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1.
Viruses ; 13(4)2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33916100

RESUMO

Sindbis virus (SINV), a positive-sense single stranded RNA virus that causes mild symptoms in humans, is transmitted by mosquito bites. SINV reverse genetics have many implications, not only in understanding alphavirus transmission, replication cycle, and virus-host interactions, but also in biotechnology and biomedical applications. The rescue of SINV infectious particles is usually achieved by transfecting susceptible cells (BHK-21) with SINV-infectious mRNA genomes generated from cDNA constructed via in vitro translation (IVT). That procedure is time consuming, costly, and relies heavily on reagent quality. Here, we constructed a novel infectious SINV cDNA construct that expresses its genomic RNA in yeast cells controlled by galactose induction. Using spheroplasts made from this yeast, we established a robust polyethylene glycol-mediated yeast: BHK-21 fusion protocol to rescue infectious SINV particles. Our approach is timesaving and utilizes common lab reagents for SINV rescue. It could be a useful tool for the rescue of large single strand RNA viruses, such as SARS-CoV-2.


Assuntos
Infecções por Alphavirus/virologia , Fusão Celular , Interações entre Hospedeiro e Microrganismos/fisiologia , Vírus Sindbis/genética , Esferoplastos , Leveduras/genética , Animais , COVID-19 , DNA Complementar , RNA Viral/genética , SARS-CoV-2 , Saccharomyces cerevisiae , Leveduras/virologia
2.
Cold Spring Harb Protoc ; 2021(2)2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33526418

RESUMO

For expression of some proteins in Escherichia coli, export to the periplasmic space is preferred over conventional expression in the cytosol. Export can be accomplished by fusing the coding sequence to DNA encoding a signal peptide (e.g., using pET-22b), which is cleaved by the bacterial signal peptidase as the protein is exported into the space between the inner and outer membranes of E. coli This protocol uses osmotic shock to release polypeptides from the periplasm. Although not quantitative, it should provide preliminary information on the cellular location of signal peptide fusion proteins.


Assuntos
Escherichia coli/metabolismo , Sinais Direcionadores de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Clonagem Molecular , Transporte Proteico , Esferoplastos/metabolismo , Frações Subcelulares/metabolismo
3.
Clin Chim Acta ; 515: 13-15, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33359495

RESUMO

BACKGROUND: In two patients under treatment with various antibiotics, spheroplasts were detected with an automated urine sediment analyzer. METHODS: Urinalysis was performed by an AutionMAX AX 4030-sediMAX platform. RESULTS: Spheroplasts can be easily misclassified as yeasts or erythrocytes, but when automated urine sediment analyzers are used by well-trained, and experienced operators they can be correctly identified and classified. CONCLUSION: Appropriate training of urine laboratory professionals in spheroplast detection and association with UTI, together with timely communication with the microbiologist and caring clinician, will provide prompt targeted treatment.


Assuntos
Eritrócitos , Urinálise , Comunicação , Humanos , Esferoplastos
4.
Biomed Res Int ; 2020: 6152356, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33083475

RESUMO

Protecting foods from contamination applying peptides produced by lactic acid bacteria is a promising strategy to increase the food quality and safety. Interacting with the pathogen membranes might produce visible changes in shape or cell wall damage. Previously, we showed that the peptides produced by Lactobacillus plantarum UTNGt2, Lactobacillus plantarum UTNCys5-4, and Lactococcus lactis subsp. lactis UTNGt28 exhibit a broad spectrum of antibacterial activity against several foodborne pathogens in vitro. In this study, their possible mode of action against the commensal microorganism Salmonella enterica subsp. enterica ATCC51741 was investigated. The target membrane permeability was determined by detection of beta-galactosidase release from ONPG (o-nitro-phenyl-L-D-galactoside) substrate and changes in the whole protein profile indicating that the peptide extracts destroy the membrane integrity and may induce breaks in membrane proteins to some extent. The release of aromatic molecules such as DNA/RNA was detected after the interaction of Salmonella with the peptide extract. Transmission electronic microscopy (TEM) micrographs depicted at least four simultaneous secondary events after the peptide extract treatment underlying their antimicrobial actions, including morphological alterations of the membrane. Spheroplast and filament formation, vacuolation, and DNA relaxation were identified as the principal events from the Gt2 and Cys5-4 peptide extracts, while Gt28 induced the formation of ghost cells by release of cytoplasmic content, filaments, and separation of cell envelope layers. Gel retarding assays indicate that the Gt2 and Gt28 peptide extracts are clearly binding the Salmonella DNA, while Cys5-4 partially interacted with Salmonella genomic DNA. These results increased our knowledge about the inhibitory mechanism employed by several peptide extracts from native lactic acid bacteria against Salmonella. Further, we shall develop peptide-based formulation and evaluate their biocontrol effect in the food chains.


