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1.
Int J Med Microbiol ; 311(4): 151511, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33975122

RESUMEN

Super-shed (SS) Escherichia coli O157 (E. coli O157) demonstrate a strong, aggregative, locus of enterocyte effacement (LEE)-independent adherence phenotype on bovine recto-anal junction squamous epithelial (RSE) cells, and harbor polymorphisms in non-LEE-adherence-related loci, including in the type 1 fimbriae operon. To elucidate the role of type 1 fimbriae in strain- and host-specific adherence, we evaluated the entire Fim operon (FimB-H) and its adhesion (FimH) deletion mutants in four E. coli O157 strains, SS17, SS52, SS77 and EDL933, and evaluated the adherence phenotype in bovine RSE and human HEp-2 adherence assays. Consistent with the prevailing dogma that fimH expression is genetically switched off in E. coli O157, the ΔfimHSS52, ΔfimB-HSS52, ΔfimB-HSS17, and ΔfimHSS77 mutants remained unchanged in adherence phenotype to RSE cells. In contrast, the ΔfimHSS17 and ΔfimB-HSS77 mutants changed from a wild-type strong and aggregative, to a moderate and diffuse adherence phenotype, while both ΔfimHEDL933 and ΔfimB-HEDL933 mutants demonstrated enhanced binding to RSE cells (p < 0.05). Additionally, both ΔfimHSS17 and ΔfimHEDL933 were non-adherent to HEp-2 cells (p < 0.05). Complementation of the mutant strains with their respective wild-type genes restored parental phenotypes. Microscopy revealed that the SS17 and EDL933 strains indeed carry type 1 fimbriae-like structures shorter than those seen in uropathogenic E. coli. Taken together, these results provide compelling evidence for a strain and host cell type-dependent role of fimH and the fim operon in E. coli O157 adherence that needs to be further evaluated.


Asunto(s)
Infecciones por Escherichia coli , Escherichia coli O157 , Proteínas de Escherichia coli , Animales , Adhesión Bacteriana , Bovinos , Proteínas de Unión al ADN , Infecciones por Escherichia coli/veterinaria , Escherichia coli O157/genética , Proteínas de Escherichia coli/genética , Fimbrias Bacterianas/genética , Humanos , Integrasas , Fenotipo
2.
Nat Commun ; 12(1): 2949, 2021 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-34011959

RESUMEN

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.


Asunto(s)
Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Resistencia al Trimetoprim/genética , Trimetoprim/análogos & derivados , Sustitución de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Cristalografía por Rayos X , Evolución Molecular Dirigida , Diseño de Fármacos , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Antagonistas del Ácido Fólico/química , Antagonistas del Ácido Fólico/farmacología , Genes Bacterianos , Genotipo , Humanos , Modelos Moleculares , Mutación , Tetrahidrofolato Deshidrogenasa/química , Tetrahidrofolato Deshidrogenasa/genética , Trimetoprim/química , Trimetoprim/farmacología
3.
Nat Commun ; 12(1): 1988, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790291

RESUMEN

Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA)3-repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA)3-repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Represoras/metabolismo , Ácidos Siálicos/metabolismo , Regulación Alostérica , Secuencia de Bases , Sitios de Unión/genética , Cristalografía por Rayos X , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Ácido N-Acetilneuramínico/metabolismo , Motivos de Nucleótidos/genética , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Represoras/genética
4.
Int J Food Microbiol ; 347: 109191, 2021 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-33838477

