RESUMEN
Vibrio vulnificus causes life-threatening wound and gastrointestinal infections, mediated primarily by the production of a Multifunctional-Autoprocessing Repeats-In-Toxin (MARTX) toxin. The most commonly present MARTX effector domain, the Makes Caterpillars Floppy-like (MCF) toxin, is a cysteine protease stimulated by host adenosine diphosphate (ADP) ribosylation factors (ARFs) to autoprocess. Here, we show processed MCF then binds and cleaves host Ras-related proteins in brain (Rab) guanosine triphosphatases within their C-terminal tails resulting in Rab degradation. We demonstrate MCF binds Rabs at the same interface occupied by ARFs. Moreover, we show MCF preferentially binds to ARF1 prior to autoprocessing and is active to cleave Rabs only subsequent to autoprocessing. We then use structure prediction algorithms to demonstrate that structural composition, rather than sequence, determines Rab target specificity. We further determine a crystal structure of aMCF as a swapped dimer, revealing an alternative conformation we suggest represents the open, activated state of MCF with reorganized active site residues. The cleavage of Rabs results in Rab1B dispersal within cells and loss of Rab1B density in the intestinal tissue of infected mice. Collectively, our work describes an extracellular bacterial mechanism whereby MCF is activated by ARFs and subsequently induces the degradation of another small host guanosine triphosphatase (GTPase), Rabs, to drive organelle damage, cell death, and promote pathogenesis of these rapidly fatal infections.
Asunto(s)
Toxinas Bacterianas , Vibrio vulnificus , Proteínas de Unión al GTP rab , Animales , Femenino , Humanos , Ratones , Factores de Ribosilacion-ADP/metabolismo , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/química , Células HEK293 , Ratones Endogámicos ICR , Proteolisis , Proteínas de Unión al GTP rab/metabolismo , Vibriosis/microbiología , Vibriosis/metabolismo , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidadRESUMEN
Antibiotics are often used to treat severe Vibrio infections, with third-generation cephalosporins and tetracyclines combined or fluoroquinolones alone being recommended by the US Centers for Disease Control and Prevention. Increases in antibiotic resistance of both environmental and clinical vibrios are of concern; however, limited longitudinal data have been generated among environmental isolates to inform how resistance patterns may be changing over time. Hence, we evaluated long-term trends in antibiotic resistance of vibrios isolated from Chesapeake Bay waters (Maryland) across two 3-year sampling periods (2009-2012 and 2019-2022). Vibrio parahaemolyticus (n = 134) and Vibrio vulnificus (n = 94) toxR-confirmed isolates were randomly selected from both sampling periods and tested for antimicrobial susceptibility against eight antibiotics using the Kirby-Bauer disk diffusion method. A high percentage (94%-96%) of V. parahaemolyticus isolates from both sampling periods were resistant to ampicillin and only 2%-6% of these isolates expressed intermediate resistance or resistance to third-generation cephalosporins, amikacin, tetracycline, and trimethoprim-sulfamethoxazole. Even lower percentages of resistant V. vulnificus isolates were observed and those were mostly recovered from 2009 to 2012, however, the presence of multiple virulence factors was observed. The frequency of multi-drug resistance was relatively low (6%-8%) but included resistance against antibiotics used to treat severe vibriosis in adults and children. All isolates were susceptible to ciprofloxacin, a fluoroquinolone, indicating its sustained efficacy as a first-line agent in the treatment of severe vibriosis. Overall, our data indicate that antibiotic resistance patterns among V. parahaemolyticus and V. vulnificus recovered from the lower Chesapeake Bay have remained relatively stable since 2009.IMPORTANCEVibrio spp. have historically been susceptible to most clinically relevant antibiotics; however, resistance and intermediate-resistance have been increasingly recorded in both environmental and clinical isolates. Our data showed that while the percentage of multi-drug resistance and resistance to antibiotics was relatively low and stable across time, some Vibrio isolates displayed resistance and intermediate resistance to antibiotics typically used to treat severe vibriosis (e.g., third-generation cephalosporins, tetracyclines, sulfamethoxazole-trimethoprim, and aminoglycosides). Also, given the high case fatality rates observed with Vibrio vulnificus infections, the presence of multiple virulence factors in the tested isolates is concerning. Nevertheless, the continued susceptibility of all tested isolates against ciprofloxacin, a fluoroquinolone, is indicative of its use as an effective first-line treatment of severe Vibrio spp. infections stemming from exposure to Chesapeake Bay waters or contaminated seafood ingestion.
