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1.
Nat Commun ; 11(1): 4906, 2020 09 30.
Artículo en Inglés | MEDLINE | ID: mdl-32999292

RESUMEN

The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the femtomolar range without any target pre-amplification step. By extending the 3'- or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discover a self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40-60 min.


Asunto(s)
Proteínas Bacterianas/metabolismo , Betacoronavirus/genética , Proteínas Asociadas a CRISPR/metabolismo , Endodesoxirribonucleasas/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , ARN Viral/aislamiento & purificación , Transactivadores/metabolismo , Betacoronavirus/aislamiento & purificación , Sistemas CRISPR-Cas , Técnicas de Laboratorio Clínico , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/virología , ADN de Cadena Simple , Pandemias , Neumonía Viral , ARN Guia/genética , ARN Viral/genética
2.
Nat Commun ; 11(1): 4947, 2020 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-33009392

RESUMEN

Pseudomonas syringae is a Gram-negative and model pathogenic bacterium that causes plant diseases worldwide. Here, we set out to identify binding motifs for all 301 annotated transcription factors (TFs) of P. syringae using HT-SELEX. We successfully identify binding motifs for 100 TFs. We map functional interactions between the TFs and their targets in virulence-associated pathways, and validate many of these interactions and functions using additional methods such as ChIP-seq, electrophoretic mobility shift assay (EMSA), RT-qPCR, and reporter assays. Our work identifies 25 virulence-associated master regulators, 14 of which had not been characterized as TFs before.


Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Pseudomonas syringae/metabolismo , Factores de Transcripción/metabolismo , Sistemas de Secreción Bacterianos , Sitios de Unión , Posición Específica de Matrices de Puntuación , Unión Proteica , Multimerización de Proteína , Pseudomonas syringae/patogenicidad , Reproducibilidad de los Resultados , Técnica SELEX de Producción de Aptámeros , Virulencia
3.
Nat Commun ; 11(1): 4501, 2020 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-32908132

RESUMEN

Streptovaricin C is a naphthalenic ansamycin antibiotic structurally similar to rifamycins with potential anti-MRSA bioactivities. However, the formation mechanism of the most fascinating and bioactivity-related methylenedioxy bridge (MDB) moiety in streptovaricins is unclear. Based on genetic and biochemical evidences, we herein clarify that the P450 enzyme StvP2 catalyzes the MDB formation in streptovaricins, with an atypical substrate inhibition kinetics. Furthermore, X-ray crystal structures in complex with substrate and structure-based mutagenesis reveal the intrinsic details of the enzymatic reaction. The mechanism of MDB formation is proposed to be an intramolecular nucleophilic substitution resulting from the hydroxylation by the heme core and the keto-enol tautomerization via a crucial catalytic triad (Asp89-His92-Arg72) in StvP2. In addition, in vitro reconstitution uncovers that C6-O-methylation and C4-O-acetylation of streptovaricins are necessary prerequisites for the MDB formation. This work provides insight for the MDB formation and adds evidence in support of the functional versatility of P450 enzymes.


Asunto(s)
Proteínas Bacterianas/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Streptomyces/metabolismo , Estreptovaricina/análogos & derivados , Acetilación , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Biocatálisis , Cristalografía por Rayos X , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/ultraestructura , Pruebas de Enzimas , Metilación , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Estreptovaricina/biosíntesis , Estreptovaricina/química , Estreptovaricina/metabolismo
4.
PLoS Pathog ; 16(8): e1008822, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32866204