RESUMEN

Fresh beef and meat products have been implicated in outbreaks of Shiga toxin-producing Escherichia coli (STEC) worldwide. This study investigated the prevalence of E. coli O157: H7 and non-O157 STEC serogroups in fresh beef in the open market and street vended meat products (n = 180) in Lagos metropolis, Nigeria. A combination of culture media and immunomagnetic separation followed by typing for associated virulence factors and serotypes was performed. Antimicrobial susceptibility testing was performed on the isolated STEC serotypes using the disk diffusion method. A total of 72 STEC serogroup isolates were detected from 61 out of 180 samples. The O157 STEC serotypes were detected in fresh beef, suya, minced meat and tsire with prevalence of 20.8% while non-O157 STEC serogroups were detected in all the samples. Molecular typing revealed 25% (n = 18) of the STEC serogroups showed presence of all the stx1, stx2, eaeA, fliCH7 and rfbEO157 virulence factors while 54.2% (n = 39) possessed a combination of two virulence genes. Multidrug resistance was discovered in 23.6% (n = 17) of the total STEC serogroups. Locally processed ready-to-eat meat products in Lagos metropolis, Nigeria harbour potentially pathogenic multi-drug resistant STEC serogroups that can constitute public health hazard.


Asunto(s)
Farmacorresistencia Bacteriana , Carne/microbiología , Escherichia coli Shiga-Toxigénica/aislamiento & purificación , Animales , Antibacterianos/farmacología , Bovinos , Escherichia coli O157/clasificación , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/genética , Escherichia coli O157/aislamiento & purificación , Proteínas de Escherichia coli/genética , Microbiología de Alimentos , Humanos , Productos de la Carne/microbiología , Nigeria , Prevalencia , Serogrupo , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/efectos de los fármacos , Escherichia coli Shiga-Toxigénica/genética , Factores de Virulencia/genética
5.
J Phys Chem B ; 125(17): 4299-4307, 2021 05 06.
Artículo en Inglés | MEDLINE | ID: mdl-33881864

RESUMEN

The DNA helicase PriA is a key protein for restarting stalled DNA replication forks in bacteria. With 3' to 5' helicase activity, PriA is important in primosome assembly. We used atomic force microscopy (AFM) and specifically employed time-lapse AFM to visualize the interaction of PriA with two DNA substrates. The results show that most of the PriA molecules are observed bound at the fork. However, PriA is capable of translocating over distances of about 400 bp. There is a preference for the long-range translocation of PriA depending on the fork type. For a fork with the nascent leading strand as single-stranded DNA (ssDNA; F4 substrate), PriA translocates preferentially on the parental arm of the fork. For the substrate F14, which contains an additional ssDNA segment between the parental and lagging arms (5 nt gap), PriA translocates on both the parental and lagging strand arms. These data suggest that transient formation of the single-stranded regions during the DNA replication can change the selection of the DNA duplex by PriA. Translocation of the helicase was directly visualized by time-lapse AFM imaging, which revealed that PriA can switch strands during translocation. These novel features of PriA shed new light on the mechanisms of PriA interaction with stalled replication forks.


Asunto(s)
Proteínas de Unión al ADN , Proteínas de Escherichia coli , ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN , ADN de Cadena Simple/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/genética
6.
Int J Food Microbiol ; 347: 109197, 2021 Jun 02.
Artículo en Inglés | MEDLINE | ID: mdl-33895597

RESUMEN

Shiga toxin-producing Escherichia coli (STEC) can cause severe human illness, which are frequently linked to the consumption of contaminated beef or dairy products. However, recent outbreaks associated with contaminated flour and undercooked dough in the United States and Canada, highlight the potential of plant based food as transmission routes for STEC. In Germany STEC has been isolated from flour, but no cases of illness have been linked to flour. In this study, we characterized 123 STEC strains isolated from flour and flour products collected between 2015 and 2019 across Germany. In addition to determination of serotype and Shiga toxin subtype, whole genome sequencing (WGS) was used for isolates collected in 2018 to determine phylogenetic relationships, sequence type (ST), and virulence-associated genes (VAGs). We found a high diversity of serotypes including those frequently associated with human illness and outbreaks, such as O157:H7 (stx2c/d, eae), O145:H28 (stx2a, eae), O146:H28 (stx2b), and O103:H2 (stx1a, eae). Serotypes O187:H28 (ST200, stx2g) and O154:H31 (ST1892, stx1d) were most prevalent, but are rarely linked to human cases. However, WGS analysis revealed that these strains, as well as, O156:H25 (ST300, stx1a) harbour high numbers of VAGs, including eae, nleB and est1a/sta1. Although STEC-contaminated flour products have yet not been epidemiologically linked to human clinical cases in Germany, this study revealed that flour can serve as a vector for STEC strains with a high pathogenic potential. Further investigation is needed to determine the sources of STEC contamination in flour and flour products particularly in regards to these rare serotypes.


