RESUMEN
Thermococcus onnurineus NA1, a hyperthermophilic carboxydotrophic archaeon, produces H2 through CO oxidation catalyzed by proteins encoded in a carbon monoxide dehydrogenase (CODH) gene cluster. TON_1525 with a DNA-binding helix-turn-helix (HTH) motif is a putative repressor regulating the transcriptional expression of the codh gene cluster. The T55I mutation in TON_1525 led to enhanced H2 production accompanied by the increased expression of genes in the codh cluster. Here, TON_1525 was demonstrated to be a dimer. Monomeric TON_1525 adopts a novel 'eighth note' symbol-like fold (referred to as 'eighth note' fold regulator, EnfR), and the dimerization mode of EnfR is unique in that it has no resemblance to structures in the Protein Data Bank. According to footprinting and gel shift assays, dimeric EnfR binds to a 36-bp pseudo-palindromic inverted repeat in the promoter region of the codh gene cluster, which is supported by an in silico EnfR/DNA complex model and mutational studies revealing the implication of N-terminal loops as well as HTH motifs in DNA recognition. The DNA-binding affinity of the T55I mutant was lowered by â¼15-fold, for which the conformational change of N-terminal loops is responsible. In addition, transcriptome analysis suggested that EnfR could regulate diverse metabolic processes besides H2 production.
RESUMEN
Instead of conventional serotyping and virulence gene combination methods, methods have been developed to evaluate the pathogenic potential of newly emerging pathogens. Among them, the machine learning (ML)-based method using whole-genome sequencing (WGS) data are getting attention because of the recent advances in ML algorithms and sequencing technologies. Here, we developed various ML models to predict the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) isolates using their WGS data. The input dataset for the ML models was generated using distinct gene repertoires from positive (pathogenic) and negative (nonpathogenic) control groups in which each STEC isolate was designated based on the source attribution, the relative risk potential of the isolation sources. Among the various ML models examined, a model using the support vector machine (SVM) algorithm, the SVM model, discriminated between the two control groups most accurately. The SVM model successfully predicted the pathogenicity of the isolates from the major sources of STEC outbreaks, the isolates with the history of outbreaks, and the isolates that cannot be assessed by conventional methods. Furthermore, the SVM model effectively differentiated the pathogenic potentials of the isolates at a finer resolution. Permutation importance analyses of the input dataset further revealed the genes important for the estimation, proposing the genes potentially essential for the pathogenicity of STEC. Altogether, these results suggest that the SVM model is a more reliable and broadly applicable method to evaluate the pathogenic potential of STEC isolates compared with conventional methods.
Asunto(s)
Proteínas de Escherichia coli/genética , Aprendizaje Automático , Toxina Shiga II/genética , Escherichia coli Shiga-Toxigénica/genética , Máquina de Vectores de Soporte , Infecciones por Escherichia coli/diagnóstico , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Humanos , Curva ROC , Reproducibilidad de los Resultados , Toxina Shiga II/metabolismo , Escherichia coli Shiga-Toxigénica/clasificación , Escherichia coli Shiga-Toxigénica/patogenicidad , Virulencia/genética , Secuenciación Completa del Genoma/métodosRESUMEN
Opportunistic bacteria strategically dampen their virulence to allow them to survive and propagate in hosts. However, the molecular mechanisms underlying virulence control are not clearly understood. Here, we found that the opportunistic pathogen Vibrio vulnificus biotype 3, which caused an outbreak of severe wound and intestinal infections associated with farmed tilapia, secretes significantly less virulent multifunctional autoprocessing repeats-in-toxin (MARTX) toxin, which is the most critical virulence factor in other clinical Vibrio strains. The biotype 3 MARTX toxin contains a cysteine protease domain (CPD) evolutionarily retaining a unique autocleavage site and a distinct ß-flap region. CPD autoproteolytic activity is attenuated following its autocleavage because of the ß-flap region. This ß-flap blocks the active site, disabling further autoproteolytic processing and release of the modularly structured effector domains within the toxin. Expression of this altered CPD consequently results in attenuated release of effectors by the toxin and significantly reduces the virulence of V. vulnificus biotype 3 in cells and in mice. Bioinformatic analysis revealed that this virulence mechanism is shared in all biotype 3 strains. Thus, these data provide new insights into the mechanisms by which opportunistic bacteria persist in an environmental reservoir, prolonging the potential to cause outbreaks.
