée par rapport au placebo. La RT-PCR quantitative a montré que, chez E. coli 21259 traité avec CFSM à 50 mg/mL, l'expression du gène faeG était significativement réprimée (P < 0,0001) par rapport à celle du témoin non traité. Conclusion: Le CFSM évalué a réduit la fréquence et la prévalence de la diarrhée sur le terrain. Le traitement in vitro a eu un effet inhibiteur sur l'expression du gène faeG chez F4+ E. coli 21259.(Traduit par Dr Serge Messier).


Assuntos
Lactococcus lactis , Animais , Suínos , Lactococcus lactis/genética , Escherichia coli , Diarreia/prevenção & controle , Diarreia/veterinária , Manejo de Espécimes/veterinária
3.
Front Cell Infect Microbiol ; 14: 1280188, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38435302

RESUMO

Human infections caused by Pseudomonas citronellolis, an environmental bacterium, are infrequent, with only two cases related to uncommon urinary tract infections and bacteremia reported in recent years. All these cases typically occurred in elderly patients with compromised or decreased immune function. Simultaneously, the epithelial barrier disruption induced by invasive biopsy procedures or gastrointestinal disorders such as gastroenteritis provided a pathway for Pseudomonas citronellolis to infiltrate the organism. In this study, we present the first report of a case where Pseudomonas citronellolis and Escherichia coli were isolated from the inflamed appendix of a patient without underlying conditions. Compared to the Escherichia coli, Pseudomonas citronellolis has never been isolated in patients with appendicitis. We identified the species using MALDI-TOF MS and genetic sequencing. Based on our findings, we highlight the perspective that Pseudomonas citronellolis can colonize the intestines of healthy individuals and may trigger infections like appendicitis.


Assuntos
Apendicite , Enterocolite , Pseudomonas , Idoso , Humanos , Escherichia coli/genética , Virulência , Intestinos , Doença Aguda , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
4.
Sci Rep ; 14(1): 5148, 2024 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429351

RESUMO

Colistin remains one of the last-resort therapies for combating infections caused by multidrug-resistant (MDR) Enterobacterales, despite its adverse nephro- and neuro-toxic effects. This study elucidates the mechanism of action of a non-antibiotic 4-anilinoquinazoline-based compound that synergistically enhances the effectiveness of colistin against Salmonella enterica. The quinazoline sensitizes Salmonella by deactivating intrinsic, mutational, and transferable resistance mechanisms that enable Salmonella to counteract the antibiotic impact colistin, together with an induced disruption to the electrochemical balance of the bacterial membrane. The attenuation of colistin resistance via the combined treatment approach also proves efficacious against E. coli, Klebsiella, and Acinetobacter strains. The dual therapy reduces the mortality of Galleria mellonella larvae undergoing a systemic Salmonella infection when compared to individual drug treatments. Overall, our findings unveil the potential of the quinazoline-colistin combined therapy as an innovative strategy against MDR bacteria.


Assuntos
Mariposas , Infecções por Salmonella , Animais , Colistina/farmacologia , Colistina/uso terapêutico , Escherichia coli , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Farmacorresistência Bacteriana Múltipla , Infecções por Salmonella/tratamento farmacológico , Testes de Sensibilidade Microbiana
5.
Microb Cell Fact ; 23(1): 72, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38429691

RESUMO

BACKGROUND: Bacterial surface glycans are assembled by glycosyltransferases (GTs) that transfer sugar monomers to long-chained lipid carriers. Most bacteria employ the 55-carbon chain undecaprenyl phosphate (Und-P) to scaffold glycan assembly. The amount of Und-P available for glycan synthesis is thought to be limited by the rate of Und-P synthesis and by competition for Und-P between phosphoglycosyl transferases (PGTs) and GTs that prime glycan assembly (which we collectively refer to as PGT/GTs). While decreasing Und-P availability disrupts glycan synthesis and promotes cell death, less is known about the effects of increased Und-P availability. RESULTS: To determine if cells can maintain higher Und-P levels, we first reduced intracellular competition for Und-P by deleting all known non-essential PGT/GTs in the Gram-negative bacterium Escherichia coli (hereafter called ΔPGT/GT cells). We then increased the rate of Und-P synthesis in ΔPGT/GT cells by overexpressing the Und-P(P) synthase uppS from a plasmid (puppS). Und-P quantitation revealed that ΔPGT/GT/puppS cells can be induced to maintain 3-fold more Und-P than wild type cells. Next, we determined how increasing Und-P availability affects glycan expression. Interestingly, increasing Und-P availability increased endogenous and recombinant glycan expression. In particular, ΔPGT/GT/puppS cells could be induced to express 7-fold more capsule from Streptococcus pneumoniae serotype 4 than traditional E. coli cells used to express recombinant glycans. CONCLUSIONS: We demonstrate that the biotechnology standard bacterium E. coli can be engineered to maintain higher levels of Und-P. The results also strongly suggest that Und-P pathways can be engineered to increase the expression of potentially any Und-P-dependent polymer. Given that many bacterial glycans are central to the production of vaccines, diagnostics, and therapeutics, increasing Und-P availability should be a foremost consideration when designing bacterial glycan expression systems.