Assuntos
Antibacterianos/farmacologia , Lactobacillales/metabolismo , Peptídeos/farmacologia , Salmonella enterica/efeitos dos fármacos , Esferoplastos/metabolismo , DNA/genética , Microbiologia de Alimentos/métodos , Salmonella enterica/genética
5.
J Biol Chem ; 295(52): 17950-17972, 2020 12 25.
Artigo em Inglês | MEDLINE | ID: mdl-32994219

RESUMO

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane-binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.


Assuntos
Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esferoplastos/metabolismo , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Células HEK293 , Humanos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Produtos do Gene gag do Vírus da Imunodeficiência Humana/genética
6.
Methods Cell Biol ; 160: 61-82, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32896333

RESUMO

Plants possess numerous ion channels that respond to a range of stimuli, including small molecules, transmembrane voltage, and mechanical force. Many in the latter category, known as mechanosensitive (MS) ion channels, open directly in response to increases in lateral membrane tension. One of the most effective techniques for characterizing ion channel properties is patch-clamp electrophysiology, in which the current through a section of membrane containing ion channels is measured. For MS channels, this technique enables the measurement of key channel properties such as tension sensitivity, conductance, and ion selectivity. These characteristics, along with the phenotypes of genetic mutants, can help reveal the physiological roles of a particular MS channel. In this protocol, we provide detailed instructions on how to study MS ion channels using single-channel patch-clamp electrophysiology in giant E. coli spheroplasts. We first present an optimized method for preparing giant spheroplasts, then describe how to measure MS channel activity using patch-clamp electrophysiology and analyze the resulting data. We also provide recommended equipment lists, setup schematics, and useful conventions.


Assuntos
Fenômenos Eletrofisiológicos , Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Mecanotransdução Celular , Técnicas de Patch-Clamp/métodos , Plantas/metabolismo , Esferoplastos/metabolismo , Animais , Galinhas
7.
Int J Mol Sci ; 21(19)2020 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-32992574

RESUMO

Cell enlargement is essential for the microinjection of various substances into bacterial cells. The cell wall (peptidoglycan) inhibits cell enlargement. Thus, bacterial protoplasts/spheroplasts are used for enlargement because they lack cell wall. Though bacterial species that are capable of gene manipulation are limited, procedure for bacterial cell enlargement does not involve any gene manipulation technique. In order to prevent cell wall resynthesis during enlargement of protoplasts/spheroplasts, incubation media are supplemented with inhibitors of peptidoglycan biosynthesis such as penicillin. Moreover, metal ion composition in the incubation medium affects the properties of the plasma membrane. Therefore, in order to generate enlarged cells that are suitable for microinjection, metal ion composition in the medium should be considered. Experiment of bacterial protoplast or spheroplast enlargement is useful for studies on bacterial plasma membrane biosynthesis. In this paper, we have summarized the factors that influence bacterial cell enlargement.