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Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Vibriosis/metabolismo , Vibrio vulnificus/patogenicidad , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/química , Toxinas Bacterianas/química , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Ratones , Modelos Moleculares , Vibrio vulnificus/fisiología , Factores de Virulencia/químicaRESUMEN
Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.
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Factores de Ribosilacion-ADP , ADP-Ribosilación , Toxinas Bacterianas , Vibrio vulnificus , Factores de Ribosilacion-ADP/química , Factores de Ribosilacion-ADP/metabolismo , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Células HEK293 , Células HeLa , Humanos , Ratones , Dominios Proteicos , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidadRESUMEN
Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are secreted by Gram-negative bacteria and function as primary virulence-promoting macromolecules that deliver multiple cytopathic and cytotoxic effector domains into the host cytoplasm. Among these effectors, Ras/Rap1-specific endopeptidase (RRSP) catalyzes the sequence-specific cleavage of the Switch I region of the cellular substrates Ras and Rap1 that are crucial for host innate immune defenses during infection. To dissect the molecular basis underpinning RRSP-mediated substrate inactivation, we determined the crystal structure of an RRSP from the sepsis-causing bacterial pathogen Vibrio vulnificus (VvRRSP). Structural and biochemical analyses revealed that VvRRSP is a metal-independent TIKI family endopeptidase composed of an N-terminal membrane-localization and substrate-recruitment domain (N lobe) connected via an inter-lobe linker to the C-terminal active site-coordinating core ß-sheet-containing domain (C lobe). Structure-based mutagenesis identified the 2His/2Glu catalytic residues in the core catalytic domain that are shared with other TIKI family enzymes and that are essential for Ras processing. In vitro KRas cleavage assays disclosed that deleting the N lobe in VvRRSP causes complete loss of enzymatic activity. Endogenous Ras cleavage assays combined with confocal microscopy analysis of HEK293T cells indicated that the N lobe functions both in membrane localization via the first α-helix and in substrate assimilation by altering the functional conformation of the C lobe to facilitate recruitment of cellular substrates. Collectively, these results indicate that RRSP is a critical virulence factor that robustly inactivates Ras and Rap1 and augments the pathogenicity of invading bacteria via the combined effects of its N and C lobes.
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Proteínas Bacterianas/metabolismo , Endopeptidasas/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Sepsis/enzimología , Sepsis/microbiología , Vibrio vulnificus/enzimología , Proteínas de Unión al GTP rap1/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Toxinas Bacterianas , Endopeptidasas/química , Endopeptidasas/genética , Células HEK293 , Interacciones Huésped-Patógeno , Humanos , Dominios Proteicos , Proteínas Proto-Oncogénicas p21(ras)/química , Proteínas Proto-Oncogénicas p21(ras)/genética , Sepsis/genética , Vibrio vulnificus/química , Vibrio vulnificus/genética , Proteínas de Unión al GTP rap1/química , Proteínas de Unión al GTP rap1/genéticaRESUMEN
The marine bacterium Vibrio vulnificus causes food-borne diseases, which may lead to life-threatening septicemia in some individuals. Therefore, identifying virulence factors in V. vulnificus is of high priority. We performed a transcriptome analysis on V. vulnificus after infection of human intestinal HT29-methotrexate cells and found induction of plpA, encoding a putative phospholipase, VvPlpA. Bioinformatics, biochemical, and genetic analyses demonstrated that VvPlpA is a phospholipase A2 secreted in a type II secretion system-dependent manner. Compared with the wild type, the plpA mutant exhibited reduced mortality, systemic infection, and inflammation in mice as well as low cytotoxicity toward the human epithelial INT-407 cells. Moreover, plpA mutation attenuated the release of actin and cytosolic cyclophilin A from INT-407 cells, indicating that VvPlpA is a virulence factor essential for causing lysis and necrotic death of the epithelial cells. plpA transcription was growth phase-dependent, reaching maximum levels during the early stationary phase. Also, transcription factor HlyU and cAMP receptor protein (CRP) mediate additive activation and host-dependent induction of plpA Molecular biological analyses revealed that plpA expression is controlled via the promoter, P plpA , and that HlyU and CRP directly bind to P plpA upstream sequences. Taken together, this study demonstrated that VvPlpA is a type II secretion system-dependent secretory phospholipase A2 regulated by HlyU and CRP and is essential for the pathogenicity of V. vulnificus.