Asunto(s)
Escherichia coli O157/genética , Escherichia coli O157/aislamiento & purificación , Harina/microbiología , Contaminación de Alimentos/análisis , Toxina Shiga/genética , Animales , Canadá , Bovinos , Brotes de Enfermedades , Infecciones por Escherichia coli/transmisión , Escherichia coli O157/patogenicidad , Proteínas de Escherichia coli/genética , Microbiología de Alimentos , Variación Genética/genética , Genoma Bacteriano/genética , Alemania , Humanos , Filogenia , Virulencia/genética , Factores de Virulencia/genética , Secuenciación Completa del Genoma
7.
Nat Commun ; 12(1): 2382, 2021 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-33888706

RESUMEN

Conventional approaches to identify secreted factors that regulate homeostasis are limited in their abilities to identify the tissues/cells of origin and destination. We established a platform to identify secreted protein trafficking between organs using an engineered biotin ligase (BirA*G3) that biotinylates, promiscuously, proteins in a subcellular compartment of one tissue. Subsequently, biotinylated proteins are affinity-enriched and identified from distal organs using quantitative mass spectrometry. Applying this approach in Drosophila, we identify 51 muscle-secreted proteins from heads and 269 fat body-secreted proteins from legs/muscles, including CG2145 (human ortholog ENDOU) that binds directly to muscles and promotes activity. In addition, in mice, we identify 291 serum proteins secreted from conditional BirA*G3 embryo stem cell-derived teratomas, including low-abundance proteins with hormonal properties. Our findings indicate that the communication network of secreted proteins is vast. This approach has broad potential across different model systems to identify cell-specific secretomes and mediators of interorgan communication in health or disease.


Asunto(s)
Ligasas de Carbono-Nitrógeno/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteómica/métodos , Proteínas Represoras/metabolismo , Coloración y Etiquetado/métodos , Animales , Animales Modificados Genéticamente , Biotina/metabolismo , Biotinilación , Ligasas de Carbono-Nitrógeno/genética , Línea Celular , Modelos Animales de Enfermedad , Drosophila , Células Madre Embrionarias , Proteínas de Escherichia coli/genética , Femenino , Humanos , Masculino , Ratones , Ingeniería de Proteínas , Transporte de Proteínas , Proteínas Represoras/genética , Espectrometría de Masas en Tándem/métodos , Teratoma/diagnóstico , Teratoma/patología
8.
Artículo en Inglés | MEDLINE | ID: mdl-33909852

RESUMEN

The plasmid-mediated colistin-resistance gene named mcr-1 has been recently described in different countries and it became a public health challenge. Of note, few studies have addressed the spread of Escherichia coli harboring the mcr-1 gene in both, community and hospital settings. A total of seven colistin-resistant E. coli carrying mcr-1, collected from 2016 to 2018, from community (n=4), healthcare-acquired infections (n=2) and colonization (n=1) were identified in three high complexity hospitals in Sao Paulo, Brazil. These colistin-resistant isolates were screened for mcr genes by PCR and all strains were submitted to Whole Genome Sequencing and the conjugation experiment. The seven strains belonged to seven distinct sequence types (ST744, ST131, ST69, ST48, ST354, ST57, ST10), and they differ regarding the resistance profiles. Transference of mcr-1 by conjugation to E. coli strain C600 was possible in five of the seven isolates. The mcr-1 gene was found in plasmid types IncX4 or IncI2. Three of the isolates have ESBL-encoding genes (blaCTX-M-2, n=2; blaCTX-M-8, n=1). We hereby report genetically distinct E. coli isolates, belonging to seven STs, harboring the mcr-1 gene, associated to community and healthcare-acquired infections, and colonization in patients from three hospitals in Sao Paulo. These findings point out for the potential spread of plasmid-mediated colistin-resistance mechanism in E. coli strains in Brazil.