RESUMEN

Shigella flexneri invades host cells by entering within a bacteria-containing vacuole (BCV). In order to establish its niche in the host cytosol, the bacterium ruptures its BCV. Contacts between S. flexneri BCV and infection-associated macropinosomes (IAMs) formed in situ have been reported to enhance BCV disintegration. The mechanism underlying S. flexneri vacuolar escape remains however obscure. To decipher the molecular mechanism priming the communication between the IAMs and S. flexneri BCV, we performed mass spectrometry-based analysis of the magnetically purified IAMs from S. flexneri-infected cells. While proteins involved in host recycling and exocytic pathways were significantly enriched at the IAMs, we demonstrate more precisely that the S. flexneri type III effector protein IpgD mediates the recruitment of the exocyst to the IAMs through the Rab8/Rab11 pathway. This recruitment results in IAM clustering around S. flexneri BCV. More importantly, we reveal that IAM clustering subsequently facilitates an IAM-mediated unwrapping of the ruptured vacuole membranes from S. flexneri, enabling the naked bacterium to be ready for intercellular spread via actin-based motility. Taken together, our work untangles the molecular cascade of S. flexneri-driven host trafficking subversion at IAMs to develop its cytosolic lifestyle, a crucial step en route for infection progression at cellular and tissue level.


Asunto(s)
Disentería Bacilar , Shigella flexneri , Transducción de Señal , Vacuolas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Disentería Bacilar/genética , Disentería Bacilar/metabolismo , Células HeLa , Humanos , Shigella flexneri/genética , Shigella flexneri/metabolismo , Shigella flexneri/patogenicidad , Vacuolas/genética , Vacuolas/metabolismo , Vacuolas/microbiología , Factores de Virulencia/genética , Factores de Virulencia/metabolismo , Proteínas de Unión al GTP rab/genética , Proteínas de Unión al GTP rab/metabolismo
5.
S Afr Med J ; 110(8): 783-790, 2020 Jul 29.
Artículo en Inglés | MEDLINE | ID: mdl-32880307

RESUMEN

BACKGROUND: Rates of healthcare-associated infections (HAIs) among babies born in developing countries are higher than among those born in resource-rich countries, as a result of suboptimal infection prevention and control (IPC) practices. Following two reported deaths of neonates with carbapenem-resistant Klebsiella pneumoniae bloodstream infections (BSIs), we conducted an outbreak investigation in a neonatal unit of a regional hospital in Gauteng Province, South Africa. OBJECTIVES: To confirm an outbreak of K. pneumoniae BSIs and assess the IPC programme in the neonatal unit. METHODS: We calculated total and organism-specific BSI incidence risks for culture-confirmed cases in the neonatal unit for baseline and outbreak periods. We conducted a clinical record review for a subset of cases with K. pneumoniae BSI that had been reported to the investigating team by the neonatal unit. An IPC audit was performed in different areas of the neonatal unit. We confirmed species identification and antimicrobial susceptibility, and used polymerase chain reaction for confirmation of carbapenemase genes and pulsed-field gel electrophoresis (PFGE) for typing of submitted clinical isolates. RESULTS: From January 2017 to August 2018, 5 262 blood cultures were submitted, of which 11% (560/5 262) were positive. Of 560 positive blood cultures, 52% (n=292) were positive for pathogenic organisms associated with healthcare-associated BSIs. K. pneumoniae comprised the largest proportion of these cases (32%; 93/292). The total incidence risk of healthcare-associated BSI for the baseline period (January 2017 - March 2018) was 6.8 cases per 100 admissions, and that for the outbreak period (April - September 2018) was 10.1 cases per 100 admissions. The incidence risk of K. pneumoniae BSI for the baseline period was 1.6 cases per 100 admissions, compared with 5.0 cases per 100 admissions during the outbreak period. Average bed occupancy for the entire period was 118% (range 101 - 133%), that for the baseline period was 117%, and that for the outbreak period was 121%. In a subset of 12 neonates with K. pneumoniae bacteraemia, the median (interquartile range (IQR)) gestational age at birth was 27 (26 - 29) weeks, and the median (IQR) birth weight was 1 100 (880 - 1 425) g. Twelve bloodstream and 31 colonising K. pneumoniae isolates were OXA-48-positive. All isolates were genetically related by PFGE analysis (89% similarity). Inadequate IPC practices were noted, including suboptimal adherence to aseptic technique and hand hygiene (57% overall score in the neonatal intensive care unit), with poor monitoring and reporting of antimicrobial use (pharmacy score 55%). CONCLUSIONS: Overcrowding and inadequate IPC and antimicrobial stewardship contributed to a large outbreak of BSIs caused by genetically related carbapenemase-producing K. pneumoniae isolates in the neonatal unit.