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Proteínas Bacterianas/metabolismo , Fosfolipasas A2/metabolismo , Vibriosis/enzimología , Vibrio vulnificus/enzimología , Vibrio vulnificus/patogenicidad , Proteínas Bacterianas/genética , Sistemas de Secreción Bacterianos/genética , Sistemas de Secreción Bacterianos/metabolismo , Línea Celular , Humanos , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Fosfolipasas A2/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vibriosis/genética , Vibriosis/patología , Vibrio vulnificus/genéticaRESUMEN
Vibrio vulnificus infects humans and causes lethal septicemia. The primary virulence factor is a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin consisting of conserved repeats-containing regions and various effector domains. Recent genomic analyses for the newly emerged V. vulnificus biotype 3 strain revealed that its MARTX toxin has two previously unknown effector domains. Herein, we characterized one of these domains, Domain X (DmXVv ). A structure-based homology search revealed that DmXVv belongs to the C58B cysteine peptidase subfamily. When ectopically expressed in cells, DmXVv was autoprocessed and induced cytopathicity including Golgi dispersion. When the catalytic cysteine or the region flanking the scissile bond was mutated, both autoprocessing and cytopathicity were significantly reduced indicating that DmXVv cytopathicity is activated by amino-terminal autoprocessing. Consistent with this, host cell protein export was affected by Vibrio cells producing a toxin with wild-type, but not catalytically inactive, DmXVv . DmXVv was found to localize to Golgi and to directly interact with Golgi-associated ADP-ribosylation factors ARF1, ARF3 and ARF4, although ARF binding was not necessary for the subcellular localization. Rather, this interaction was found to induce autoprocessing of DmXVv . These data demonstrate that the V. vulnificus hijacks the host ARF proteins to activate the cytopathic DmXVv effector domain of MARTX toxin.
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Factores de Ribosilacion-ADP/metabolismo , Toxinas Bacterianas/metabolismo , Aparato de Golgi/enzimología , Vibriosis/microbiología , Vibrio vulnificus/fisiología , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Aparato de Golgi/microbiología , Células HEK293 , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Fenotipo , Transporte de ProteínasRESUMEN
Pathogenic and commensal bacteria that experience limited nutrient availability in their host have evolved sophisticated systems to catabolize the mucin sugar N-acetylneuraminic acid, thereby facilitating their survival and colonization. The correct function of the associated catabolic machinery is particularly crucial for the pathogenesis of enteropathogenic bacteria during infection, although the molecular mechanisms involved with the regulation of the catabolic machinery are unknown. This study reports the complex structure of NanR, a repressor of the N-acetylneuraminate (nan) genes responsible for N-acetylneuraminic acid catabolism, and its regulatory ligand, N-acetylmannosamine 6-phosphate (ManNAc-6P), in the human pathogenic bacterium Vibrio vulnificus. Structural studies combined with electron microscopic, biochemical, and in vivo analysis demonstrated that NanR forms a dimer in which the two monomers create an arched tunnel-like DNA-binding space, which contains positively charged residues that interact with the nan promoter. The interaction between the NanR dimer and DNA is alleviated by the ManNAc-6P-mediated relocation of residues in the ligand-binding domain of NanR, which subsequently relieves the repressive effect of NanR and induces the transcription of the nan genes. Survival studies in which mice were challenged with a ManNAc-6P-binding-defective mutant strain of V. vulnificus demonstrated that this relocation of NanR residues is critical for V. vulnificus pathogenesis. In summary, this study presents a model of the mechanism that regulates sialic acid catabolism via NanR in V. vulnificus.