Asunto(s)
Colistina , Proteínas de Escherichia coli , Antibacterianos/farmacología , Brasil , Colistina/farmacología , Prestación de Atención de Salud , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Humanos , Pruebas de Sensibilidad Microbiana
9.
Nat Commun ; 12(1): 1986, 2021 03 31.
Artículo en Inglés | MEDLINE | ID: mdl-33790266

RESUMEN

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.


Asunto(s)
Proteínas Bacterianas/metabolismo , GMP Cíclico/análogos & derivados , Proteínas de Escherichia coli/metabolismo , Liasas de Fósforo-Oxígeno/metabolismo , Pseudomonas aeruginosa/metabolismo , Sistemas de Mensajero Secundario/fisiología , Transducción de Señal/fisiología , Algoritmos , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Cromatografía Liquida , GMP Cíclico/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Espectrometría de Masas , Liasas de Fósforo-Oxígeno/genética , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/fisiología , Temperatura
10.
Int J Mol Sci ; 22(6)2021 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-33802163

RESUMEN

Bacteria access iron, a key nutrient, by producing siderophores or using siderophores produced by other microorganisms. The pathogen Pseudomonas aeruginosa produces two siderophores but is also able to pirate enterobactin (ENT), the siderophore produced by Escherichia coli. ENT-Fe complexes are imported across the outer membrane of P. aeruginosa by the two outer membrane transporters PfeA and PirA. Iron is released from ENT in the P. aeruginosa periplasm by hydrolysis of ENT by the esterase PfeE. We show here that pfeE gene deletion renders P. aeruginosa unable to grow in the presence of ENT because it is unable to access iron via this siderophore. Two-species co-cultures under iron-restricted conditions show that P. aeruginosa strongly represses the growth of E. coli as long it is able to produce its own siderophores. Both strains are present in similar proportions in the culture as long as the siderophore-deficient P. aeruginosa strain is able to use ENT produced by E. coli to access iron. If pfeE is deleted, E. coli has the upper hand in the culture and P. aeruginosa growth is repressed. Overall, these data show that PfeE is the Achilles' heel of P. aeruginosa in communities with bacteria producing ENT.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Esterasas/metabolismo , Hierro/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Portadoras/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Esterasas/genética , Pseudomonas aeruginosa/genética
11.
Int J Mol Sci ; 22(5)2021 Mar 09.
Artículo en Inglés | MEDLINE | ID: mdl-33803409

RESUMEN

Protein engineering is actively pursued in industrial and laboratory settings for high thermostability. Among the many protein engineering methods, rational design by bioinformatics provides theoretical guidance without time-consuming experimental screenings. However, most rational design methods either rely on protein tertiary structure information or have limited accuracies. We proposed a primary-sequence-based algorithm for increasing the heat resistance of a protein while maintaining its functions. Using adenylate kinase (ADK) family as a model system, this method identified a series of amino acid sites closely related to thermostability. Single- and double-point mutants constructed based on this method increase the thermal denaturation temperature of the mesophilic Escherichia coli (E. coli) ADK by 5.5 and 8.3 °C, respectively, while preserving most of the catalytic function at ambient temperatures. Additionally, the constructed mutants have improved enzymatic activity at higher temperature.