Assuntos
Escherichia coli , Fosfatos de Poli-Isoprenil , Escherichia coli/genética , Polissacarídeos , Biotecnologia
6.
Actas Esp Psiquiatr ; 52(1): 1-9, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38454896

RESUMO

BACKGROUND: Depression has become one of the most common mood disorders in adolescents, with an increasing incidence each year. Abnormal activation of peripheral immunity causes an increase in pro-inflammatory factors, which in turn affects neuroendocrine dysfunction and alters neurobiochemistry, leading to depression. In this study, we aimed to explore the relationship between inflammatory immune function and intestinal flora in adolescents with first-episode depression. METHODS: A total of 170 cases of adolescent patients with first-episode depression who attended our hospital from January 2020 to March 2023 were retrospectively selected as the observation group. Simultaneously, 170 individuals who underwent a healthy physical examination during the same period were chosen as the control group. The enzyme-linked immunosorbent assay (ELISA) was employed to quantify the levels of monoamine neurotransmitters 5-hydroxytryptamine (5-HT), substance P (SP), neuropeptide Y (NPY), serum tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-6 in the patients. Flow cytometry was utilized to assess the levels of T-lymphocytes CD3+, CD4+, and CD8+ cells. The levels of 16S ribosomal RNA (16SrRNA) method were used to determine the intestinal flora of the subjects in both groups. Inflammatory factor levels, immune function, and intestinal flora expression were observed, and correlation analysis was performed. RESULTS: The levels of 5-HT and NPY in the observation group were lower than those in the control group. The SP level was significantly higher in the observation group compared to the control group (p < 0.05). The observation group demonstrated significantly higher TNF-α, IL-1ß, and IL-6 levels than the control group (p < 0.05). The values of CD3+, CD4+, CD4+/CD8+ in the observation group were lower than those in the control group (p < 0.05), whereas the CD8+ values were notably higher (p < 0.05). Bifidobacterium, Escherichia coli, Lactobacillus, and Bacteroides in the observation group were less than those in the control group (p < 0.05). The content of Bifidobacterium was negatively correlated with the level of TNF-α (r = -0.358, p < 0.001), positively correlated with the level of CD3+, CD4+, CD4+/CD8+ (r = 0.490, 0.169, 0.165, p < 0.05), and negatively correlated with the level of CD8+ (r = -0.154, p < 0.05). The level of Escherichia coli content was negatively correlated with the levels of IL-6, CD3+, CD4+, CD4+/CD8+ (r = -0.483, -0.548, -0.317, -0.328, p < 0.001), and positively correlated with the levels of CD8+ (r = 0.325, p < 0.001). The content of Lactobacillus was positively correlated with the levels of CD3+, CD4+, CD4+/CD8+ (r = 0.552, 0.188, 0.194, p < 0.05), and negatively correlated with the level of CD8+ (r = -0.186, p < 0.05). The content of Bacteroides was positively correlated with the level of CD3+, CD4+, CD4+/CD8+ (r = -0.570, -0.183, -0.193, p < 0.05), and negatively correlated with the level of CD8+ levels were positively correlated (r = 0.187, p < 0.05). CONCLUSIONS: The intestinal flora is related to the level of inflammatory factors and immune function. Further study on the relationship between intestinal flora, inflammatory immune function, and depression could offer novel insights for the prevention and treatment of depressive disorders.