Assuntos
Bactérias/citologia , Crescimento Celular , Meios de Cultura/química , Protoplastos/fisiologia , Esferoplastos/crescimento & desenvolvimento , Membrana Celular/metabolismo , Parede Celular/efeitos dos fármacos , Íons/química , Metais/química , Pressão Osmótica , Penicilinas/farmacologia , Peptidoglicano/biossíntese , Biossíntese de Proteínas/efeitos dos fármacos
8.
Cold Spring Harb Protoc ; 2020(10)2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32763978

RESUMO

In this protocol, yeast DNA is prepared by digestion of the cell wall and lysis of the resulting spheroplasts with SDS. This method reproducibly yields several micrograms of yeast DNA that can be efficiently cleaved by restriction enzymes and used as a template in polymerase chain reaction (PCR). Note that yeast colonies can also be used directly in PCR, without purifying yeast DNA.


Assuntos
Parede Celular/química , DNA Fúngico/genética , Reação em Cadeia da Polimerase/métodos , Saccharomyces cerevisiae/genética , Dodecilsulfato de Sódio/química , Esferoplastos/química , Clonagem Molecular/métodos , Meios de Cultura/química , Enzimas de Restrição do DNA/metabolismo , DNA Fúngico/isolamento & purificação , DNA Fúngico/metabolismo , Biblioteca Genômica , Saccharomyces cerevisiae/crescimento & desenvolvimento
9.
Annu Rev Biochem ; 89: 77-101, 2020 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-32569517

RESUMO

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.


Assuntos
DNA/genética , Edição de Genes/métodos , Técnicas de Transferência de Genes , Genes Sintéticos , Engenharia Genética/métodos , Genoma , Sistemas CRISPR-Cas , DNA/química , DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Oligonucleotídeos/síntese química , Oligonucleotídeos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Poliovirus/genética , Poliovirus/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esferoplastos/genética , Esferoplastos/metabolismo
10.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510483

RESUMO

The powerful tools available to edit yeast genomes have made this microbe a valuable platform for engineering. While it is now possible to construct libraries of millions of genetically distinct strains, screening for a desired phenotype remains a significant obstacle. With existing screening techniques, there is a tradeoff between information output and throughput, with high-throughput screening typically being performed on one product of interest. Therefore, we present an approach to accelerate strain screening by adapting single cell RNA sequencing to isogenic picoliter colonies of genetically engineered yeast strains. To address the unique challenges of performing RNA sequencing on yeast cells, we culture isogenic yeast colonies within hydrogels and spheroplast prior to performing RNA sequencing. The RNA sequencing data can be used to infer yeast phenotypes and sort out engineered pathways. The scalability of our method addresses a critical obstruction in microbial engineering.


Assuntos
Engenharia Genética/métodos , Ensaios de Triagem em Larga Escala/métodos , RNA Fúngico/análise , Saccharomyces cerevisiae/genética , Análise de Sequência de RNA/métodos , Esferoplastos/genética , Fenótipo , Saccharomyces cerevisiae/classificação , Saccharomyces cerevisiae/metabolismo
11.
Elife ; 92020 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-32420869

RESUMO

Current methods for single-cell RNA sequencing (scRNA-seq) of yeast cells do not match the throughput and relative simplicity of the state-of-the-art techniques that are available for mammalian cells. In this study, we report how 10x Genomics' droplet-based single-cell RNA sequencing technology can be modified to allow analysis of yeast cells. The protocol, which is based on in-droplet spheroplasting of the cells, yields an order-of-magnitude higher throughput in comparison to existing methods. After extensive validation of the method, we demonstrate its use by studying the dynamics of the response of isogenic yeast populations to a shift in carbon source, revealing the heterogeneity and underlying molecular processes during this shift. The method we describe opens new avenues for studies focusing on yeast cells, as well as other cells with a degradable cell wall.