Asunto(s)
Adenilato Quinasa/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Calor , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo
12.
Nat Commun ; 12(1): 2287, 2021 04 16.
Artículo en Inglés | MEDLINE | ID: mdl-33863894

RESUMEN

Both adenine base editors (ABEs) and cytosine base editors (CBEs) have been recently revealed to induce transcriptome-wide RNA off-target editing in a guide RNA-independent manner. Here we construct a reporter system containing E.coli Hokb gene with a tRNA-like motif for robust detection of RNA editing activities as the optimized ABE, ABEmax, induces highly efficient A-to-I (inosine) editing within an E.coli tRNA-like structure. Then, we design mutations to disrupt the potential interaction between TadA and tRNAs in structure-guided principles and find that Arginine 153 (R153) within TadA is essential for deaminating RNAs with core tRNA-like structures. Two ABEmax or mini ABEmax variants (TadA* fused with Cas9n) with deletion of R153 within TadA and/or TadA* (named as del153/del153* and mini del153) are successfully engineered, showing minimized RNA off-targeting, but comparable DNA on-targeting activities. Moreover, R153 deletion in recently reported ABE8e or ABE8s can also largely reduce their RNA off-targeting activities. Taken together, we develop a strategy to generate engineered ABEs (eABEs) with minimized RNA off-targeting activities.


Asunto(s)
Adenosina Desaminasa/genética , Proteína 9 Asociada a CRISPR/genética , ADN/genética , Proteínas de Escherichia coli/genética , Edición Génica/métodos , Adenina/metabolismo , Adenosina Desaminasa/metabolismo , Toxinas Bacterianas/genética , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Citosina/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Genes Reporteros , Células HEK293 , Humanos , Inosina/genética , Inosina/metabolismo , Ingeniería de Proteínas , Edición de ARN/genética , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , RNA-Seq , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo
13.
Nat Commun ; 12(1): 2249, 2021 04 21.
Artículo en Inglés | MEDLINE | ID: mdl-33883550

RESUMEN

The RNA chaperone Hfq, acting as a hexamer, is a known mediator of post-transcriptional regulation, expediting basepairing between small RNAs (sRNAs) and their target mRNAs. However, the intricate details associated with Hfq-RNA biogenesis are still unclear. Previously, we reported that the stringent response regulator, RelA, is a functional partner of Hfq that facilitates Hfq-mediated sRNA-mRNA regulation in vivo and induces Hfq hexamerization in vitro. Here we show that RelA-mediated Hfq hexamerization requires an initial binding of RNA, preferably sRNA to Hfq monomers. By interacting with a Shine-Dalgarno-like sequence (GGAG) in the sRNA, RelA stabilizes the initially unstable complex of RNA bound-Hfq monomer, enabling the attachment of more Hfq subunits to form a functional hexamer. Overall, our study showing that RNA binding to Hfq monomers is at the heart of RelA-mediated Hfq hexamerization, challenges the previous concept that only Hfq hexamers can bind RNA.


Asunto(s)
Proteínas de Escherichia coli/metabolismo , GTP Pirofosfoquinasa/metabolismo , Proteína de Factor 1 del Huésped/metabolismo , ARN Bacteriano/metabolismo , Sustitución de Aminoácidos , Secuencia de Bases , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , GTP Pirofosfoquinasa/química , GTP Pirofosfoquinasa/genética , Proteína de Factor 1 del Huésped/química , Modelos Biológicos , Unión Proteica , Multimerización de Proteína , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Subunidades de Proteína , ARN Bacteriano/química , ARN Bacteriano/genética , ARN Pequeño no Traducido/química , ARN Pequeño no Traducido/genética , ARN Pequeño no Traducido/metabolismo , Eliminación de Secuencia
14.
Nat Commun ; 12(1): 2363, 2021 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-33888690

RESUMEN

Cell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.