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Ácido N-Acetilneuramínico/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Vibrio vulnificus/metabolismo , Microscopía Electrónica , Modelos MolecularesRESUMEN
Quorum sensing allows bacteria to sense and respond to changes in population density. Acyl-homoserine lactones serve as quorum-sensing signals for many Proteobacteria, and acyl-homoserine lactone signaling is known to control cooperative activities. Quorum-controlled activities vary from one species to another. Quorum-sensing controls a constellation of genes in the opportunistic pathogen Pseudomonas aeruginosa, which thrives in a number of habitats ranging from soil and water to animal hosts. We hypothesized that there would be significant variation in quorum-sensing regulons among strains of P. aeruginosa isolated from different habitats and that differences in the quorum-sensing regulons might reveal insights about the ecology of P. aeruginosa. As a test of our hypothesis we used RNA-seq to identify quorum-controlled genes in seven P. aeruginosa isolates of diverse origins. Although our approach certainly overlooks some quorum-sensing-regulated genes we found a shared set of genes, i.e., a core quorum-controlled gene set, and we identified distinct, strain-variable sets of quorum-controlled genes, i.e., accessory genes. Some quorum-controlled genes in some strains were not present in the genomes of other strains. We detected a correlation between traits encoded by some genes in the strain-variable subsets of the quorum regulons and the ecology of the isolates. These findings indicate a role for quorum sensing in extension of the range of habitats in which a species can thrive. This study also provides a framework for understanding the molecular mechanisms by which quorum-sensing systems operate, the evolutionary pressures by which they are maintained, and their importance in disparate ecological contexts.
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Variación Genética , Pseudomonas aeruginosa/genética , Percepción de Quorum/genética , Regulón , Análisis por Conglomerados , Fibrosis Quística/microbiología , Microbiología Ambiental , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Genoma Bacteriano/genética , Humanos , Solanum lycopersicum/microbiología , Datos de Secuencia Molecular , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Infecciones por Pseudomonas/microbiología , Pseudomonas aeruginosa/clasificación , Pseudomonas aeruginosa/aislamiento & purificación , Análisis de Secuencia de ADN , Especificidad de la EspecieRESUMEN
Bacteriophages (phages) have gained considerable attention as effective antimicrobial agents that infect and kill pathogenic bacteria. Based on this feature, phages have been increasingly used to achieve food safety. They are stored in a medium or buffer to ensure stability; however, they cannot be directly applied to food under these conditions due to reasons such as regulatory considerations and concerns about marketability. This study developed a stabilizing solution that allowed the maintenance of phage activity for extended periods at room temperature while being directly applicable to food. The stability of phages stored in distilled water was relatively low. However, adding a stabilizer composed of sugars and salts improved the survival rates of phages significantly, resulting in stability for up to 48 weeks at room temperature. When Escherichia coli O157:H7-contaminated vegetables were washed with tap water containing phages, the phages reduced the pathogenic E. coli count by over 90% compared with washing with tap water alone. Additionally, when pathogenic E. coli-contaminated vegetables were placed in a phage-coated container and exposed to water, the coating of the container dissolved, releasing phages and lysing the pathogenic E. coli. This led to a significant 90% reduction in pathogenic E. coli contamination compared to that after water rinsing. These results suggest an effective and economical method for maintaining phage activity and establishing the potential for commercialization through application in the food industry.