Assuntos
Metabolismo Energético/genética , Glucose/metabolismo , Maltose/metabolismo , RNA-Seq/métodos , Análise de Célula Única/métodos , Carbono/metabolismo , Metabolismo Energético/fisiologia , Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , RNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Esferoplastos , Transcrição Genética/genética , Transcriptoma/genética
12.
Prion ; 14(1): 118-128, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32306832

RESUMO

Semi-denaturing detergent agarose gel electrophoresis (SDD-AGE) was proposed by Vitaly V. Kushnirov in the Michael D. Ter-Avanesyan's laboratory as a method to compare sizes of amyloid aggregates. Currently, this method is widely used for amyloid investigation, but mostly as a qualitative approach. In this work, we assessed the possibilities and limitations of the quantitative analysis of amyloid aggregate size distribution using SDD-AGE results. For this purpose, we used aggregates of two well-characterized yeast amyloid-forming proteins, Sup35 and Rnq1, and developed a protocol to standardize image analysis and process the result. A detailed investigation of factors that may affect the results of SDD-AGE revealed that both the cell lysis method and electrophoresis conditions can substantially affect the estimation of aggregate size. Despite this, quantitative analysis of SDD-AGE results is possible when one needs to estimate and compare the size of aggregates on the same gel, or even in different experiments, if the experimental conditions are tightly controlled and additional standards are used.


Assuntos
Amiloide/análise , Detergentes/química , Eletroforese em Gel de Ágar , Agregados Proteicos , Desnaturação Proteica , Amiloide/ultraestrutura , Tampões (Química) , Fracionamento Celular , Concentração de Íons de Hidrogênio , Peso Molecular , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Esferoplastos/metabolismo
13.
mSphere ; 5(1)2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-32024705

RESUMO

The determination of the exact location of a protein in the cell is essential to the understanding of biological processes. Here, we report for the first time the visualization of a protein of interest in Saccharomyces cerevisiae using focused ion beam scanning electron microscopy (FIB-SEM). As a proof of concept, the integral endoplasmic reticulum (ER) membrane protein Erg11 has been C-terminally tagged with APEX2, which is an engineered peroxidase that catalyzes an electron-dense deposition of 3,3'-diaminobenzidine (DAB), as such marking the location of the fused protein of interest in electron microscopic images. As DAB is unable to cross the yeast cell wall to react with APEX2, cell walls have been partly removed by the formation of spheroplasts. This has resulted in a clear electron-dense ER signal for the Erg11 protein using FIB-SEM. With this study, we have validated the use of the APEX2 tag for visualization of yeast proteins in electron microscopy. Furthermore, we have introduced a methodology that enables precise and three-dimensional (3D) localization studies in yeast, with nanometer resolution and without the need for antibody staining. Because of these properties, the described technique can offer valuable information on the molecular functions of studied proteins.IMPORTANCE With this study, we have validated the use of the APEX2 tag to define the localization of proteins in the model yeast S. cerevisiae As such, FIB-SEM can identify the exact 3D location of a protein of interest in the cell with nanometer-scale resolution. Such detailed imaging could provide essential information on the elucidation of various biological processes. APEX2, which adds electron density to a fused protein of interest upon addition of the substrate DAB, originally was used in mammalian studies. With this study, we expand its use to protein localization studies in one of the most important models in molecular biology.


Assuntos
Sistema Enzimático do Citocromo P-450/ultraestrutura , Imageamento Tridimensional/métodos , Proteínas de Saccharomyces cerevisiae/ultraestrutura , Saccharomyces cerevisiae/ultraestrutura , Esferoplastos/ultraestrutura , Parede Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Microscopia Eletrônica de Varredura , Saccharomyces cerevisiae/fisiologia
14.
Biochim Biophys Acta Biomembr ; 1862(4): 183176, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31923411

RESUMO

Bacterial mechanosensitive channels gate in response to membrane tension, driven by shifts in environmental osmolarity. The mechanosensitive channels of small conductance (MscS) and large conductance (MscL) from Escherichia coli (Ec) gate in response to mechanical force applied to the membrane. Ec-MscS is the foundational member of the MscS superfamily of ion channels, a diverse family with at least fifteen subfamilies identified by homology to the pore lining helix of Ec-MscS, as well as significant diversity on the N- and C-termini. The MscL family of channels are homologous to Ec-MscL. In a rhizosphere associated bacterium, Paraburkholderia graminis C4D1M, mechanosensitive channels are essential for cell survival during changing osmotic environments such as a rainstorm. Utilizing bioinformatics, we predicted six MscS superfamily members and a single MscL homologue. The MscS superfamily members fall into at least three subfamilies: bacterial cyclic nucleotide gated, multi-TM, and extended N-terminus. Osmotic downshock experiments show that wildtype P. graminis cells contain a survival mechanism that prevents cell lysis in response to hypoosmotic shock. To determine if this rescue is due to mechanosensitive channels, we developed a method to create giant spheroplasts of P. graminis to explore the single channel response to applied mechanical tension. Patch clamp electrophysiology on these spheroplasts shows two unique conductances: MscL-like and MscS-like. These conductances are due to likely three unique proteins. This indicates that channels that gate in response to mechanical tension are present in the membrane. Here, we report the first single channel evidence of mechanosensitive ion channels from P. graminis membranes.