Assuntos
Microbioma Gastrointestinal , Fator de Necrose Tumoral alfa , Humanos , Adolescente , Interleucina-6 , Depressão , Estudos Retrospectivos , Serotonina , Escherichia coli , Imunidade
7.
Microb Biotechnol ; 17(3): e14427, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38465475

RESUMO

Optimal transcriptional regulatory circuits are expected to exhibit stringent control, maintaining silence in the absence of inducers while exhibiting a broad induction dynamic range upon the addition of effectors. In the Plac /LacI pair, the promoter of the lac operon in Escherichia coli is characterized by its leakiness, attributed to the moderate affinity of LacI for its operator target. In response to this limitation, the LacI regulatory protein underwent engineering to enhance its regulatory properties. The M7 mutant, carrying I79T and N246S mutations, resulted in the lac promoter displaying approximately 95% less leaky expression and a broader induction dynamic range compared to the wild-type LacI. An in-depth analysis of each mutation revealed distinct regulatory profiles. In contrast to the wild-type LacI, the M7 mutant exhibited a tighter binding to the operator sequence, as evidenced by surface plasmon resonance studies. Leveraging the capabilities of the M7 mutant, a high-value sugar biosensor was constructed. This biosensor facilitated the selection of mutant galactosidases with approximately a seven-fold improvement in specific activity for transgalactosylation. Consequently, this advancement enabled enhanced biosynthesis of galacto-oligosaccharides (GOS).


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Repressores Lac/genética , Repressores Lac/química , Repressores Lac/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Regiões Promotoras Genéticas , Proteínas de Bactérias/genética
8.
Methods Mol Biol ; 2760: 147-155, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468087

RESUMO

Microbial genome editing can be achieved by donor DNA-directed mutagenesis and CRISPR-Cas12a-mediated negative selection. Single-nucleotide-level genome editing enables the manipulation of microbial cells exactly as designed. Here, we describe single-nucleotide substitutions/indels in the target DNA of E. coli genome using a mutagenic DNA oligonucleotide donor and truncated crRNA/Cas12a system. The maximal truncation of nucleotides at the 3'-end of the crRNA enables Cas12a-mediated single-nucleotide-level precise editing at galK targets in the genome of E. coli.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Sistemas CRISPR-Cas/genética , RNA Guia de Sistemas CRISPR-Cas , Nucleotídeos , Escherichia coli/genética , Genoma Microbiano , DNA
9.
Methods Mol Biol ; 2760: 253-265, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468093

RESUMO

Positive selection screens are high-throughput assays to characterize novel enzymes from environmental samples and enrich for more powerful variants from libraries in applications such as biodiversity mining and directed evolution. However, overly stringent selection can limit the power of these screens due to a high false-negative rate. To create a more flexible and less restrictive screen for novel programmable DNA endonucleases, we developed a novel I-SceI-based platform. In this system, mutant E. coli genomes are cleaved upon induction of I-SceI to inhibit cell growth. Growth is rescued in an activity-dependent manner by plasmid curing or cleavage of the I-SceI expression plasmid via endonuclease candidates. More active candidates more readily proliferate and overtake growth of less active variants leading to enrichment. While demonstrated here with Cas9, this protocol can be readily adapted to any programmable DNA endonuclease and used to characterize single candidates or to enrich more powerful variants from pooled candidates or libraries.


Assuntos
Proteínas de Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos/genética , Endonucleases/genética
10.
Methods Mol Biol ; 2760: 345-369, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468098

RESUMO

The identification of essential genes is a key challenge in systems and synthetic biology, particularly for engineering metabolic pathways that convert feedstocks into valuable products. Assessment of gene essentiality at a genome scale requires large and costly growth assays of knockout strains. Here we describe a strategy to predict the essentiality of metabolic genes using binary classification algorithms. The approach combines elements from genome-scale metabolic models, directed graphs, and machine learning into a predictive model that can be trained on small knockout data. We demonstrate the efficacy of this approach using the most complete metabolic model of Escherichia coli and various machine learning algorithms for binary classification.


Assuntos
Algoritmos , Aprendizado de Máquina , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Essenciais , Redes e Vias Metabólicas/genética
11.
Methods Mol Biol ; 2760: 479-507, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468105

RESUMO

Small regulatory RNAs (sRNAs) are short non-coding RNAs in bacteria capable of post-transcriptional regulation. sRNAs have recently gained attention as tools in basic and applied sciences, for example, to fine-tune genetic circuits or biotechnological processes. Even though sRNAs often have a rather simple and modular structure, the design of functional synthetic sRNAs is not necessarily trivial. This protocol outlines how to use computational predictions and synthetic biology approaches to design, construct, and validate synthetic sRNA functionality for their application in bacteria. The computational tool, SEEDling, matches the optimal seed region with the user-selected sRNA scaffold for repression of target mRNAs. The synthetic sRNAs are assembled using Golden Gate cloning and their functionality is subsequently validated. The protocol uses the acrA mRNA as an exemplary proof-of-concept target in Escherichia coli. Since AcrA is part of a multidrug efflux pump, acrA repression can be revealed by assessing oxacillin susceptibility in a phenotypic screen. However, in case target repression does not result in a screenable phenotype, an alternative validation of synthetic sRNA functionality based on a fluorescence reporter is described.