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Bacteriófagos , Escherichia coli O157 , Microbiología de Alimentos , Temperatura , Verduras , Bacteriófagos/fisiología , Verduras/microbiología , Verduras/virología , Escherichia coli O157/virología , Enfermedades Transmitidas por los Alimentos/prevención & control , Enfermedades Transmitidas por los Alimentos/microbiología , Inocuidad de los AlimentosRESUMEN
Multiple bacterial genera take advantage of the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin to invade host cells. Secretion of the MARTX toxin by Vibrio vulnificus, a deadly opportunistic pathogen that causes primary septicemia, the precursor of sepsis, is a major driver of infection; however, the molecular mechanism via which the toxin contributes to septicemia remains unclear. Here, we report the crystal and cryo-electron microscopy (EM) structures of a toxin effector duet comprising the domain of unknown function in the first position (DUF1)/Rho inactivation domain (RID) complexed with human targets. These structures reveal how the duet is used by bacteria as a potent weapon. The data show that DUF1 acts as a RID-dependent transforming NADase domain (RDTND) that disrupts NAD+ homeostasis by hijacking calmodulin. The cryo-EM structure of the RDTND-RID duet complexed with calmodulin and Rac1, together with immunological analyses in vitro and in mice, provide mechanistic insight into how V. vulnificus uses the duet to suppress ROS generation by depleting NAD(P)+ and modifying Rac1 in a mutually-reinforcing manner that ultimately paralyzes first line immune responses, promotes dissemination of invaders, and induces sepsis. These data may allow development of tools or strategies to combat MARTX toxin-related human diseases.
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Toxinas Bacterianas , Microscopía por Crioelectrón , Vibrio vulnificus , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidad , Animales , Humanos , Ratones , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Femenino , NAD/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Sepsis/microbiología , Dominios Proteicos , Vibriosis/microbiología , NAD+ Nucleosidasa/metabolismo , NAD+ Nucleosidasa/química , Cristalografía por Rayos XRESUMEN
Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that produces attaching and effacing lesions on the large intestine and causes hemorrhagic colitis. It is primarily transmitted through the consumption of contaminated meat or fresh produce. Similar to other bacterial pathogens, antibiotic resistance is of concern for EHEC. Furthermore, since the production of Shiga toxin by this pathogen is enhanced after antibiotic treatment, alternative agents that control EHEC are necessary. This study aimed to discover alternative treatments that target virulence factors and reduce EHEC toxicity. The locus of enterocyte effacement (LEE) is essential for EHEC attachment to host cells and virulence, and most of the LEE genes are positively regulated by the transcriptional regulator, Ler. GrlA protein, a transcriptional activator of ler, is thus a potential target for virulence inhibitors of EHEC. To identify the GrlA inhibitors, an in vivo high-throughput screening (HTS) system consisting of a GrlA-expressing plasmid and a reporter plasmid was constructed. Since the reporter luminescence gene was fused to the ler promoter, the bioluminescence would decrease if inhibitors affected the GrlA. By screening 8,201 compounds from the Korea Chemical Bank, we identified a novel GrlA inhibitor named Grlactin [3-[(2,4-dichlorophenoxy)methyl]-4-(3-methylbut-2-en-1-yl)-4,5-dihydro-1,2,4-oxadiazol-5-one], which suppresses the expression of LEE genes. Grlactin significantly diminished the adhesion of EHEC strain EDL933 to human epithelial cells without inhibiting bacterial growth. These findings suggest that the developed screening system was effective at identifying GrlA inhibitors, and Grlactin has potential for use as a novel anti-adhesion agent for EHEC while reducing the incidence of resistance.