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Antibacterianos , Bahías , Vibrio parahaemolyticus , Vibrio vulnificus , Vibrio parahaemolyticus/efectos de los fármacos , Vibrio parahaemolyticus/aislamiento & purificación , Vibrio vulnificus/efectos de los fármacos , Vibrio vulnificus/aislamiento & purificación , Vibrio vulnificus/crecimiento & desarrollo , Bahías/microbiología , Antibacterianos/farmacología , Estudios Longitudinales , Maryland , Pruebas de Sensibilidad Microbiana , Farmacorresistencia Bacteriana , Vibriosis/microbiología , HumanosRESUMEN
CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) is a gene knockdown method that uses a deactivated Cas9 protein (dCas9) that binds a specific gene target locus dictated by an encoded guide RNA (sgRNA) to block transcription. Mobile-CRISPRi is a suite of modular vectors that enable CRISPRi knockdowns in diverse bacteria by integrating IPTG-inducible dcas9 and sgRNA genes into the genome using Tn7 transposition. Here, we show that the Mobile-CRISPRi system functions robustly and specifically in multiple Vibrio species: Vibrio cholerae, Vibrio fischeri, Vibrio vulnificus, Vibrio parahaemolyticus, and Vibrio campbellii. We demonstrate efficacy by targeting both essential and non-essential genes that function to produce defined, measurable phenotypes: bioluminescence, quorum sensing, cell division, and growth arrest. We anticipate that Mobile-CRISPRi will be used in Vibrio species to systematically probe gene function and essentiality in various behaviors and native environments.IMPORTANCEThe genetic manipulation of bacterial genomes is an invaluable tool in experimental microbiology. The development of CRISPRi (Clustered Regularly Interspaced Palindromic Repeats interference) tools has revolutionized genetics in many organisms, including bacteria. Here, we optimized the use of Mobile-CRISPRi in five Vibrio species, each of which has significant impacts on marine environments and organisms that include squid, shrimp, shellfish, finfish, corals, and multiple of which pose direct threats to human health. The Mobile-CRISPRi technology is easily adaptable, moveable from strain to strain, and enables researchers to selectively turn off gene expression. Our experiments demonstrate Mobile-CRISPRi is effective and robust at repressing gene expression of both essential and non-essential genes in Vibrio species.
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Vibrio vulnificus , Vibrio , Vibrio/genética , Vibrio vulnificus/genética , Vibrio parahaemolyticus/genética , Regulación Bacteriana de la Expresión Génica , Sistemas CRISPR-Cas , Vibrio cholerae/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Técnicas de Silenciamiento del Gen , Aliivibrio fischeri/genéticaRESUMEN
BACKGROUND: Vibrio vulnificus exists as one of the most serious foodborne pathogens for humans, and rapid and sensitive detection methods are needed to control its infections. As an emerging method, The Loop-Mediated Isothermal Amplification (LAMP) assay has been applied to the early detection of various foodborne pathogens due to its high efficiency, but sample preprocessing still prolongs the complete detection. To optimize the detection process, our study established a novel sample preprocessing method that was more efficient compared to common methods. RESULT: Using V. vulnificus as the detecting pathogen, the water-lysis-based detecting LAMP method shortened the preprocessing time to ≤ 1 min with 100% LAMP specificity; the detection limits of the LAMP assay were decreased to 1.20 × 102 CFU/mL and 1.47 × 103 CFU/g in pure culture and in oyster, respectively. Furthermore, the 100% LAMP specificity and high sensitivity of the water-lysis method were also obtained on detecting V. parahaemolyticus, V. alginolyticus, and P. mirabilis, revealing its excellent LAMP adaption with improvement in sensitivity and efficiency. CONCLUSION: Our study provided a novel LAMP preprocessing method that was more efficient compared to common methods and possessed the practical potential for LAMP application in the future.