Assuntos
Pequeno RNA não Traduzido , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/química , Bactérias/genética , RNA Mensageiro/genética , Escherichia coli/genética , RNA Bacteriano/genética , RNA Bacteriano/química , Regulação Bacteriana da Expressão Gênica
12.
Methods Mol Biol ; 2760: 447-461, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38468103

RESUMO

Cell-free transcription-translation (TXTL) enables achieving an ever-growing number of applications, ranging from the rapid characterization of DNA parts to the production of biologics. As TXTL systems gain in versatility and efficacy, larger DNAs can be expressed in vitro extending the scope of cell-free biomanufacturing to new territories. The demonstration that complex entities such as infectious bacteriophages can be synthesized from their genomes in TXTL reactions opens new opportunities, especially for biomedical applications. Over the last century, phages have been instrumental in the discovery of many ground-breaking biotechnologies including CRISPR. The primary function of phages is to infect bacteria. In that capacity, phages are considered an alternative approach to tackling current societal problems such as the rise of antibiotic-resistant microbes. TXTL provides alternative means to produce phages and with several advantages over in vivo synthesis methods. In this chapter, we describe the basic procedures to purify phage genomes, cell-free synthesize phages, and quantitate them using an all-E. coli TXTL system.


Assuntos
Bacteriófagos , Bacteriófagos/genética , Escherichia coli/genética , DNA , Biotecnologia , Antibacterianos
13.
Front Cell Infect Microbiol ; 14: 1343858, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38469349

RESUMO

Introduction: The emergence of extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae is an urgent and alarming One Health problem. This study aimed to investigate duplications of plasmid-encoded ESBL genes and their impact on antimicrobial resistance (AMR) phenotypes in clinical and screening isolates. Methods: Multi-drug-resistant bacteria from hospitalized patients were collected during routine clinical surveillance from January 2022 to June 2023, and their antimicrobial susceptibility patterns were determined. Genotypes were extracted from long-read whole-genome sequencing data. Furthermore, plasmids and other mobile genetic elements associated with ESBL genes were characterized, and the ESBL genes were correlated to ceftazidime minimal inhibitory concentration (MIC). Results: In total, we identified four cases of plasmid-encoded ESBL gene duplications that match four genetically similar plasmids during the 18-month surveillance period: five Escherichia coli and three Klebsiella pneumoniae isolates. As the ESBL genes were part of transposable elements, the surrounding sequence regions were duplicated as well. In-depth analysis revealed insertion sequence (IS)-mediated transposition mechanisms. Isolates with duplicated ESBL genes exhibited a higher MIC for ceftazidime in comparison to isolates with a single gene copy (3-256 vs. 1.5-32 mg/L, respectively). Conclusion: ESBL gene duplications led to an increased phenotypic resistance against ceftazidime. Our data suggest that ESBL gene duplications by an IS-mediated transposition are a relevant mechanism for how AMR develops in the clinical setting and is part of the microevolution of plasmids.


Assuntos
Antibacterianos , Ceftazidima , Humanos , Ceftazidima/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , beta-Lactamases/genética , Duplicação Gênica , Escherichia coli , Plasmídeos/genética , Enterobacteriaceae/genética , Klebsiella pneumoniae , Testes de Sensibilidade Microbiana
14.
Cancer Cell ; 42(3): 487-496.e6, 2024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38471458

RESUMO

Co-culture of intestinal organoids with a colibactin-producing pks+E. coli strain (EcC) revealed mutational signatures also found in colorectal cancer (CRC). E. coli Nissle 1917 (EcN) remains a commonly used probiotic, despite harboring the pks operon and inducing double strand DNA breaks. We determine the mutagenicity of EcN and three CRC-derived pks+E. coli strains with an analytical framework based on sequence characteristic of colibactin-induced mutations. All strains, including EcN, display varying levels of mutagenic activity. Furthermore, a machine learning approach attributing individual mutations to colibactin reveals that patients with colibactin-induced mutations are diagnosed at a younger age and that colibactin can induce a specific APC mutation. These approaches allow the sensitive detection of colibactin-induced mutations in ∼12% of CRC genomes and even in whole exome sequencing data, representing a crucial step toward pinpointing the mutagenic activity of distinct pks+E. coli strains.


