RESUMEN
Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180 ± 80 M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590 ± 20 M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4 ± 4.1 × 104 M-1s-1 to 20.3 ± 4.3 × 104 M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7 ± 5.6 × 104 M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3 ± 1.9 × 105 M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform in vivo function. Furthermore, purified LapX and Lap restore the timing of the V. cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.
Asunto(s)
Proteínas Bacterianas , Leucil Aminopeptidasa , Serina Proteasas , Vibrio cholerae , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Leucil Aminopeptidasa/química , Leucil Aminopeptidasa/genética , Leucil Aminopeptidasa/fisiología , Péptidos , Serina Proteasas/química , Serina Proteasas/genética , Serina Proteasas/fisiología , Especificidad por Sustrato , Vibrio cholerae/enzimología , Vibrio cholerae/genética , Vibrio cholerae/fisiología , CatálisisRESUMEN
Bacteria orchestrate collective behaviors using the cell-cell communication process called quorum sensing (QS). QS relies on the synthesis, release, and group-wide detection of small molecules called autoinducers. In Vibrio cholerae, a multicellular community aggregation program occurs in liquid, during the stationary phase, and in the high-cell-density QS state. Here, we demonstrate that this aggregation program consists of two subprograms. In one subprogram, which we call void formation, structures form that contain few cells but provide a scaffold within which cells can embed. The other subprogram relies on flagellar machinery and enables cells to enter voids. A genetic screen for factors contributing to void formation, coupled with companion molecular analyses, showed that four extracellular proteases, Vca0812, Vca0813, HapA, and PrtV, control the onset timing of both void formation and aggregation; moreover, proteolytic activity is required. These proteases, or their downstream products, can be shared between void-producing and non-void-forming cells and can elicit aggregation in a normally nonaggregating V. cholerae strain. Employing multiple proteases to control void formation and aggregation timing could provide a redundant and irreversible path to commitment to this community lifestyle. IMPORTANCE Bacteria can work as collectives to form multicellular communities. Vibrio cholerae, the bacterium that causes the disease cholera in humans, forms aggregated communities in liquid. Aggregate formation relies on a chemical communication process called quorum sensing. Here, we show that, beyond overarching control by quorum sensing, there are two aggregation subprograms. One subprogram, which we call void formation, creates a scaffold within which cells can embed. The second subprogram, which allows bacteria to enter the scaffold, requires motility. We discovered that four extracellular proteases control the timing of both void formation and aggregation. We argue that, by using redundant proteases, V. cholerae ensures the reliable execution of this community formation process. These findings may provide insight into how V. cholerae persists in the marine environment or colonizes the human host, as both lifestyles are central to the spread of the disease cholera.
Asunto(s)
Cólera/microbiología , Metaloendopeptidasas/metabolismo , Péptido Hidrolasas/metabolismo , Vibrio cholerae/enzimología , Vibrio cholerae/crecimiento & desarrollo , Biopelículas , Humanos , Metaloendopeptidasas/genética , Operón , Péptido Hidrolasas/genética , Percepción de Quorum , Vibrio cholerae/genéticaRESUMEN
Quorum sensing is a process of cell-to-cell communication that bacteria use to orchestrate collective behaviors. Quorum sensing depends on the production, release, and detection of extracellular signal molecules called autoinducers (AIs) that accumulate with increasing cell density. While most AIs are species specific, the AI called AI-2 is produced and detected by diverse bacterial species, and it mediates interspecies communication. We recently reported that mammalian cells produce an AI-2 mimic that can be detected by bacteria through the AI-2 receptor LuxP, potentially expanding the role of the AI-2 system to interdomain communication. Here, we describe a second molecule capable of interdomain signaling through LuxP, 4-hydroxy-5-methylfuran-3(2H)-one (MHF), that is produced by the yeast Saccharomyces cerevisiae Screening the S. cerevisiae deletion collection revealed Cff1p, a protein with no known role, to be required for MHF production. Cff1p is proposed to be an enzyme, with structural similarity to sugar isomerases and epimerases, and substitution at the putative catalytic residue eliminated MHF production in S. cerevisiae Sequence analysis uncovered Cff1p homologs in many species, primarily bacterial and fungal, but also viral, archaeal, and higher eukaryotic. Cff1p homologs from organisms from all domains can complement a cff1ΔS. cerevisiae mutant and restore MHF production. In all cases tested, the identified catalytic residue is conserved and required for MHF to be produced. These findings increase the scope of possibilities for interdomain interactions via AI-2 and AI-2 mimics, highlighting the breadth of molecules and organisms that could participate in quorum sensing.IMPORTANCE Quorum sensing is a cell-to-cell communication process that bacteria use to monitor local population density. Quorum sensing relies on extracellular signal molecules called autoinducers (AIs). One AI called AI-2 is broadly made by bacteria and used for interspecies communication. Here, we describe a eukaryotic AI-2 mimic, 4-hydroxy-5-methylfuran-3(2H)-one, (MHF), that is made by the yeast Saccharomyces cerevisiae, and we identify the Cff1p protein as essential for MHF production. Hundreds of viral, archaeal, bacterial, and eukaryotic organisms possess Cff1p homologs. This finding, combined with our results showing that homologs from all domains can replace S. cerevisiae Cff1p, suggests that like AI-2, MHF is widely produced. Our results expand the breadth of organisms that may participate in quorum-sensing-mediated interactions.