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Técnicas de Diagnóstico Molecular , Vibrio vulnificus , Humanos , Vibrio vulnificus/genética , Técnicas de Amplificación de Ácido Nucleico/métodos , Agua , Manejo de Especímenes , Sensibilidad y EspecificidadRESUMEN
Vibrio vulnificus is a commonly pathogenic bacterium in cultivated eels, but its pathogenicity to American eel (Anguilla rostrata) and the molecular mechanism of host anti-V. vulnificus infection remains uncertain. In this study, American eels were infected with different dose of V. vulnificus to determine the LD50. Then, bacterial load in the liver and kidney histopathology were assessed post the LD50 of V. vulnificus infection. Additionally, gene expressions of 18 immune related genes in the liver, spleen and kidney were detected. Furthermore, transcriptome sequencing and enrichment of differentially expressed genes (DEGs) were analyzed in the eel spleens between pre-infection (Con_0), post-36 h (Vv_36), and post-60 h (Vv_60) infection. The results showed that LD50 of V. vulnificus to American eels was determined to be 5.0 × 105 cfu/g body weight, and the bacterial load peaked at 24 and 12 h post the infection (hpi) in the kidney and liver, respectively. The histopathology was highlighted by necrotic hepatocytes and splenic cells, congestion blood vessels in liver and spleen, atrophied glomeruli and vacuolization of renal tubular epithelial cells. The results of RT-PCR revealed that 18 host immune-related genes showed significantly up or downregulated expression post-infection compare to that of pre-infection. Finally, results of the RNA-seq revealed 16 DEGs play essential role to the immunosuppression in American eels, and the protein-protein interactions shed light on the widespread upregulation GEGs related to metabolism and immune response maintained the host cell homeostasis post the V. vulnificus infection, shedding new light on our understanding of the V. vulnificus pathogenesis towards understudied American eel and the host anti-V. vulnificus infection strategies in gene transcript.
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Anguilla , Enfermedades de los Peces , Vibriosis , Vibrio vulnificus , Animales , Vibrio vulnificus/genética , Anguilla/genética , Anguilla/microbiología , Virulencia/genética , RNA-Seq , Enfermedades de los Peces/microbiologíaRESUMEN
Climate change-induced stressors are contributing to the emergence of infectious diseases, including those caused by marine bacterial pathogens such as Vibrio spp. These stressors alter Vibrio temporal and geographical distribution, resulting in increased spread, exposure, and infection rates, thus facilitating greater Vibrio-human interactions. Concurrently, wildfires are increasing in size, severity, frequency, and spread in the built environment due to climate change, resulting in the emission of contaminants of emerging concern. This study aimed to understand the potential effects of urban interface wildfire ashes on Vibrio vulnificus (V. vulnificus) growth and gene expression using transcriptomic approaches. V. vulnificus was exposed to structural and vegetation ashes and analyzed to identify differentially expressed genes using the HTSeq-DESeq2 strategy. Exposure to wildfire ash altered V. vulnificus growth and gene expression, depending on the trace metal composition of the ash. The high Fe content of the vegetation ash enhanced bacterial growth, while the high Cu, As, and Cr content of the structural ash suppressed growth. Additionally, the overall pattern of upregulated genes and pathways suggests increased virulence potential due to the selection of metal- and antibiotic-resistant strains. Therefore, mixed fire ashes transported and deposited into coastal zones may lead to the selection of environmental reservoirs of Vibrio strains with enhanced antibiotic resistance profiles, increasing public health risk.