Assuntos
Neoplasias Colorretais , Escherichia coli , Peptídeos , Policetídeos , Humanos , Escherichia coli/genética , Mutação , Dano ao DNA , Mutagênicos , Organoides
15.
Biotechnol J ; 19(3): e2300642, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38472088

RESUMO

The biosynthesis of cadaverine from lysine is an environmentally promising technology, that could contribute to a more sustainable approach to manufacturing bio-nylon 5X. However, the titer of biosynthesized cadaverine has still not reached a sufficient level for industrial production. A powerful green cell factory was developed to enhance cadaverine production by regulating lipopolysaccharide (LPS) genes and improving membrane permeability. Firstly, 10 LPS mutant strains were constructed and the effect on the growth was investigated. Then, the lysine decarboxylase (CadA) was overexpressed in 10 LPS mutant strains of Escherichia coli MG1655 and the ability to produce cadaverine was compared. Using 20.0 g L-1 of L-lysine hydrochloride (L-lysine-HCl) as the substrate for the biotransformation reaction, Cad02 and Cad06 strains exhibited high production levels of cadaverine, with 8.95 g L-1 and 7.55 g L-1 respectively while the control strain Cad00 only 4.92 g L-1 . Directed evolution of CadA was also used to improve its stability under alkaline conditions. The cadaverine production of the Cad02-M mutant stain increased by 1.86 times at pH 8.0. Finally, the production process was scaled up using recombinant whole cells as catalysts, achieving a high titer of 211 g L-1 cadaverine (96.8%) by fed-batch bioconversion. This study demonstrates the potential role of LPS in enhancing the efficiency of mass transfer between substrate and enzymes in vivo by increasing cell permeability. The results indicate that the argumentation of cell permeability could not only significantly enhance the biotransformation efficiency of cadaverine, but also provide a universally applicable, straightforward, environment-friendly, and cost-effective method for the biosynthesis of other high-value chemicals.


Assuntos
Escherichia coli , Lipopolissacarídeos , Escherichia coli/genética , Cadaverina/metabolismo , Lipopolissacarídeos/metabolismo , Catálise , Biotransformação , Lisina/metabolismo
16.
Arch Microbiol ; 206(4): 152, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472371

RESUMO

Producing recombinant proteins is a major accomplishment of biotechnology in the past century. Heterologous hosts, either eukaryotic or prokaryotic, are used for the production of these proteins. The utilization of microbial host systems continues to dominate as the most efficient and affordable method for biotherapeutics and food industry productions. Hence, it is crucial to analyze the limitations and advantages of microbial hosts to enhance the efficient production of recombinant proteins on a large scale. E. coli is widely used as a host for the production of recombinant proteins. Researchers have identified certain obstacles with this host, and given the growing demand for recombinant protein production, there is an immediate requirement to enhance this host. The following review discusses the elements contributing to the manifestation of recombinant protein. Subsequently, it sheds light on innovative approaches aimed at improving the expression of recombinant protein. Lastly, it delves into the obstacles and optimization methods associated with translation, mentioning both cis-optimization and trans-optimization, producing soluble recombinant protein, and engineering the metal ion transportation. In this context, a comprehensive description of the distinct features will be provided, and this knowledge could potentially enhance the expression of recombinant proteins in E. coli.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Biotecnologia/métodos , Proteínas de Escherichia coli/metabolismo
17.
Int J Nanomedicine ; 19: 2429-2440, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38476285

RESUMO

Purpose: COVID-19 is rampant throughout the world, which has caused great damage to human lives and seriously hindered the development of the global economy. Aiming at the treatment of SARS-CoV-2, in this study, we proposed a novel fenobody strategy based on ferritin (Fe) self-assembly technology. Methods: The neutralizing nanobody H11-D4 of SARS-CoV-2 fused to the C-terminus of end-modified human ferritin was expressed in E. coli and silkworm baculovirus expression systems. A large number of nanoparticles were successfully self-assembled in silkworms, while relatively few nanoparticles can be observed in the treated products from E. coli by electron microscopy. Subsequently, the fenobody's expression level and neutralizing activity were then evaluated. Results: The results showed that the IC50 of H11-D4 and fenobody Fe-H11-D4 expressed in E. coli were 171.1 nmol L-1 and 20.87 nmol L-1, respectively. However, the IC50 of Fe-HD11-D4 expressed in silkworms was 1.46 nmol L-1 showing better neutralization activity. Conclusion: Therefore, fenobodies can be well self-assembled in silkworm baculovirus expression system, and ferritin self-assembly technology can effectively improve nanobody neutralization activity.