Asunto(s)
Bacteriemia/microbiología , Infección Hospitalaria/microbiología , Brotes de Enfermedades , Unidades Hospitalarias , Infecciones por Klebsiella/epidemiología , Programas de Optimización del Uso de los Antimicrobianos , Bacteriemia/epidemiología , Proteínas Bacterianas/metabolismo , Auditoría Clínica , Infección Hospitalaria/epidemiología , Aglomeración , Humanos , Incidencia , Recién Nacido , Control de Infecciones , Klebsiella pneumoniae/enzimología , Klebsiella pneumoniae/aislamiento & purificación , Programas Médicos Regionales , Sudáfrica/epidemiología , beta-Lactamasas/metabolismo
6.
Mol Genet Genomics ; 295(6): 1529-1535, 2020 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-32894358

RESUMEN

Lanthipeptides are a subgroup of ribosomally encoded and post-translationally modified peptides (RiPPs) which frequently possess potent biological activity. Here we provide the first comprehensive bioinformatic analysis of the lanthipeptide-producing capability of the Salinispora genus, a marine actinomycete. One hundred twenty-two Salinispora arenicola, tropica, and pacifica genomic sequences were analyzed for lanthipeptide gene clusters, and the resulting 182 clusters were divided into seven groups based on sequence similarities. Group boundaries were defined based on LanB and LanM sequences with greater than 80% similarity within groups. Of the seven groups, six are predicted to encode class I lanthipeptides while only one group is predicted to encode class II lanthipeptides. Leader and core peptides were predicted for each cluster along with the number of possible lanthionine bridges. Notably, all of the predicted products of these clusters would represent novel lanthipeptide scaffolds. Of the 122 Salinispora genomes analyzed in this study, 92% contained at least one lanthipeptide gene cluster suggesting that Salinispora is a rich, yet untapped, source of lanthipeptides.


Asunto(s)
Alanina/análogos & derivados , Proteínas Bacterianas/metabolismo , Genoma Bacteriano , Micromonosporaceae/metabolismo , Fragmentos de Péptidos/metabolismo , Sulfuros/metabolismo , Alanina/aislamiento & purificación , Alanina/metabolismo , Proteínas Bacterianas/genética , Genómica , Micromonosporaceae/genética , Micromonosporaceae/crecimiento & desarrollo , Fragmentos de Péptidos/aislamiento & purificación , Sulfuros/aislamiento & purificación
7.
PLoS Pathog ; 16(9): e1008852, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32960931

RESUMEN

Enzymatic inactivation of Rho-family GTPases by the glucosyltransferase domain of Clostridioides difficile Toxin B (TcdB) gives rise to various pathogenic effects in cells that are classically thought to be responsible for the disease symptoms associated with C. difficile infection (CDI). Recent in vitro studies have shown that TcdB can, under certain circumstances, induce cellular toxicities that are independent of glucosyltransferase (GT) activity, calling into question the precise role of GT activity. Here, to establish the importance of GT activity in CDI disease pathogenesis, we generated the first described mutant strain of C. difficile producing glucosyltransferase-defective (GT-defective) toxin. Using allelic exchange (AE) technology, we first deleted tcdA in C. difficile 630Δerm and subsequently introduced a deactivating D270N substitution in the GT domain of TcdB. To examine the role of GT activity in vivo, we tested each strain in two different animal models of CDI pathogenesis. In the non-lethal murine model of infection, the GT-defective mutant induced minimal pathology in host tissues as compared to the profound caecal inflammation seen in the wild-type and 630ΔermΔtcdA (ΔtcdA) strains. In the more sensitive hamster model of CDI, whereas hamsters in the wild-type or ΔtcdA groups succumbed to fulminant infection within 4 days, all hamsters infected with the GT-defective mutant survived the 10-day infection period without primary symptoms of CDI or evidence of caecal inflammation. These data demonstrate that GT activity is indispensable for disease pathogenesis and reaffirm its central role in disease and its importance as a therapeutic target for small-molecule inhibition.


Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas , Clostridium difficile , Enterocolitis Seudomembranosa , Glucosiltransferasas , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridium difficile/enzimología , Clostridium difficile/genética , Clostridium difficile/patogenicidad , Cricetinae , Modelos Animales de Enfermedad , Enterocolitis Seudomembranosa/enzimología , Enterocolitis Seudomembranosa/genética , Enterocolitis Seudomembranosa/patología , Femenino , Eliminación de Gen , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Masculino , Ratones
8.
Nat Commun ; 11(1): 4817, 2020 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-32968056

RESUMEN

Lysozymes are among the best-characterized enzymes, acting upon the cell wall substrate peptidoglycan. Here, examining the invasive bacterial periplasmic predator Bdellovibrio bacteriovorus, we report a diversified lysozyme, DslA, which acts, unusually, upon (GlcNAc-) deacetylated peptidoglycan. B. bacteriovorus are known to deacetylate the peptidoglycan of the prey bacterium, generating an important chemical difference between prey and self walls and implying usage of a putative deacetyl-specific "exit enzyme". DslA performs this role, and ΔDslA strains exhibit a delay in leaving from prey. The structure of DslA reveals a modified lysozyme superfamily fold, with several adaptations. Biochemical assays confirm DslA specificity for deacetylated cell wall, and usage of two glutamate residues for catalysis. Exogenous DslA, added ex vivo, is able to prematurely liberate B. bacteriovorus from prey, part-way through the predatory lifecycle. We define a mechanism for specificity that invokes steric selection, and use the resultant motif to identify wider DslA homologues.


Asunto(s)
Bdellovibrio bacteriovorus/enzimología , Bdellovibrio bacteriovorus/metabolismo , Muramidasa/química , Muramidasa/metabolismo , Periplasma/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Bdellovibrio bacteriovorus/genética , Pared Celular , Escherichia coli , Regulación Bacteriana de la Expresión Génica , Modelos Moleculares , Muramidasa/genética , Mutación , Peptidoglicano/metabolismo , Fenotipo , Conformación Proteica , Especificidad por Sustrato
9.
Nat Commun ; 11(1): 4522, 2020 09 09.
Artículo en Inglés | MEDLINE | ID: mdl-32908144

RESUMEN

A unique, protective cell envelope contributes to the broad drug resistance of the nosocomial pathogen Acinetobacter baumannii. Here we use transposon insertion sequencing to identify A. baumannii mutants displaying altered susceptibility to a panel of diverse antibiotics. By examining mutants with antibiotic susceptibility profiles that parallel mutations in characterized genes, we infer the function of multiple uncharacterized envelope proteins, some of which have roles in cell division or cell elongation. Remarkably, mutations affecting a predicted cell wall hydrolase lead to alterations in lipooligosaccharide synthesis. In addition, the analysis of altered susceptibility signatures and antibiotic-induced morphology patterns allows us to predict drug synergies; for example, certain beta-lactams appear to work cooperatively due to their preferential targeting of specific cell wall assembly machineries. Our results indicate that the pathogen may be effectively inhibited by the combined targeting of multiple pathways critical for envelope growth.