Asunto(s)
Vibrio vulnificus , Vibrio vulnificus/genética , Incendios Forestales , Expresión GénicaRESUMEN
BACKGROUND: Vibrio spp. naturally occur in warm water with moderate salinity. Infections with non-cholera Vibrio (vibriosis) cause an estimated 80,000 illnesses and 100 fatalities each year in the United States. Climate associated changes to environmental parameters in aquatic ecosystems are largely promoting Vibrio growth, and increased incidence of vibriosis is being reported globally. However, vibriosis trends in the northeastern U.S. (e.g., Maryland) have not been evaluated since 2008. METHODS: Vibriosis case data for Maryland (2006-2019; n = 611) were obtained from the COVIS database. Incidence rates were calculated using U.S. Census Bureau population estimates for Maryland. A logistic regression model, including region, age group, race, gender, occupation, and exposure type, was used to estimate the likelihood of hospitalization. RESULTS: Comparing the 2006-2012 and 2013-2019 periods, there was a 39% (p = 0.01) increase in the average annual incidence rate (per 100,000 population) of vibriosis, with V. vulnificus infections seeing the greatest percentage increase (53%, p = 0.01), followed by V. parahaemolyticus (47%, p = 0.05). The number of hospitalizations increased by 58% (p = 0.01). Since 2010, there were more reported vibriosis cases with a hospital duration ≥10 days. Patients from the upper eastern shore region and those over the age of 65 were more likely (OR = 6.8 and 12.2) to be hospitalized compared to other patients. CONCLUSIONS: Long-term increases in Vibrio infections, notably V. vulnificus wound infections, are occurring in Maryland. This trend, along with increased rates in hospitalizations and average hospital durations, underscore the need to improve public awareness, water monitoring, post-harvest seafood interventions, and environmental forecasting ability.
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Vibriosis , Vibrio parahaemolyticus , Vibrio vulnificus , Estados Unidos/epidemiología , Humanos , Maryland/epidemiología , Incidencia , Ecosistema , Vibriosis/epidemiología , AguaRESUMEN
INTRODUCTION: Gallic acid (GA) has a good therapeutic effect in bacteriological inhibition and plays a variety of functions in maintaining the stability of the immune system. The aim of the present study was to investigate the effect of GA on the bactericidal activity of macrophages against Vibrio vulnificus (Vv). METHODS: A cell counting kit-8 (CCK-8) assay was carried out to test the cytotoxicity of GA on J774A.1 cells. Concentration of proinflammatory cytokines in J774A.1 cells were evaluated by ELISA. The internalization and degradation of Vv in the phagosomes were observed by transmission electron microscopy (TEM). The phagosome acidification and phagolysosome formation were detected to evaluate the bacteria-clearing function of J774A.1 cells. The bactericidal activity of GA in vivo was also investigated by collecting the survival time of Vv infected mice and observing the inflammatory infiltration of organs. RESULTS: Our results demonstrated that GA at 50 µM significantly inhibited the proinflammatory cytokines levels, promoted phagosome acidification and phagolysosome formation in J774A.1 cells with Vv infection. This may be related to the activation of NLRP3/mTOR signaling pathway. Additionally, GA treatment improves the survival and bactericidal activity of mice infected with Vv. CONCLUSIONS: In summary, GA exerts bactericidal activity against Vv infection by regulating the formation and acidification of phagocytic lysosomes in macrophages.
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Ácido Gálico , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Fagosomas , Transducción de Señal , Serina-Treonina Quinasas TOR , Vibrio vulnificus , Ácido Gálico/farmacología , Animales , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ratones , Transducción de Señal/efectos de los fármacos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Fagosomas/efectos de los fármacos , Fagosomas/metabolismo , Vibrio vulnificus/efectos de los fármacos , Línea Celular , Citocinas/metabolismo , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , FemeninoRESUMEN
Vibrio vulnificus is a hazardous foodborne pathogen responsible for approximately 95% of seafood-related deaths. This highlights the urgent requirement for specialized detection tools to be developed and used by food enterprises and food safety authorities. The DETECTR (DNA endonuclease targeted CRISPR trans reporter) system that combines CRISPR/Cas and recombinase polymerase amplification (RPA) has been utilized to develop a molecular detection assay for V. vulnificus. However, because the incompatibility between RPA and Cas12a cleavage has not been addressed, it is a two-step assay that lacks convenience and presents contamination risk. Here, we developed a one-step RPA-CRISPR assay for V. vulnificus using a special crRNA targeting a sequence with a suboptimal protospacer adjacent motif (PAM). The entire assay, conducted at 37°C, takes only 40-60 min, yields results visualized under blue light, and exhibits exceptional specificity and sensitivity (detecting 4 pathogen genome copies per reaction). This study offers a valuable tool for detecting V. vulnificus, aiding in foodborne infection prevention, and exemplifies one-step RPA-CRISPR assays managing Cas-cleavage activity through PAM adjustments.