Asunto(s)
Micropartículas Derivadas de Células/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Glicoproteínas/biosíntesis , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Biosíntesis de Proteínas , Membrana Celular/genética , Membrana Celular/metabolismo , Micropartículas Derivadas de Células/genética , Cromatografía Líquida de Alta Presión/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/aislamiento & purificación , Glicoproteínas/aislamiento & purificación , Hexosiltransferasas/genética , Hexosiltransferasas/aislamiento & purificación , Espectrometría de Masas/métodos , Proteínas de la Membrana/genética , Proteínas de la Membrana/aislamiento & purificación , Oligosacáridos/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
15.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artículo en Inglés | MEDLINE | ID: mdl-33799639

RESUMEN

Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a 'gate' for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells.


Asunto(s)
Proteínas Asociadas a CRISPR/metabolismo , ADN Helicasas/metabolismo , ADN de Cadena Simple/metabolismo , ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Triptófano/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Alanina/química , Alanina/genética , Alanina/metabolismo , Secuencia de Aminoácidos , Proteínas Asociadas a CRISPR/química , Proteínas Asociadas a CRISPR/genética , Sistemas CRISPR-Cas , Dominio Catalítico/genética , Dicroismo Circular , ADN/química , ADN/genética , ADN Helicasas/química , ADN Helicasas/genética , ADN de Cadena Simple/química , ADN de Cadena Simple/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Edición Génica/métodos , Humanos , Mutación Missense , Conformación Proteica , Homología de Secuencia de Aminoácido , Temperatura , Triptófano/química , Triptófano/genética
16.
Nat Commun ; 12(1): 2116, 2021 04 09.
Artículo en Inglés | MEDLINE | ID: mdl-33837188

RESUMEN

Nicotinamide adenine dinucleotide (NAD) and its reduced form are indispensable cofactors in life. Diverse NAD mimics have been developed for applications in chemical and biological sciences. Nicotinamide cytosine dinucleotide (NCD) has emerged as a non-natural cofactor to mediate redox transformations, while cells are fed with chemically synthesized NCD. Here, we create NCD synthetase (NcdS) by reprograming the substrate binding pockets of nicotinic acid mononucleotide (NaMN) adenylyltransferase to favor cytidine triphosphate and nicotinamide mononucleotide over their regular substrates ATP and NaMN, respectively. Overexpression of NcdS alone in the model host Escherichia coli facilitated intracellular production of NCD, and higher NCD levels up to 5.0 mM were achieved upon further pathway regulation. Finally, the non-natural cofactor self-sufficiency was confirmed by mediating an NCD-linked metabolic circuit to convert L-malate into D-lactate. NcdS together with NCD-linked enzymes offer unique tools and opportunities for intriguing studies in chemical biology and synthetic biology.


Asunto(s)
Coenzimas/biosíntesis , Proteínas de Escherichia coli/genética , Niacinamida/biosíntesis , Nicotinamida-Nucleótido Adenililtransferasa/genética , Ingeniería de Proteínas , Coenzimas/química , Citidina Trifosfato/metabolismo , Pruebas de Enzimas , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Ensayos Analíticos de Alto Rendimiento , Ácido Láctico/metabolismo , Malatos/metabolismo , Niacinamida/química , Mononucleótido de Nicotinamida/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo , Oxidación-Reducción , Especificidad por Sustrato/genética , Biología Sintética/métodos
17.
Int J Mol Sci ; 22(9)2021 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-33926096

RESUMEN

The SOS response is induced upon DNA damage and the inhibition of Z ring formation by the product of the sulA gene, which is one of the LexA-regulated genes, allows time for repair of damaged DNA. On the other hand, severely DNA-damaged cells are eliminated from cell populations. Overexpression of sulA leads to cell lysis, suggesting SulA eliminates cells with unrepaired damaged DNA. Transcriptome analysis revealed that overexpression of sulA leads to up-regulation of numerous genes, including soxS. Deletion of soxS markedly reduced the extent of cell lysis by sulA overexpression and soxS overexpression alone led to cell lysis. Further experiments on the SoxS regulon suggested that LpxC is a main player downstream from SoxS. These findings suggested the SulA-dependent cell lysis (SDCL) cascade as follows: SulA→SoxS→LpxC. Other tests showed that the SDCL cascade pathway does not overlap with the apoptosis-like and mazEF cell death pathways.