Asunto(s)
Infecciones por Acinetobacter/tratamiento farmacológico , Acinetobacter baumannii/genética , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Infección Hospitalaria/tratamiento farmacológico , Farmacorresistencia Bacteriana Múltiple/genética , Infecciones por Acinetobacter/microbiología , Acinetobacter baumannii/efectos de los fármacos , Antibacterianos/uso terapéutico , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Pared Celular/efectos de los fármacos , Pared Celular/genética , Pared Celular/metabolismo , Infección Hospitalaria/microbiología , Análisis Mutacional de ADN , Elementos Transponibles de ADN/genética , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Mutación
10.
Nat Commun ; 11(1): 4554, 2020 09 11.
Artículo en Inglés | MEDLINE | ID: mdl-32917865

RESUMEN

Non-ribosomal peptide synthetase (NRPS) enzymes form modular assembly-lines, wherein each module governs the incorporation of a specific monomer into a short peptide product. Modules are comprised of one or more key domains, including adenylation (A) domains, which recognise and activate the monomer substrate; condensation (C) domains, which catalyse amide bond formation; and thiolation (T) domains, which shuttle reaction intermediates between catalytic domains. This arrangement offers prospects for rational peptide modification via substitution of substrate-specifying domains. For over 20 years, it has been considered that C domains play key roles in proof-reading the substrate; a presumption that has greatly complicated rational NRPS redesign. Here we present evidence from both directed and natural evolution studies that any substrate-specifying role for C domains is likely to be the exception rather than the rule, and that novel non-ribosomal peptides can be generated by substitution of A domains alone. We identify permissive A domain recombination boundaries and show that these allow us to efficiently generate modified pyoverdine peptides at high yields. We further demonstrate the transferability of our approach in the PheATE-ProCAT model system originally used to infer C domain substrate specificity, generating modified dipeptide products at yields that are inconsistent with the prevailing dogma.


Asunto(s)
Adenosina Monofosfato/metabolismo , Péptidos/química , Péptidos/metabolismo , Dominios Proteicos , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Barajamiento de ADN , Modelos Moleculares , Familia de Multigenes , Oligopéptidos/química , Oligopéptidos/metabolismo , Péptido Sintasas/genética , Péptido Sintasas/metabolismo , Conformación Proteica , Pseudomonas , Especificidad por Sustrato
11.
Adv Exp Med Biol ; 1267: 45-58, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32894476

RESUMEN

In this chapter, we will focus on ParABS: an apparently simple, three-component system, required for the segregation of bacterial chromosomes and plasmids. We will specifically describe how biophysical measurements combined with physical modeling advanced our understanding of the mechanism of ParABS-mediated complex assembly, segregation and positioning.


Asunto(s)
Proteínas Bacterianas/metabolismo , Segregación Cromosómica , Cromosomas Bacterianos/metabolismo , Posicionamiento de Cromosoma , ADN Bacteriano/metabolismo , Plásmidos/metabolismo
12.
Nat Commun ; 11(1): 4851, 2020 09 25.
Artículo en Inglés | MEDLINE | ID: mdl-32978386

RESUMEN

Cell factories converting bio-based precursors to chemicals present an attractive avenue to a sustainable economy, yet screening of genetically diverse strain libraries to identify the best-performing whole-cell biocatalysts is a low-throughput endeavor. For this reason, transcriptional biosensors attract attention as they allow the screening of vast libraries when used in combination with fluorescence-activated cell sorting (FACS). However, broad ligand specificity of transcriptional regulators (TRs) often prohibits the development of such ultra-high-throughput screens. Here, we solve the structure of the TR LysG of Corynebacterium glutamicum, which detects all three basic amino acids. Based on this information, we follow a semi-rational engineering approach using a FACS-based screening/counterscreening strategy to generate an L-lysine insensitive LysG-based biosensor. This biosensor can be used to isolate L-histidine-producing strains by FACS, showing that TR engineering towards a more focused ligand spectrum can expand the scope of application of such metabolite sensors.


Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/química , Proteínas Bacterianas/química , Técnicas Biosensibles/métodos , Ligandos , Ingeniería Metabólica/métodos , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Proteínas Bacterianas/metabolismo , Corynebacterium glutamicum/metabolismo , Cristalografía , Citometría de Flujo/métodos , Ensayos Analíticos de Alto Rendimiento/métodos , Lisina/metabolismo , Técnicas Analíticas Microfluídicas , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Termodinámica
13.
PLoS Pathog ; 16(9): e1008867, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32925969

RESUMEN

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the downstream toxins that mediate surface-dependent P. aeruginosa virulence remain unclear, as do the signaling pathways that lead to their activation. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and act directly on host cells to cause cytotoxicity. Surface-induced AQ cytotoxicity is independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. We further show that packaging of AQs in outer-membrane vesicles (OMVs) increases their cytotoxicity to host cells but not their ability to stimulate downstream quorum sensing pathways in bacteria. OMVs lacking AQs are significantly less cytotoxic, suggesting these molecules play a role in OMV cytotoxicity, in addition to their previously characterized role in OMV biogenesis. AQ reporters also enabled us to dissect the signal transduction pathways downstream of the two known regulators of surface-dependent virulence, the quorum sensing receptor, LasR, and the putative mechanosensor, PilY1. Specifically, we show that PilY1 regulates surface-induced AQ production by repressing the AlgR-AlgZ two-component system. AlgR then induces RhlR, which can induce the AQ biosynthesis operon under specific conditions. These findings collectively suggest that the induction of AQs upon surface association is both necessary and sufficient to explain surface-induced P. aeruginosa virulence.


Asunto(s)
Proteínas Bacterianas/metabolismo , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Quinolonas/farmacología , Percepción de Quorum/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Factores de Virulencia/metabolismo , Células A549 , Animales , Humanos , Ratones , Infecciones por Pseudomonas/patología , Pseudomonas aeruginosa/metabolismo , Pseudomonas aeruginosa/patogenicidad
14.
PLoS Pathog ; 16(9): e1008878, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32946535

RESUMEN

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.


Asunto(s)
Proteínas Bacterianas , Membrana Celular/metabolismo , Chlamydia trachomatis , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Membrana Celular/genética , Membrana Celular/patología , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidad , Células HeLa , Humanos , Mutación , Dominios Proteicos , Seudópodos/genética , Seudópodos/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética
15.
Mol Cell ; 79(5): 758-767.e6, 2020 09 03.
Artículo en Inglés | MEDLINE | ID: mdl-32755596

RESUMEN

During proteotoxic stress, bacteria maintain critical processes like DNA replication while removing misfolded proteins, which are degraded by the Lon protease. Here, we show that in Caulobacter crescentus Lon controls deoxyribonucleoside triphosphate (dNTP) pools during stress through degradation of the transcription factor CcrM. Elevated dNTP/nucleotide triphosphate (NTP) ratios in Δlon cells protects them from deletion of otherwise essential deoxythymidine triphosphate (dTTP)-producing pathways and shields them from hydroxyurea-induced loss of dNTPs. Increased dNTP production in Δlon results from higher expression of ribonucleotide reductase driven by increased CcrM. We show that misfolded proteins can stabilize CcrM by competing for limited protease and that Lon-dependent control of dNTPs improves fitness during protein misfolding conditions. We propose that linking dNTP production with availability of Lon allows Caulobacter to maintain replication capacity when misfolded protein burden increases, such as during rapid growth. Because Lon recognizes misfolded proteins regardless of the stress, this mechanism allows for response to a variety of unanticipated conditions.