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Sistemas CRISPR-Cas , Vibrio vulnificus , Vibrio vulnificus/aislamiento & purificación , Vibrio vulnificus/genética , Microbiología de Alimentos , Alimentos Marinos/microbiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Recombinasas/metabolismo , Técnicas de Amplificación de Ácido Nucleico/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/prevención & control , Proteínas Asociadas a CRISPR/genética , Proteínas Asociadas a CRISPR/metabolismo , Sensibilidad y EspecificidadRESUMEN
Citral has attracted much attention as a safe and effective plant-derived bacteriostatic agent. However, the ability of citral to induce the formation of VBNC state in Vibrio vulnificus has not been evaluated. In the present study, V. vulnificus was shown to be induced to form the VBNC state at 4.5 h and 3 h of citral treatment at 4MIC and 6MIC. Moreover, the citral-induced VBNC state of V. vulnificus maintained some respiratory chain activity and was able to recover well in both APW media, APW media supplemented with 5 % (v/v) Tween 80 and 2 mg/mL sodium pyruvate. Field emission and transmission electron microscopy showed that the external structure of the citral-induced VBNC V. vulnificus cells was shortened to short rods, with folded cell membrane, rough cell surface, and dense cytoplasm and loose nuclear material in the internal cell structure. In addition, the possible molecular mechanisms of citral-induced formation and recovery of V. vulnificus in the VBNC state were explored by transcriptomics. Transcriptome analyses revealed that 1118 genes were significantly altered upon entry into the VBNC state, and 1052 genes were changed after resuscitation. Most of the physiological activities related to energy production were inhibited in the citral-induced VBNC state of V. vulnificus; however, the bacteria retained its pathogenicity. The citral-induced resuscitation of V. vulnificus in the VBNC state selectively restored the activity of some genes related to bacterial growth and reproduction. Meanwhile, the expression levels of other genes may have been influenced by citral-induced resuscitation after the formation of the VBNC state. In conclusion, this study evaluated and analyzed the ability and possible mechanism of citral on the formation of VBNC state and the recovery of VBNC state of V. vulnificus, and made a comprehensive assessment for the safety of citral application in food production.
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Monoterpenos Acíclicos , Vibrio vulnificus , Perfilación de la Expresión GénicaRESUMEN
Vibrio vulnificus (V. vulnificus) is a halophilic gram-negative bacterium that inhabits coastal warm water and induce severe diseases such as primary septicemia. To investigate the mechanisms of rapid bacterial translocation on intestinal infection, we focused on outer membrane vesicles (OMVs), which are extracellular vesicles produced by Gram-negative bacteria and deliver virulence factors. However, there are very few studies on the pathogenicity or contents of V. vulnificus OMVs (Vv-OMVs). In this study, we investigated the effects of Vv-OMVs on host cells. Epithelial cells INT407 were stimulated with purified OMVs and morphological alterations and levels of lactate dehydrogenase (LDH) release were observed. In cells treated with OMVs, cell detachment without LDH release was observed, which exhibited different characteristics from cytotoxic cell detachment observed in V. vulnificus infection. Interestingly, OMVs from a Vibrio Vulnificus Hemolysin (VVH) and Multifunctional-autoprocessing repeats-in -toxin (MARTX) double-deletion mutant strain also caused cell detachment without LDH release. Our results suggested that the proteolytic function of a serine protease contained in Vv-OMVs may contribute to pathogenicity of V. vulnificus by assisting bacterial translocation. This study reveals a new pathogenic mechanism during V. vulnificus infections. J. Med. Invest. 71 : 102-112, February, 2024.