Asunto(s)
Daño del ADN/fisiología , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Amidohidrolasas/metabolismo , Apoptosis/genética , Proteínas Bacterianas/metabolismo , División Celular/genética , Daño del ADN/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Genes Bacterianos/genética , Serina Endopeptidasas/metabolismo , Transactivadores/metabolismo
18.
Int J Food Microbiol ; 346: 109164, 2021 May 16.
Artículo en Inglés | MEDLINE | ID: mdl-33813365

RESUMEN

The aim of the study was to assess the presence of genes in ESBL-producing E. coli (ESBL-Ec) isolated from retail raw food in Nha Trang, Vietnam. A total of 452 food samples comprising chicken (n = 116), pork (n = 112), fish (n = 112) and shrimp (n = 112) collected between 2015 and 2017 were examined for the prevalence of ESBL-Ec. ESBL-Ec were detected in 46.0% (208/452) of retail food samples, particularly in 66.4% (77/116), 55.4% (62/112), 42.0% (47/112) 19.6% (22/112) of chicken, pork, fish and shrimp, respectively. Sixty-five out of the 208 (31.3%) ESBL-Ec isolates were positive for mcr genes including mcr-1, mcr-3 and both mcr-1 and mcr-3 genes in 56/208 (26.9%), 1/208 (0.5%) and 8/208 (3.9%) isolates, respectively. Particularly, there was higher prevalence of mcr-1 in ESBL-Ec isolates from chicken (53.2%, 41/77) in comparison to shrimp (22.7%, 5/22), pork (11.3%, 7/62) and fish (6.4%, 3/47). mcr-3 gene was detected in co-existence with mcr-1 in ESBL-Ec isolates from shrimp (9.1%, 2/22), pork (8.1%, 5/62) and fish (2.1%, 1/47) but not chicken. The 65 mcr-positive ESBL-Ec (mcr-ESBL-Ec) were colistin-resistant with the MICs of 4-8 µg/mL. All mcr-3 gene-positive isolates belonged to group A, whereas phylogenetic group distribution of isolates harboring only mcr-1 was B1 (44.6%), A (28.6%) and D (26.8%). PFGE analysis showed diverse genotypes, although some isolates demonstrated nearly clonal relationships. S1-PFGE and Southern hybridization illustrated that the mcr-1 and mcr-3 genes were located either on chromosomes or on plasmids. However, the types of mcr genes were harbored on different plasmids with varied sizes of 30-390 kb. Besides, the ESBL genes of CTX-M-1 or CTX-M-9 were also detected to be located on plasmids. Noteworthy, co-location of CTX-M-1 with mcr-1 or mcr-3 genes on the same plasmid was identified. The conjugation experiment indicated that the mcr-1 or mcr-3 was horizontally transferable. All mcr-ESBL-Ec isolates were multidrug resistance (resistance to ≥3 antimicrobial classes). Moreover, ß-Lactamase-encoding genes of the CTX-M-1 (78.5%), CTX-M-9 (21.5%), TEM (61.5%) groups were found in mcr-ESBL-Ec. The astA gene was detected in 27 (41.5%) mcr-ESBL-Ec isolates demonstrating their potential virulence. In conclusion, mcr-1 and mcr-3 genes existed individually or concurrently in ESBL-Ec isolates recovered from retail raw food in Nha Trang city, which might further complicate the antimicrobial-resistant situation in Vietnam, and is a possible health risk for human.