Asunto(s)
Caulobacter crescentus/metabolismo , Nucleótidos/metabolismo , Proteasa La/metabolismo , Pliegue de Proteína , Proteínas Bacterianas/metabolismo , Caulobacter crescentus/enzimología , Elementos Transponibles de ADN , Didesoxinucleósidos/metabolismo , Regulación Bacteriana de la Expresión Génica , Nucleótido Desaminasas/genética , Nucleótido Desaminasas/metabolismo , Ribonucleótido Reductasas/metabolismo , Estrés Fisiológico , Factores de Transcripción/metabolismo , Regulación hacia Arriba
16.
Nat Commun ; 11(1): 4097, 2020 08 14.
Artículo en Inglés | MEDLINE | ID: mdl-32796861

RESUMEN

Staphylococcus aureus is generally thought to divide in three alternating orthogonal planes over three consecutive division cycles. Although this mode of division was proposed over four decades ago, the molecular mechanism that ensures this geometry of division has remained elusive. Here we show, for three different strains, that S. aureus cells do not regularly divide in three alternating perpendicular planes as previously thought. Imaging of the divisome shows that a plane of division is always perpendicular to the previous one, avoiding bisection of the nucleoid, which segregates along an axis parallel to the closing septum. However, one out of the multiple planes perpendicular to the septum which divide the cell in two identical halves can be used in daughter cells, irrespective of its orientation in relation to the penultimate division plane. Therefore, division in three orthogonal planes is not the rule in S. aureus.


Asunto(s)
Proteínas Bacterianas/metabolismo , Staphylococcus aureus/citología , Staphylococcus aureus/metabolismo , Proteínas Bacterianas/genética , Microbiología , Imagen de Lapso de Tiempo
17.
Nat Commun ; 11(1): 4126, 2020 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-32807804

RESUMEN

Neisseria gonorrhoeae is an urgent public health threat due to rapidly increasing incidence and antibiotic resistance. In contrast with the trend of increasing resistance, clinical isolates that have reverted to susceptibility regularly appear, prompting questions about which pressures compete with antibiotics to shape gonococcal evolution. Here, we used genome-wide association to identify loss-of-function (LOF) mutations in the efflux pump mtrCDE operon as a mechanism of increased antibiotic susceptibility and demonstrate that these mutations are overrepresented in cervical relative to urethral isolates. This enrichment holds true for LOF mutations in another efflux pump, farAB, and in urogenitally-adapted versus typical N. meningitidis, providing evidence for a model in which expression of these pumps in the female urogenital tract incurs a fitness cost for pathogenic Neisseria. Overall, our findings highlight the impact of integrating microbial population genomics with host metadata and demonstrate how host environmental pressures can lead to increased antibiotic susceptibility.


Asunto(s)
Proteínas Bacterianas/metabolismo , Cuello del Útero/microbiología , Neisseria gonorrhoeae/efectos de los fármacos , Neisseria gonorrhoeae/genética , Animales , Proteínas Bacterianas/genética , Farmacorresistencia Microbiana/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Estudio de Asociación del Genoma Completo , Humanos , Pruebas de Sensibilidad Microbiana , Mutación/genética , Neisseria gonorrhoeae/metabolismo , Operón/genética , Regiones Promotoras Genéticas/genética
18.
Nat Commun ; 11(1): 3970, 2020 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-32769975

RESUMEN

The rise of antibiotic resistance in many bacterial pathogens has been driven by the spread of a few successful strains, suggesting that some bacteria are genetically pre-disposed to evolving resistance. Here, we test this hypothesis by challenging a diverse set of 222 isolates of Staphylococcus aureus with the antibiotic ciprofloxacin in a large-scale evolution experiment. We find that a single efflux pump, norA, causes widespread variation in evolvability across isolates. Elevated norA expression potentiates evolution by increasing the fitness benefit provided by DNA topoisomerase mutations under ciprofloxacin treatment. Amplification of norA provides a further mechanism of rapid evolution in isolates from the CC398 lineage. Crucially, chemical inhibition of NorA effectively prevents the evolution of resistance in all isolates. Our study shows that pre-existing genetic diversity plays a key role in shaping resistance evolution, and it may be possible to predict which strains are likely to evolve resistance and to optimize inhibitor use to prevent this outcome.