Assuntos
Burkholderiaceae/genética , Mecanotransdução Celular/genética , Concentração Osmolar , Esferoplastos/genética , Burkholderiaceae/metabolismo , Sobrevivência Celular/genética , Microambiente Celular/genética , Biologia Computacional , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Canais Iônicos/genética , Canais Iônicos de Abertura Ativada por Ligante/genética , Pressão Osmótica , Rizosfera , Homologia de Sequência de Aminoácidos
15.
Biochim Biophys Acta Biomembr ; 1862(5): 183203, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31981589

RESUMO

Mechanosensitive (MS) channels have an intimate relationship with membrane lipids that underlie their mechanosensitivity. Membrane lipids may influence channel activity by directly interacting with MS channels or by influencing the global properties of the membrane such as elastic area expansion modulus or bending rigidity. Previous work has implicated membrane stiffness as a potential determinant of the mechanosensitivity of E. coli (Ec)MscS. Here we systematically tested this hypothesis using patch fluorometry of azolectin liposomes doped with lipids of increasing elastic area expansion modulus. Increasing dioleoylphosphatidylethanolamine (DOPE) content of azolectin liposomes made it more difficult to activate EcMscS by membrane tension (i.e. increased gating threshold). This effect was exacerbated by stiffer forms of phosphatidylethanolamine such as the branched chain lipid diphytanoylphosphoethanolamine (DPhPE) or the fully saturated lipid distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). Furthermore, a comparison of the branched chain lipid diphytanoylphosphocholine (DPhPC) to the stiffer DPhPE indicated again that it was harder to activate EcMscS in the presence of the stiffer DPhPE. We show that these effects are not due to changes in membrane bending rigidity as the membrane tension threshold of EcMscS in membranes doped with PC18:1 and PC18:3 remained the same, despite a two-fold difference in their bending rigidity. We also show that after prolonged pressure application sudden removal of force in softer membranes caused a rebound reactivation of EcMscS and we discuss the relevance of this phenomenon to bacterial osmoregulation. Collectively, our data suggests that membrane stiffness (elastic area expansion modulus) is one of the key determinants of the mechanosensitivity of EcMscS.


Assuntos
Proteínas de Escherichia coli/metabolismo , Canais Iônicos/metabolismo , Bicamadas Lipídicas/química , Mecanotransdução Celular/fisiologia , Transporte Biológico , Fenômenos Biomecânicos/fisiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Ativação do Canal Iônico/fisiologia , Canais Iônicos/química , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Lipídeos de Membrana/metabolismo , Membranas/metabolismo , Técnicas de Patch-Clamp/métodos , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas , Esferoplastos/metabolismo
16.
Sci Rep ; 9(1): 19558, 2019 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-31862951

RESUMO

The treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to ß-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to ß-lactam antibiotics are activated only when the bacterium encounters a ß-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the ß-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how ß-lactam binding culminates in the proteolytic degradation of MecI.