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Vesículas Extracelulares , Vibrio vulnificus , Vibrio vulnificus/patogenicidad , Vibrio vulnificus/metabolismo , Humanos , Vesículas Extracelulares/metabolismo , Proteínas Hemolisinas/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Membrana Externa Bacteriana/metabolismo , Células Epiteliales/microbiologíaRESUMEN
Human exposure to Vibrio vulnificus, a gram-negative, halophilic environmental pathogen, is increasing. Despite this, the mechanisms of its pathogenicity and virulence remain largely unknown. Each year, hundreds of infections related to V. vulnificus occur, leading to hospitalization in 92% of cases and a mortality rate of 35%. The infection is severe, typically contracted through the consumption of contaminated food or exposure of an open wound to contaminated water. This can result in necrotizing fasciitis and the need for amputation of the infected tissue. Although several genes (rtxA1, vvpE, and vvhA) have been implicated in the pathogenicity of this organism, a defined mechanism has not been discovered. In this study, we examine environmentally isolated V. vulnificus strains using a zebrafish model (Danio rerio) to investigate their virulence capabilities. We found significant variation in virulence between individual strains. The commonly used marker gene of disease-causing strains, vcgC, did not accurately predict the more virulent strains. Notably, the least virulent strain in the study, V. vulnificus Sept WR1-BW6, which tested positive for vcgC, vvhA, and rtxA1, did not cause severe disease in the fish and was the only strain that did not result in any mortality. Our study demonstrates that virulence varies greatly among different environmental strains and cannot be accurately predicted based solely on genotype.
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Vibriosis , Vibrio vulnificus , Pez Cebra , Vibrio vulnificus/patogenicidad , Vibrio vulnificus/genética , Vibrio vulnificus/aislamiento & purificación , Animales , Pez Cebra/microbiología , Virulencia/genética , Vibriosis/microbiología , Factores de Virulencia/genética , Modelos Animales de Enfermedad , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Humanos , Microbiología AmbientalRESUMEN
The incidence of Vibrio vulnificus infections, with high mortality rates in humans and aquatic animals, has escalated, highlighting a significant public health challenge. Currently, reliable markers to identify strains with high virulence potential are lacking, and the understanding of evolutionary drivers behind the emergence of pathogenic strains is limited. In this study, we analyzed the distribution of virulent genotypes and phenotypes to discern the infectious potential of V. vulnificus strains isolated from three distinct sources. Most isolates, traditionally classified as biotype 1, possessed the virulence-correlated gene-C type. Environmental isolates predominantly exhibited YJ-like alleles, while clinical and diseased fish isolates were significantly associated with the nanA gene and pathogenicity region XII. Hemolytic activity was primarily observed in the culture supernatants of clinical and diseased fish isolates. Genetic relationships, as determined by multiple-locus variable-number tandem repeat analysis, suggested that strains originating from the same source tended to cluster together. However, multilocus sequence typing revealed considerable genetic diversity across clusters and sources. A phylogenetic analysis using single nucleotide polymorphisms of diseased fish strains alongside publicly available genomes demonstrated a high degree of evolutionary relatedness within and across different isolation sources. Notably, our findings reveal no direct correlation between phylogenetic patterns, isolation sources, and virulence capabilities. This underscores the necessity for proactive risk management strategies to address pathogenic V. vulnificus strains emerging from environmental reservoirs.IMPORTANCEAs the global incidence of Vibrio vulnificus infections rises, impacting human health and marine aquacultures, understanding the pathogenicity of environmental strains remains critical yet underexplored. This study addresses this gap by evaluating the virulence potential and genetic relatedness of V. vulnificus strains, focusing on environmental origins. We conduct an extensive genotypic analysis and phenotypic assessment, including virulence testing in a wax moth model. Our findings aim to uncover genetic and evolutionary factors that drive pathogenic strain emergence in the environment. This research advances our ability to identify reliable virulence markers and understand the distribution of pathogenic strains, offering significant insights for public health and environmental risk management.