Asunto(s)
Bacterias/metabolismo , Furanos/metabolismo , Homoserina/análogos & derivados , Lactonas/metabolismo , Percepción de Quorum , Saccharomyces cerevisiae/metabolismo , Proteínas Bacterianas/metabolismo , Furanos/análisis , Homoserina/genética , Homoserina/metabolismo , Saccharomyces cerevisiae/genética , Transducción de SeñalRESUMEN
Pseudomonas aeruginosa is a leading cause of hospital-acquired infections in the United States. PqsE, a thioesterase enzyme, is vital for virulence of P. aeruginosa, making PqsE an attractive target for inhibition. Neither the substrate nor the product of PqsE catalysis has been identified. A library of 550 million DNA-encoded drug-like small molecules was screened for those that bind to the purified PqsE protein. The structures of the bound molecules were identified by high throughput sequencing of the attached DNA barcodes. Putative PqsE binders with the strongest affinity features were examined for inhibition of PqsE thioesterase activity in vitro. The most potent inhibitors were resynthesized off DNA and examined for the ability to alter PqsE thermal melting and for PqsE thioesterase inhibition. Here, we report the synthesis, biological activity, mechanism of action, and early structure-activity relationships of a series of 2-(phenylcarbamoyl)benzoic acids that noncompetitively inhibit PqsE. A small set of analogs designed to probe initial structure-activity relationships showed increases in potency relative to the original hits, the best of which has an IC50 = 5 µM. Compound refinement is required to assess their in vivo activities as the current compounds do not accumulate in the P. aeruginosa cytosol. Our strategy validates DNA-encoded compound library screening as a rapid and effective method to identify catalytic inhibitors of the PqsE protein, and more generally, for discovering binders to bacterial proteins revealed by genetic screening to have crucial in vivo activities but whose biological functions have not been well-defined.
Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , ADN/química , Inhibidores Enzimáticos/farmacología , Pseudomonas aeruginosa/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Tioléster Hidrolasas/antagonistas & inhibidores , Benzamidas/síntesis química , Benzamidas/farmacología , Inhibidores Enzimáticos/síntesis química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Ácidos Ftálicos/síntesis química , Ácidos Ftálicos/farmacología , Pseudomonas aeruginosa/enzimología , Bibliotecas de Moléculas Pequeñas/síntesis química , Relación Estructura-ActividadRESUMEN
Host-microbial symbioses are vital to health; nonetheless, little is known about the role crosskingdom signaling plays in these relationships. In a process called quorum sensing, bacteria communicate with one another using extracellular signal molecules called autoinducers. One autoinducer, AI-2, is proposed to promote interspecies bacterial communication, including in the mammalian gut. We show that mammalian epithelia produce an AI-2 mimic activity in response to bacteria or tight-junction disruption. This AI-2 mimic is detected by the bacterial AI-2 receptor, LuxP/LsrB, and can activate quorum-sensing-controlled gene expression, including in the enteric pathogen Salmonella typhimurium. AI-2 mimic activity is induced when epithelia are directly or indirectly exposed to bacteria, suggesting that a secreted bacterial component(s) stimulates its production. Mutagenesis revealed genes required for bacteria to both detect and stimulate production of the AI-2 mimic. These findings uncover a potential role for the mammalian AI-2 mimic in fostering crosskingdom signaling and host-bacterial symbioses.