Assuntos
COVID-19 , SARS-CoV-2 , Humanos , Ferritinas , Escherichia coli , Anticorpos Neutralizantes , Anticorpos Antivirais
18.
RNA Biol ; 21(1): 1-18, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-38469716

RESUMO

RNA degradation is critical for synchronising gene expression with changing conditions in prokaryotic and eukaryotic organisms. In bacteria, the preference of the central ribonucleases RNase E, RNase J and RNase Y for 5'-monophosphorylated RNAs is considered important for RNA degradation. For RNase E, the underlying mechanism is termed 5' sensing, contrasting to the alternative 'direct entry' mode, which is independent of monophosphorylated 5' ends. Cyanobacteria, such as Synechocystis sp. PCC 6803 (Synechocystis), encode RNase E and RNase J homologues. Here, we constructed a Synechocystis strain lacking the 5' sensing function of RNase E and mapped on a transcriptome-wide level 283 5'-sensing-dependent cleavage sites. These included so far unknown targets such as mRNAs encoding proteins related to energy metabolism and carbon fixation. The 5' sensing function of cyanobacterial RNase E is important for the maturation of rRNA and several tRNAs, including tRNAGluUUC. This tRNA activates glutamate for tetrapyrrole biosynthesis in plant chloroplasts and in most prokaryotes. Furthermore, we found that increased RNase activities lead to a higher copy number of the major Synechocystis plasmids pSYSA and pSYSM. These results provide a first step towards understanding the importance of the different target mechanisms of RNase E outside Escherichia coli.


Assuntos
Endorribonucleases , Synechocystis , Endorribonucleases/genética , Endorribonucleases/metabolismo , RNA , Ribonucleases , Escherichia coli/genética , Escherichia coli/metabolismo , Synechocystis/genética , RNA de Transferência
19.
Nat Commun ; 15(1): 2223, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472230

RESUMO

Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.


Assuntos
Bacteriófagos , Bacteriófagos/genética , Escherichia coli/genética , Genoma , Engenharia
20.
Int J Mol Sci ; 25(5)2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38473724

RESUMO

Although the SARS-CoV-2 vaccination is the primary preventive intervention, there are still few antiviral therapies available, with current drugs decreasing viral replication once the virus is intracellular. Adding novel drugs to target additional points in the viral life cycle is paramount in preventing future pandemics. The purpose of this study was to create and test a novel protein to decrease SARS-CoV-2 replication. We created the recombinant rod domain of vimentin (rhRod) in E. coli and used biolayer interferometry to measure its affinity to the SARS-CoV-2 S1S2 spike protein and the ability to block the SARS-CoV-2-ACE2 interaction. We performed plaque assays to measure rhRod's effect on SARS-CoV-2 replication in Vero E6 cells. Finally, we measured lung inflammation in SARS-CoV-2-exposed K18-hACE transgenic mice given intranasal and intraperitoneal rhRod. We found that rhRod has a high affinity for the S1S2 protein with a strong ability to block S1S2-ACE2 interactions. The daily addition of rhRod decreased viral replication in Vero E6 cells starting at 48 h at concentrations >1 µM. Finally, SARS-CoV-2-infected mice receiving rhRod had decreased lung inflammation compared to mock-treated animals. Based on our data, rhRod decreases SARS-CoV-2 replication in vitro and lung inflammation in vivo. Future studies will need to evaluate the protective effects of rhRod against additional viral variants and identify the optimal dosing scheme that both prevents viral replication and host lung injury.


Assuntos
COVID-19 , Pneumonia , Humanos , Camundongos , Animais , SARS-CoV-2/metabolismo , Enzima de Conversão de Angiotensina 2/farmacologia , Vimentina , Glicoproteína da Espícula de Coronavírus/metabolismo , Vacinas contra COVID-19/farmacologia , Escherichia coli/metabolismo , Replicação Viral
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