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Enfermedades de los Peces , Variación Genética , Filogenia , Vibriosis , Vibrio vulnificus , Vibrio vulnificus/genética , Vibrio vulnificus/patogenicidad , Vibrio vulnificus/aislamiento & purificación , Vibrio vulnificus/clasificación , Animales , Vibriosis/microbiología , Vibriosis/veterinaria , Humanos , Virulencia/genética , Enfermedades de los Peces/microbiología , Peces/microbiología , Tipificación de Secuencias Multilocus , Factores de Virulencia/genética , Genotipo , Genoma Bacteriano/genéticaRESUMEN
Vibrio parahaemolyticus and Vibrio vulnificus are bacteria with a significant public health impact. Identifying factors impacting their presence and concentrations in food sources could enable the identification of significant risk factors and prevent incidences of foodborne illness. In recent years, machine learning has shown promise in modeling microbial presence based on prevalent external and internal variables, such as environmental variables and gene presence/absence, respectively, particularly with the generation and availability of large amounts and diverse sources of data. Such analyses can prove useful in predicting microbial behavior in food systems, particularly under the influence of the constant changes in environmental variables. In this study, we tested the efficacy of six machine learning regression models (random forest, support vector machine, elastic net, neural network, k-nearest neighbors, and extreme gradient boosting) in predicting the relationship between environmental variables and total and pathogenic V. parahaemolyticus and V. vulnificus concentrations in seawater and oysters. In general, environmental variables were found to be reliable predictors of total and pathogenic V. parahaemolyticus and V. vulnificus concentrations in seawater, and pathogenic V. parahaemolyticus in oysters (Acceptable Prediction Zone >70 %) when analyzed using our machine learning models. SHapley Additive exPlanations, which was used to identify variables influencing Vibrio concentrations, identified chlorophyll a content, seawater salinity, seawater temperature, and turbidity as influential variables. It is important to note that different strains were differentially impacted by the same environmental variable, indicating the need for further research to study the causes and potential mechanisms of these variations. In conclusion, environmental variables could be important predictors of Vibrio growth and behavior in seafood. Moreover, the models developed in this study could prove invaluable in assessing and managing the risks associated with V. parahaemolyticus and V. vulnificus, particularly in the face of a changing environment.
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Aprendizaje Automático , Ostreidae , Agua de Mar , Vibrio parahaemolyticus , Vibrio vulnificus , Ostreidae/microbiología , Agua de Mar/microbiología , Vibrio parahaemolyticus/aislamiento & purificación , Vibrio parahaemolyticus/crecimiento & desarrollo , Animales , Vibrio vulnificus/aislamiento & purificación , Vibrio vulnificus/crecimiento & desarrollo , Microbiología de Alimentos , Contaminación de Alimentos/análisis , Mariscos/microbiología , Alimentos Marinos/microbiología , Temperatura , Vibrio/aislamiento & purificaciónRESUMEN
Vibrio is a genus of halophilic, gram-negative bacteria found in estuaries around the globe. Integral parts of coastal cultures often involve contact with vectors of pathogenic Vibrio spp. (e.g., consuming raw shellfish). High rates of mortality from certain Vibrio spp. infections demonstrate the need for an improved understanding of Vibrio spp. dynamics in estuarine regions. Our study assessed meteorological, hydrographic, and biological correlates of Vibrio parahaemolyticus and V. vulnificus at 10 sites in the Eastern Mississippi Sound System (EMSS) from April to October 2019. During the sampling period, median abundances of V. parahaemolyticus and V. vulnificus were 2.31 log MPN/L and 2.90 log MPN/L, respectively. Vibrio spp. dynamics were largely driven by site-based variation, with sites closest to freshwater inputs having the highest abundances. The E-W wind scalar, which affects Ekman transport, was a novel Vibrio spp. correlate observed. A potential salinity effect on bacterial-particle associations was identified, where V. vulnificus was associated with larger particles in conditions outside of their optimal salinity. Additionally, V. vulnificus abundances were correlated to those of harmful algal species that did not dominate community chlorophyll. Correlates from this study may be used to inform the next iteration of regionally predictive Vibrio models and may lend additional insight to Vibrio spp. ecology in similar systems. IMPORTANCE: Vibrio spp. are bacteria found in estuaries worldwide; some species can cause illness and infections in humans. Relationships between Vibrio spp. abundance, salinity, and temperature are well documented, but correlations to other environmental parameters are less understood. This study identifies unique correlates (e.g., E-W wind scalar and harmful algal species) that could potentially inform the next iteration of predictive Vibrio models for the EMSS region. Additionally, these correlates may allow existing environmental monitoring efforts to be leveraged in providing data inputs for future Vibrio risk models. An observed correlation between salinity and V. vulnificus/particle-size associations suggests that predicted environmental changes may affect the abundance of Vibrio spp. in certain reservoirs, which may alter which vectors present the greatest vibrio risk.