Asunto(s)
Antibacterianos/farmacología , Colistina/farmacología , Proteínas de Escherichia coli/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/aislamiento & purificación , Carne/microbiología , Alimentos Crudos/microbiología , beta-Lactamasas/genética , Animales , Pollos , Farmacorresistencia Bacteriana , Escherichia coli/clasificación , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Peces , Contaminación de Alimentos/análisis , Contaminación de Alimentos/estadística & datos numéricos , Genotipo , Humanos , Pruebas de Sensibilidad Microbiana , Filogenia , Plásmidos/genética , Plásmidos/metabolismo , Prevalencia , Alimentos Crudos/economía , Porcinos , Vietnam , beta-Lactamasas/metabolismo
19.
Int J Mol Sci ; 22(8)2021 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-33921535

RESUMEN

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459-PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459-PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa.


Asunto(s)
Adaptación Fisiológica/genética , Farmacorresistencia Bacteriana/genética , Infecciones por Pseudomonas/genética , Pseudomonas aeruginosa/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Humanos , Operón/genética , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/patogenicidad , Proteínas Represoras/genética , Proteínas Represoras/aislamiento & purificación
20.
BMC Infect Dis ; 21(1): 361, 2021 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-33865334

RESUMEN

BACKGROUND: Urinary Tract Infection (UTI) is one of the most common bacterial infectious diseases which causes considerable morbidity and costly health problems. Uropathogenic Escherichia coli (UPEC), the most common pathogen causing UTI, is a highly heterogeneous group of extraintestinal pathogenic E. coli (ExPEC) which may carry a variety of virulence factors and belonging to different phylogenetic backgrounds. The current study aimed to investigate the frequency and association between various virulence factors (VFs) and phylogenetic groups of UPEC and commensal isolates. METHODS: UPEC and commensal E. coli strains isolated from UTI and feces of healthy humans were compared for the presence of VFs and phylogenetic groups. Association between virulence genes was investigated and cluster analysis was employed. RESULTS: According to the results, among a 30 virulence markers tested, the pathogenicity-associated island (PAI), papAH, papEF, fimH, fyuA, and traT genes prevalence were statistically significant in UPEC isolates. A strong association was found between the B2 and D phylogenetic groups and clinical isolates of UPEC; while, commensal isolates were mostly associated with phylogenetic group A. The aggregated VFs scores were more than twice higher in the UPEC isolates in comparison with the commensal isolates. Interestingly, the B2 group in both UPEC and commensal isolates had the highest VF scores. A strong positive association was found between several virulence genes. The clustering results demonstrated that UPEC or commensal E. coli isolates were highly heterogeneous due to different composition of their virulence gene pool and pathogenicity islands. CONCLUSION: Genetic structure and VFs of UPEC strains vary from region to region; therefore, to control the UTI, the epidemiological aspects and characterization of the UPEC isolates need to be investigated in different regions. Since UPEC isolates are generally originate from the commensal strains, it may be feasible to reduce the UTI burden by interfering the intestinal colonization, particularly in the highly pathogenic clonal lineages such as B2.


Asunto(s)
Infecciones por Escherichia coli/microbiología , Infecciones Urinarias/microbiología , Escherichia coli Uropatógena , Factores de Virulencia/genética , Virulencia/genética , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Niño , Preescolar , Infecciones por Escherichia coli/epidemiología , Proteínas de Escherichia coli/genética , Escherichia coli Patógena Extraintestinal/genética , Escherichia coli Patógena Extraintestinal/aislamiento & purificación , Escherichia coli Patógena Extraintestinal/patogenicidad , Femenino , Islas Genómicas/genética , Humanos , Lactante , Recién Nacido , Irán/epidemiología , Masculino , Persona de Mediana Edad , Filogenia , Prevalencia , Infecciones Urinarias/epidemiología , Escherichia coli Uropatógena/clasificación , Escherichia coli Uropatógena/genética , Escherichia coli Uropatógena/aislamiento & purificación , Escherichia coli Uropatógena/patogenicidad , Adulto Joven
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