Asunto(s)
Homoserina/análogos & derivados , Interacciones Huésped-Patógeno , Lactonas/metabolismo , Percepción de Quorum , Infecciones por Salmonella/metabolismo , Salmonella typhimurium/fisiología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células Epiteliales/metabolismo , Células Epiteliales/microbiología , Regulación Bacteriana de la Expresión Génica , Homoserina/metabolismo , Humanos , Infecciones por Salmonella/genética , Infecciones por Salmonella/microbiología , Salmonella typhimurium/genéticaRESUMEN
Five homologous noncoding small RNAs (sRNAs), called the Qrr1-5 sRNAs, function in the Vibrio harveyi quorum-sensing cascade to drive its operation. Qrr1-5 use four different regulatory mechanisms to control the expression of â¼ 20 mRNA targets. Little is known about the roles individual nucleotides play in mRNA target selection, in determining regulatory mechanism, or in defining Qrr potency and dynamics of target regulation. To identify the nucleotides vital for Qrr function, we developed a method we call RSort-Seq that combines saturating mutagenesis, fluorescence-activated cell sorting, high-throughput sequencing, and mutual information theory to explore the role that every nucleotide in Qrr4 plays in regulation of two mRNA targets, luxR and luxO. Companion biochemical assays allowed us to assign specific regulatory functions/underlying molecular mechanisms to each important base. This strategy yielded a regional map of nucleotides in Qrr4 vital for stability, Hfq interaction, stem-loop formation, and base pairing to both luxR and luxO, to luxR only, and to luxO only. In terms of nucleotides critical for sRNA function, the RSort-Seq analysis provided strikingly different results from those predicted by commonly used regulatory RNA-folding algorithms. This approach is applicable to any RNA-RNA interaction, including sRNAs in other bacteria and regulatory RNAs in higher organisms.
Asunto(s)
Escherichia coli/fisiología , Nucleótidos/fisiología , Percepción de Quorum , ARN no Traducido/fisiología , Vibrio/fisiología , Escherichia coli/genética , Vibrio/genéticaRESUMEN
Synucleinopathies are neurodegenerative diseases associated with toxicity of the lipid-binding protein α-synuclein (α-syn). When expressed in yeast, α-syn associates with membranes at the endoplasmic reticulum and traffics with vesicles out to the plasma membrane. At higher levels it elicits a number of phenotypes, including blocking vesicle trafficking. The expression of α-syn splice isoforms varies with disease, but how these isoforms affect protein function is unknown. We investigated two of the most abundant isoforms, resulting in deletion of exon four (α-synΔ4) or exon six (α-synΔ6). α-SynΔ4, missing part of the lipid-binding domain, had reduced toxicity and membrane binding. α-SynΔ6, missing part of the protein-protein interaction domain, had reduced toxicity but no reduction in membrane binding. To compare the mechanism by which the splice isoforms exert toxicity, equally toxic strains were probed with genetic modifiers of α-syn-induced toxicity. Most modifiers equally altered the toxicity induced by the splice isoforms and full-length α-syn (α-synFL). However, the splice isoform strains responded differently to a sterol-binding protein, leading us to examine the effect of sterols on α-syn-induced toxicity. Upon inhibition of sterol synthesis, α-synFL and α-synΔ6, but not α-synΔ4, showed decreased plasma membrane association, increased vesicular association, and increased cellular toxicity. Thus, higher membrane sterol concentrations favor plasma membrane binding of α-synFL and α-synΔ6 and may be protective of synucleinopathy progression. Given the common use of cholesterol-reducing statins and these potential effects on membrane binding proteins, further investigation of how sterol concentration and α-syn splice isoforms affect vesicular trafficking in synucleinopathies is warranted.
Asunto(s)
Isoformas de Proteínas/metabolismo , Eliminación de Secuencia/genética , Esteroles/metabolismo , alfa-Sinucleína/genética , Western Blotting , Membrana Celular/metabolismo , Cartilla de ADN/genética , Retículo Endoplásmico/metabolismo , Fluconazol , Modelos Lineales , Microscopía Fluorescente , Isoformas de Proteínas/genética , Isoformas de Proteínas/toxicidad , Reacción en Cadena en Tiempo Real de la Polimerasa , Levaduras , alfa-Sinucleína/toxicidadRESUMEN
For a protein to function appropriately, it must first achieve its proper conformation and location within the crowded environment inside the cell. Multiple chaperone systems are required to fold proteins correctly. In addition, degradation pathways participate by destroying improperly folded proteins. The intricacy of this multisystem process provides many opportunities for error. Furthermore, mutations cause misfolded, nonfunctional forms of proteins to accumulate. As a result, many pathological conditions are fundamentally rooted in the protein-folding problem that all cells must solve to maintain their function and integrity. Here, to illustrate the breadth of this phenomenon, we describe five examples of protein-misfolding events that can lead to disease: improper degradation, mislocalization, dominant-negative mutations, structural alterations that establish novel toxic functions, and amyloid accumulation. In each case, we will highlight current therapeutic options for battling such diseases.