Assuntos
Proteínas de Bactérias/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/metabolismo , beta-Lactamas/farmacologia , Proteínas de Bactérias/genética , Western Blotting , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Simulação de Acoplamento Molecular , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Espectrometria de Fluorescência , Esferoplastos/efeitos dos fármacos , Esferoplastos/genética , Resistência beta-Lactâmica/genética
17.
Biochem Med (Zagreb) ; 29(3): 030801, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31624465

RESUMO

Introduction: It has already been reported that subinhibitory concentrations of ß-lactam antibiotics can cause abnormal changes of bacterial forms, such as spheroplasts. Herein we report a case of Croatian male patient with Escherichia coli spheroplasts present in urine after treatment with tazobactam, on the tenth day of hospitalization. The aim of this report is to emphasize the inability of imaging based automated urine analysers to recognize some relatively uncommon forms of bacterial presentation in urine sediment. Materials and methods: During routine urine analysis, unusual particles were observed in patient urine. Urine sediment was examined by two urine analysers: Atellica 1500 (Siemens, Germany) and Iris iQ200 (Beckman Coulter, USA). Additionally, urine was sent for culture testing to Microbiology department. Results: Both urine analysers didn't indicate presence of bacteria in urine sediment. Unusual particles observed on the tenth day were classified as erythrocytes by both instruments. Dipstick test showed blood trace and microscopic analysis revealed bacteria in urine. Urine culture was positive for Escherichia coli. Careful examination of urine sediment has confirmed that shapes present in urine were abnormal bacterial forms called spheroplasts. Conclusions: Imaging based automated urine analysers are not able to recognize bacterial spheroplasts in urine sediment misclassifying it as erythrocytes. Microscopic examination remains the gold standard for urines with blood trace or negative blood, in which erythrocytes are reported by urine analyser in urine sediment. Failure to identify and follow up such cases may lead to inaccurate treatment decisions and puts patient safety at risk.


Assuntos
Eritrócitos , Escherichia coli/isolamento & purificação , Esferoplastos/isolamento & purificação , Urinálise/métodos , Urinálise/normas , Croácia , Humanos , Masculino , Pessoa de Meia-Idade
18.
J Bacteriol ; 202(1)2019 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-31636107

RESUMO

Antimicrobial treatment can induce many bacterial pathogens to enter a cell wall-deficient state that contributes to persistent infections. The effect of this physiological state on the assembly of transenvelope-anchored organelles is not well understood. The type VI secretion system (T6SS) is a widespread molecular weapon for interspecies interactions and virulence, comprising a long double tubular structure and a transenvelope/baseplate complex. Here, we report that cell wall-deficient spheroplasts assembled highly flexible and elastic T6SS structures forming U, O, or S shapes. Upon contacting the inner membrane, the T6SS tubes did not contract but rather continued to grow along the membrane. Such deformation likely results from continual addition of sheath/tube subunits at the distal end. Induction of TagA repressed curved sheath formation. Curved sheaths could also contract and deliver T6SS substrates and were readily disassembled by the ClpV ATPase after contraction. Our data highlight the dramatic effect of cell wall deficiency on the shape of the T6SS structures and reveal the elastic nature of this double tubular contractile injection nanomachine.IMPORTANCE The cell wall is a physical scaffold that all transenvelope complexes have to cross for assembly. However, the cell wall-deficient state has been described as a common condition found in both Gram-negative and Gram-positive pathogens during persistent infections. Loss of cell wall is known to have pleiotropic physiological effects, but how membrane-anchored large cellular organelles adapt to this unique state is less completely understood. Our study examined the assembly of the T6SS in cell wall-deficient spheroplast cells. We report the elastic nature of contractile T6SS tubules under such conditions, providing key insights for understanding how large intracellular structures such as the T6SS accommodate the multifaceted changes in cell wall-deficient cells.


Assuntos
Sistemas de Secreção Tipo VI/fisiologia , Proteínas de Bactérias/fisiologia , Parede Celular/química , Parede Celular/fisiologia , Elasticidade , Lipoproteínas/fisiologia , Esferoplastos/fisiologia , Sistemas de Secreção Tipo VI/química
19.
Sci Rep ; 9(1): 13127, 2019 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-31511600