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Escherichia coli O157 , Proteínas de Escherichia coli , Humanos , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Transactivadores/metabolismo , Proteínas de Escherichia coli/metabolismo , Factores de Transcripción/genética , Regiones Promotoras Genéticas , Regulación Bacteriana de la Expresión GénicaRESUMEN
Sialic acids consist of nine-carbon keto sugars that are commonly found at the terminal end of mucins. This positional feature of sialic acids contributes to host cell interactions but is also exploited by some pathogenic bacteria in evasion of host immune system. Moreover, many commensals and pathogens use sialic acids as an alternative energy source to survive within the mucus-covered host environments, such as the intestine, vagina, and oral cavity. Among the various biological events mediated by sialic acids, this review will focus on the processes necessary for the catabolic utilization of sialic acid in bacteria. First of all, transportation of sialic acid should be preceded before its catabolism. There are four types of transporters that are used for sialic acid uptake; the major facilitator superfamily (MFS), the tripartite ATP-independent periplasmic C4-dicarboxilate (TRAP) multicomponent transport system, the ATP binding cassette (ABC) transporter, and the sodium solute symporter (SSS). After being moved by these transporters, sialic acid is degraded into an intermediate of glycolysis through the well-conserved catabolic pathway. The genes encoding the catabolic enzymes and transporters are clustered into an operon(s), and their expression is tightly controlled by specific transcriptional regulators. In addition to these mechanisms, we will cover some researches about sialic acid utilization by oral pathogens.
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Proteínas Bacterianas , Ácido N-Acetilneuramínico , Ácido N-Acetilneuramínico/metabolismo , Proteínas Bacterianas/genética , Bacterias/genética , Bacterias/metabolismo , Ácidos Siálicos/metabolismo , Proteínas de Transporte de Membrana/genética , Adenosina Trifosfato/metabolismoRESUMEN
16S rRNA short amplicon sequencing-based microbiota profiling has been thought of and suggested as a feasible method to assess food safety. However, even if a comprehensive microbial information can be obtained by microbiota profiling, it would not be necessarily sufficient for all circumstances. To prove this, the feasibility of the most widely used V3-V4 amplicon sequencing method for food safety assessment was examined here. We designed a pathogen (Vibrio parahaemolyticus) contamination and/or V. parahaemolyticus-specific phage treatment model of raw oysters under improper storage temperature and monitored their microbial structure changes. The samples stored at refrigerator temperature (negative control, NC) and those that were stored at room temperature without any treatment (no treatment, NT) were included as control groups. The profiling results revealed that no statistical difference exists between the NT group and the pathogen spiked- and/or phage treated-groups even when the bacterial composition was compared at the possible lowest-rank taxa, family/genus level. In the beta-diversity analysis, all the samples except the NC group formed one distinct cluster. Notably, the samples with pathogen and/or phage addition did not form each cluster even though the enumerated number of V. parahaemolyticus in those samples were extremely different. These discrepant results indicate that the feasibility of 16S rRNA short amplicon sequencing should not be overgeneralized in microbiological safety assessment of food samples, such as raw oyster.
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Bacteriófagos , Microbiota , Ostreidae , Animales , ARN Ribosómico 16S/genética , Estudios de Factibilidad , Microbiota/genética , Alimentos Marinos , Ostreidae/genética , Ostreidae/microbiología , Bacteriófagos/genéticaRESUMEN
The emergence and spread of antibiotic-resistant pathogenic bacteria have necessitated finding new control alternatives. Under these circumstances, lytic bacteriophages offer a viable and promising option. This review focuses on Vibrio-infecting bacteriophages and the characteristics that make them suitable for application in the food and aquaculture industries. Bacteria, particularly Vibrio spp., can produce biofilms under stress conditions. Therefore, this review summarizes several anti-biofilm mechanisms that phages have, such as stimulating the host bacteria to produce biofilm-degrading enzymes, utilizing tail depolymerases, and penetrating matured biofilms through water channels. Additionally, the advantages of bacteriophages over antibiotics, such as a lower probability of developing resistance and the ability to infect dormant cells, are discussed. Finally, this review presents future research prospects related to further utilization of phages in diverse fields.