Asunto(s)
Pliegue de Proteína , Deficiencias en la Proteostasis/fisiopatología , Amiloide/metabolismo , Fibrosis Quística/fisiopatología , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Descubrimiento de Drogas , Enfermedad de Gaucher/fisiopatología , Genes Dominantes , Humanos , Chaperonas Moleculares/metabolismo , Mutación , Enfermedades Neurodegenerativas/fisiopatología , Conformación Proteica , Desnaturalización ProteicaRESUMEN
Aß (beta-amyloid peptide) is an important contributor to Alzheimer's disease (AD). We modeled Aß toxicity in yeast by directing the peptide to the secretory pathway. A genome-wide screen for toxicity modifiers identified the yeast homolog of phosphatidylinositol binding clathrin assembly protein (PICALM) and other endocytic factors connected to AD whose relationship to Aß was previously unknown. The factors identified in yeast modified Aß toxicity in glutamatergic neurons of Caenorhabditis elegans and in primary rat cortical neurons. In yeast, Aß impaired the endocytic trafficking of a plasma membrane receptor, which was ameliorated by endocytic pathway factors identified in the yeast screen. Thus, links between Aß, endocytosis, and human AD risk factors can be ascertained with yeast as a model system.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Endocitosis , Fragmentos de Péptidos/metabolismo , Saccharomyces cerevisiae , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/metabolismo , Citoesqueleto/metabolismo , Susceptibilidad a Enfermedades , Estudios de Asociación Genética , Pruebas Genéticas , Glutamatos/metabolismo , Humanos , Proteínas de Ensamble de Clatrina Monoméricas/genética , Proteínas de Ensamble de Clatrina Monoméricas/metabolismo , Neuronas/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Multimerización de Proteína , Transporte de Proteínas , Ratas , Factores de Riesgo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Vías SecretorasRESUMEN
Early-onset torsion dystonia is a severe, life-long disease that leads to loss of motor control and involuntary muscle contractions. While the molecular etiology of the disease is not fully understood, a mutation in an AAA+ ATPase, torsinA, has been linked to disease onset. Previous work on torsinA has shown that it localizes to the endoplasmic reticulum, where there is evidence that it plays roles in protein trafficking, and potentially also protein folding. Given the high level of evolutionary conservation among proteins involved in these processes, the ability of human such proteins to function effectively in yeast, as well as the previous successes achieved in examining other proteins involved in complex human diseases in yeast, we hypothesized that Saccharomyces cerevisiae might represent a useful model system for studying torsinA function and the effects of its mutants. Since torsinA is proposed to function in protein homeostasis, we tested cells for their ability to respond to various stressors, using a fluorescent reporter to measure the unfolded protein response, as well as their rate of protein secretion. TorsinA did not impact these processes, even after co-expression of its recently identified interacting partner, printor. In light of these findings, we propose that yeast may lack an additional cofactor necessary for torsinA function or proteins required for essential post-translational modifications of torsinA. Alternatively, torsinA may not function in endoplasmic reticulum protein homeostasis. The strains and assays we describe may provide useful tools for identifying and investigating these possibilities and are freely available.
Asunto(s)
Proteínas Portadoras/metabolismo , Estrés del Retículo Endoplásmico , Chaperonas Moleculares/metabolismo , Saccharomyces cerevisiae/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Fenotipo , Unión Proteica/efectos de los fármacos , Señales de Clasificación de Proteína , Transporte de Proteínas/efectos de los fármacos , Saccharomyces cerevisiae/citología , Respuesta de Proteína Desplegada/efectos de los fármacos , alfa-Sinucleína/toxicidadRESUMEN
Cells respond to stimuli by changes in various processes, including signaling pathways and gene expression. Efforts to identify components of these responses increasingly depend on mRNA profiling and genetic library screens. By comparing the results of these two assays across various stimuli, we found that genetic screens tend to identify response regulators, whereas mRNA profiling frequently detects metabolic responses. We developed an integrative approach that bridges the gap between these data using known molecular interactions, thus highlighting major response pathways. We used this approach to reveal cellular pathways responding to the toxicity of alpha-synuclein, a protein implicated in several neurodegenerative disorders including Parkinson's disease. For this we screened an established yeast model to identify genes that when overexpressed alter alpha-synuclein toxicity. Bridging these data and data from mRNA profiling provided functional explanations for many of these genes and identified previously unknown relations between alpha-synuclein toxicity and basic cellular pathways.