RESUMO

The killer toxin K1 is a virally encoded fungal A/B toxin acting by disrupting plasma membrane integrity. The connection of α and ß constitutes a critical feature for toxin biology and for decades the formation of three disulphide bonds linking the major toxin subunits was accepted as status quo. Due to the absence of experimental evidence, the involvement of each cysteine in heterodimer formation, K1 lethality and immunity was systematically analysed. Substitution of any cysteine in α led to a complete loss of toxin dimer secretion and toxicity, whereas K1 toxin derivatives carrying mutations of C248, C312 or the double mutation C248-312 were active against spheroplasted cells. Importantly, substitution of the C95 and C107 in the toxin precursor completely abolished the mediation of functional immunity. In contrast, K1 toxicity, i.e. its ionophoric effect, does not depend on the cysteine residues at all. In contrast to the literature, our data imply the formation of a single disulphide bond involving C92 in α and C239 in ß. This finding not only refines the current model stated for decades but also provides new opportunities to elucidate the mechanisms underlying K1 toxicity and immunity at the molecular level.


Assuntos
Cisteína/metabolismo , Fatores Matadores de Levedura/química , Mutação , Precursores de Proteínas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Esferoplastos/imunologia , Transporte Biológico , Membrana Celular/imunologia , Membrana Celular/metabolismo , Cisteína/química , Cisteína/genética , Fatores Matadores de Levedura/genética , Fatores Matadores de Levedura/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Esferoplastos/metabolismo
20.
Appl Environ Microbiol ; 85(20)2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31420339

RESUMO

A viability quantitative PCR (qPCR) utilizing propidium monoazide (PMA) is presented for rapid quantification of viable cells using the foodborne pathogen Campylobacter coli as a bacterial model. It includes optimized spheroplast formation via lysozyme and EDTA, induction of a mild osmotic shock for enhancing the selective penetration of PMA into dead cells, and exploitation of an internal sample process control (ISPC) involving cell inactivation to assess residual false-positive signals within each sample. Spheroplasting of bacteria in exponential phase did not permit PMA entrance into viable cells since a strong linear relationship was detected between simple qPCR and PMA-qPCR quantification, and no differences were observed regardless of whether spheroplasting was utilized. The PMA-qPCR signal suppression of dead cells was elevated using spheroplast formation. With regard to the ISPC, cell inactivation by hydrogen peroxide resulted in higher signal suppression during qPCR than heat inactivation did. Viability quantification of C. coli cells by optimized spheroplasting-PMA-qPCR with ISPC was successfully applied in an aging pure culture under aerobic conditions and artificially inoculated meat. The same method exhibited a high linear range of quantification (1.5 to 8.5 log10 viable cells ml-1), and results were highly correlated with culture-based enumeration. PMA-qPCR quantification of viable cells can be affected by their rigidity, age, culture media, and niches, but spheroplast formation along with osmotic shock and the use of a proper ISPC can address such variations. The developed methodology could detect cells in a viable-but-nonculturable state and might be utilized for the quantification of other Gram-negative bacteria.IMPORTANCE There is need for rapid and accurate methods to detect viable bacterial cells of foodborne pathogens. Conventional culture-based methods are time-consuming and unable to detect bacteria in a viable-but-nonculturable state. The high sensitivity and specificity of the quantitative PCR (qPCR) are negated by its inability to differentiate the DNAs from viable and dead cells. The combination of propidium monoazide (PMA), a DNA-intercalating dye, with qPCR assays is promising for detection of viable cells. Despite encouraging results, these assays still encounter various challenges, such as false-positive signals by dead cells and the lack of an internal control identifying these signals per sample. The significance of our research lies in enhancing the selective entrance of PMA into dead Campylobacter coli cells via spheroplasting and in developing an internal sample process control, thus delivering reliable results in pure cultures and meat samples, approaches that can be applicable to other Gram-negative pathogens.


Assuntos
Azidas/química , Campylobacter coli/isolamento & purificação , Microbiologia de Alimentos/métodos , Carne/microbiologia , Viabilidade Microbiana , Propídio/análogos & derivados , Reação em Cadeia da Polimerase em Tempo Real/métodos , Esferoplastos/isolamento & purificação , Microbiologia de Alimentos/instrumentação , Propídio/química , Reação em Cadeia da Polimerase em Tempo Real/instrumentação
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