RESUMEN
Bacillus licheniformis has been widely utilized in the food industry as well as various agricultural industries. In particular, it is a main microorganism of fermented soybeans. In this study, the changes of the metabolome and transcriptome of B. licheniformis KACC15844, which had been isolated from fermented soybeans, were investigated depending on alkaline pH (BP) and a high salt concentration (BS) using an integrated-omics technology, focusing on leucine metabolism. Overall, carbohydrate (glycolysis, sugar transport, and overflow) and amino acid (proline, glycine betaine, and serine) metabolisms were strongly associated with BS, while fatty acid metabolism, malate utilization, and branched-chain amino acid-derived volatiles were closely related to BP, in both gene and metabolic expressions. In particular, in leucine metabolism, the formation of 3-methylbutanoic acid, which has strong cheesy odor notes, was markedly increased in BP compared to the other samples. This study provided information on how specific culture conditions can affect gene expressions and metabolite formations in B. licheniformis using an integrated-omics approach.
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Bacillus licheniformis , Alimentos Fermentados , Bacillus licheniformis/genética , Transcriptoma , Glycine max/genética , Glycine max/metabolismo , Presión Osmótica , Leucina/metabolismo , Concentración de Iones de HidrógenoRESUMEN
The nan cluster of Vibrio vulnificus, a food-borne pathogen, consists of two divergently transcribed operons, nanT(PSL)AR and nanEK nagA, required for transport and catabolism of N-acetylneuraminic acid (Neu5Ac). A mutation of nanR abolished the extensive lag phase observed for the bacteria growing on Neu5Ac and increased transcription of nanT(P) and nanE, suggesting that NanR is a transcriptional repressor of both nan operons. Intracellular accumulation of Neu5Ac was dependent on the carbon source, implying that the nan operons are also subject to catabolite repression. Hence, cAMP receptor protein (CRP) appeared to activate and repress transcription of nanT(PSL)AR and nanEK nagA, respectively. Direct bindings of NanR and CRP to the nanT(P)-nanE intergenic DNA were demonstrated by EMSA. Two adjacent NanR-binding sites centered at +44.5 and -10 and a CRP-binding site centered at -60.5 from the transcription start site of nanT(P) were identified by DNase I protection assays. Mutagenesis approaches, in vitro transcription, and isothermal titration calorimetry experiments demonstrated that N-acetylmannosamine 6-phosphate specifically binds to NanR and functions as the inducer of the nan operons. The combined results propose a model in which NanR, CRP, and N-acetylmannosamine 6-phosphate cooperate for precise adjustment of the expression level of the V. vulnificus nan cluster.
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Proteínas Bacterianas/metabolismo , Proteína Receptora de AMP Cíclico/metabolismo , Genes Bacterianos/genética , Hexosaminas/metabolismo , Familia de Multigenes/genética , Fosfatos/metabolismo , Fosfatos de Azúcar/metabolismo , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Proteína Receptora de AMP Cíclico/genética , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Desoxirribonucleasa I/metabolismo , Mutación , Ácido N-Acetilneuramínico/metabolismo , Oligodesoxirribonucleótidos/genética , Oligodesoxirribonucleótidos/metabolismo , Operón/genética , Regiones Promotoras Genéticas/genética , Especificidad por Sustrato , Transcripción GenéticaRESUMEN
Opportunistic pathogen Vibrio vulnificus causes severe systemic infection in humans with high mortality. Although multiple exotoxins have been characterized in V. vulnificus, their interactions and potential synergistic roles in pathogen-induced host cell death have not been investigated previously. By employing a series of multiple exotoxin deletion mutants, we investigated whether specific exotoxins of the pathogen functioned together to achieve severe and rapid necrotic cell death. Human epithelial cells treated with V. vulnificus with a plpA deletion background exhibited an unusually prolonged cell blebbing, suggesting the importance of PlpA, a phospholipase A2, in rapid necrotic cell death by this pathogen. Additional deletion of the rtxA gene encoding the multifunctional autoprocessing repeats-in-toxin (MARTX) toxin did not result in necrotic cell blebs. However, if the rtxA gene was engineered to produce an effector-free MARTX toxin, the cell blebbing was observed, indicating that the pore forming activity of the MARTX toxin is sufficient, but the MARTX toxin effector domains are not necessary, for the blebbing. When a recombinant PlpA was treated on the blebbed cells, the blebs were completely disrupted. Consistent with this, MARTX toxin-pendent rapid release of cytosolic lactate dehydrogenase was significantly delayed in the plpA deletion background. Mutations in other exotoxins such as elastase, cytolysin/hemolysin, and/or extracellular metalloprotease did not affect the bleb formation or disruption. Together, these findings indicate that the pore forming MARTX toxin and the phospholipase A2, PlpA, cooperate sequentially to achieve rapid necrotic cell death by inducing cell blebbing and disrupting the blebs, respectively.