Asunto(s)
Proteínas Bacterianas/metabolismo , Farmacorresistencia Microbiana , Evolución Molecular , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación , Ciprofloxacino/farmacología , Farmacorresistencia Microbiana/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Genoma Bacteriano , Mutación/genética , Filogenia , Staphylococcus aureus/efectos de los fármacos , Transcriptoma/efectos de los fármacos , Transcriptoma/genética
19.
Nat Commun ; 11(1): 3974, 2020 08 07.
Artículo en Inglés | MEDLINE | ID: mdl-32769995

RESUMEN

Bacillus thuringiensis Vip3 (Vegetative Insecticidal Protein 3) toxins are widely used in biotech crops to control Lepidopteran pests. These proteins are produced as inactive protoxins that need to be activated by midgut proteases to trigger cell death. However, little is known about their three-dimensional organization and activation mechanism at the molecular level. Here, we have determined the structures of the protoxin and the protease-activated state of Vip3Aa at 2.9 Å using cryo-electron microscopy. The reconstructions show that the protoxin assembles into a pyramid-shaped tetramer with the C-terminal domains exposed to the solvent and the N-terminal region folded into a spring-loaded apex that, after protease activation, drastically remodels into an extended needle by a mechanism akin to that of influenza haemagglutinin. These results provide the molecular basis for Vip3 activation and function, and serves as a strong foundation for the development of more efficient insecticidal proteins.


Asunto(s)
Bacillus thuringiensis/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/ultraestructura , Modelos Moleculares , Dominios Proteicos , Estructura Secundaria de Proteína , Tripsina/metabolismo
20.
PLoS Comput Biol ; 16(8): e1007898, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32797038

RESUMEN

New treatments for diseases caused by antimicrobial-resistant microorganisms can be developed by identifying unexplored therapeutic targets and by designing efficient drug screening protocols. In this study, we have screened a library of compounds to find ligands for the flavin-adenine dinucleotide synthase (FADS) -a potential target for drug design against tuberculosis and pneumonia- by implementing a new and efficient virtual screening protocol. The protocol has been developed for the in silico search of ligands of unexplored therapeutic targets, for which limited information about ligands or ligand-receptor structures is available. It implements an integrative funnel-like strategy with filtering layers that increase in computational accuracy. The protocol starts with a pharmacophore-based virtual screening strategy that uses ligand-free receptor conformations from molecular dynamics (MD) simulations. Then, it performs a molecular docking stage using several docking programs and an exponential consensus ranking strategy. The last filter, samples the conformations of compounds bound to the target using MD simulations. The MD conformations are scored using several traditional scoring functions in combination with a newly-proposed score that takes into account the fluctuations of the molecule with a Morse-based potential. The protocol was optimized and validated using a compound library with known ligands of the Corynebacterium ammoniagenes FADS. Then, it was used to find new FADS ligands from a compound library of 14,000 molecules. A small set of 17 in silico filtered molecules were tested experimentally. We identified five inhibitors of the activity of the flavin adenylyl transferase module of the FADS, and some of them were able to inhibit growth of three bacterial species: C. ammoniagenes, Mycobacterium tuberculosis, and Streptococcus pneumoniae, where the last two are human pathogens. Overall, the results show that the integrative VS protocol is a cost-effective solution for the discovery of ligands of unexplored therapeutic targets.


Asunto(s)
Antibacterianos , Proteínas Bacterianas , Nucleotidiltransferasas , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Corynebacterium/efectos de los fármacos , Corynebacterium/enzimología , Diseño de Fármacos , Farmacorresistencia Bacteriana/efectos de los fármacos , Ligandos , Simulación de Dinámica Molecular , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/enzimología , Nucleotidiltransferasas/antagonistas & inhibidores , Nucleotidiltransferasas/química , Nucleotidiltransferasas/metabolismo
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