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Estuarios , Vibrio parahaemolyticus , Vibrio vulnificus , Vibrio parahaemolyticus/aislamiento & purificación , Vibrio parahaemolyticus/crecimiento & desarrollo , Vibrio vulnificus/aislamiento & purificación , Vibrio vulnificus/crecimiento & desarrollo , Alabama , Dinámica Poblacional , Salinidad , Vibriosis/microbiología , Vibriosis/epidemiología , Agua de Mar/microbiología , Microbiología del AguaRESUMEN
The multifunctional autoprocessing repeat-in-toxin (MARTX) toxin is the primary virulence factor of Vibrio vulnificus displaying cytotoxic and hemolytic properties. The cysteine protease domain (CPD) is responsible for activating the MARTX toxin by cleaving the toxin precursor and releasing the mature toxin fragments. To investigate the structural determinants for inositol hexakisphosphate (InsP6)-mediated activation of the CPD, we determined the crystal structures of unprocessed and ß-flap truncated MARTX CPDs of Vibrio vulnificus strain MO6-24/O in complex with InsP6 at 1.3 and 2.2Å resolution, respectively. The CPD displays a conserved domain with a central seven-stranded ß-sheet flanked by three α-helices. The scissile bond Leu3587-Ala3588 is bound in the catalytic site of the InsP6-loaded form of the Cys3727Ala mutant. InsP6 interacts with the conserved basic cleft and the ß-flap inducing the active conformation of catalytic residues. The ß-flap of the post-CPD is flexible in the InsP6-unbound state. The structure of the CPD Δß-flap showed an inactive conformation of the catalytic residues due to the absence of interaction between the active site and the ß-flap. This study confirms the InsP6-mediated activation of the MARTX CPDs in which InsP6-binding induces conformational changes of the catalytic residues and the ß-flap that holds the N terminus of the CPD in the active site, facilitating hydrolysis of the scissile bond.
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Ácido Fítico , Vibrio vulnificus , Vibrio vulnificus/enzimología , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Ácido Fítico/metabolismo , Dominio Catalítico , Proteasas de Cisteína/metabolismo , Proteasas de Cisteína/química , Proteasas de Cisteína/genética , Cristalografía por Rayos X , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/genética , Dominios Proteicos , Modelos Moleculares , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Secuencia de AminoácidosRESUMEN
Vibrio vulnificus (vv) is a multidrug-resistant human bacterial pathogen whose prevalence is expected to increase over the years. Transketolases (TK), transferases catalyzing two reactions of the nonoxidative branch of the pentose-phosphate pathway and therefore linked to several crucial metabolic pathways, are potential targets for new drugs against this pathogen. Here, the vvTK is crystallized and its structure is solved at 2.1 Å. A crown of 6 histidyl residues is observed in the active site and expected to participate in the thiamine pyrophosphate (cofactor) activation. Docking of fructose-6-phosphate and ferricyanide used in the activity assay, suggests that both substrates can bind vvTK simultaneously. This is confirmed by steady-state kinetics showing a sequential mechanism, on the contrary to the natural transferase reaction which follows a substituted mechanism. Inhibition by the I38-49 inhibitor (2-(4-ethoxyphenyl)-1-(pyrimidin-2-yl)-1H-pyrrolo[2,3-b]pyridine) reveals for the first time a cooperative behavior of a TK and docking experiments suggest a previously undescribed binding site at the interface between the pyrophosphate and pyridinium domains.
Asunto(s)
Transcetolasa , Vibrio vulnificus , Humanos , Transcetolasa/química , Transcetolasa/metabolismo , Vibrio vulnificus/metabolismo , Cinética , Conducta Cooperativa , Tiamina Pirofosfato/metabolismo , Transferasas/metabolismoRESUMEN
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.