Asunto(s)
Toxinas Bacterianas/genética , Exotoxinas/genética , Fosfolipasas A2/genética , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Células 3T3-L1 , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/metabolismo , Muerte Celular , Exotoxinas/metabolismo , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Ratones , Fosfolipasas A2/metabolismo , Eliminación de Secuencia , Vibriosis/microbiología , Vibrio vulnificus/patogenicidad , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
V. vulnificus-infected patients suffer from hemolytic anemia and circulatory lesions, often accompanied by venous thrombosis. However, the pathophysiological mechanism of venous thrombosis associated with V. vulnificus infection remains largely unknown. Herein, V. vulnificus infection at the sub-hemolytic level induced shape change of human red blood cells (RBCs) accompanied by phosphatidylserine exposure, and microvesicle generation, leading to the procoagulant activation of RBCs and ultimately, acquisition of prothrombotic activity. Of note, V. vulnificus exposed to RBCs substantially upregulated the rtxA gene encoding multifunctional autoprocessing repeats-in-toxin (MARTX) toxin. Mutant studies showed that V. vulnificus-induced RBC procoagulant activity was due to the pore forming region of the MARTX toxin causing intracellular Ca2+ influx in RBCs. In a rat venous thrombosis model triggered by tissue factor and stasis, the V. vulnificus wild type increased thrombosis while the ΔrtxA mutant failed to increase thrombosis, confirming that V. vulnificus induces thrombosis through the procoagulant activation of RBCs via the mediation of the MARTX toxin.
Asunto(s)
Toxinas Bacterianas , Trombosis , Vibrio vulnificus , Animales , Toxinas Bacterianas/genética , Eritrocitos , Humanos , Fosfatidilserinas , Ratas , Trombosis/genética , Vibrio vulnificus/genéticaRESUMEN
Little is known about the molecular mechanism for autolysis of Gram-negative bacteria. In the present study, we identified the vvpS gene encoding a serine protease, VvpS, from Vibrio vulnificus, a Gram-negative food-borne pathogen. The amino acid sequence predicted that VvpS consists of two functional domains, an N-terminal protease catalytic domain (PCD) and a C-terminal carbohydrate binding domain (CBD). A null mutation of vvpS significantly enhanced viability during stationary phase, as measured by enumerating CFU and differentially staining viable cells. The vvpS mutant reduced the release of cytoplasmic ß-galactosidase and high-molecular-weight extracellular chromosomal DNA into the culture supernatants, indicating that VvpS contributes to the autolysis of V. vulnificus during stationary phase. VvpS is secreted via a type II secretion system (T2SS), and it exerts its effects on autolysis through intracellular accumulation during stationary phase. Consistent with this, a disruption of the T2SS accelerated intracellular accumulation of VvpS and thereby the autolysis of V. vulnificus. VvpS also showed peptidoglycan-hydrolyzing activity, indicating that the autolysis of V. vulnificus is attributed to the self-digestion of the cell wall by VvpS. The functions of the VvpS domains were assessed by C-terminal deletion analysis and demonstrated that the PCD indeed possesses a proteolytic activity and that the CBD is required for hydrolyzing peptidoglycan effectively. Finally, the vvpS mutant exhibited reduced virulence in the infection of mice. In conclusion, VvpS is a serine protease with a modular structure and plays an essential role in the autolysis and pathogenesis of V. vulnificus.