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1.
Microbiome ; 9(1): 88, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33845910

RESUMO

BACKGROUND: Acute hepatopancreatic necrosis disease (AHPND) is an important shrimp bacterial disease caused by some Vibrio species. The severity of the impact of this disease on aquaculture worldwide has made it necessary to develop alternatives to prophylactic antibiotics use, such as the application of probiotics. To assess the potential to use probiotics in order to limit the detrimental effects of AHNPD, we evaluated the effect of the ILI strain, a Vibrio sp. bacterium and efficient shrimp probiotic, using metabarcoding (16S rRNA gene) on the gastrointestinal microbiota of shrimp after being challenged with AHPND-causing V. parahaemolyticus. RESULTS: We showed how the gastrointestinal microbiome of shrimp varied between healthy and infected organisms. Nevertheless, a challenge of working with AHPND-causing Vibrio pathogens and Vibrio-related bacteria as probiotics is the potential risk of the probiotic strain becoming pathogenic. Consequently, we evaluated whether ILI strain can acquire the plasmid pV-AHPND via horizontal transfer and further cause the disease in shrimp. Conjugation assays were performed resulting in a high frequency (70%) of colonies harboring the pv-AHPND. However, no shrimp mortality was observed when transconjugant colonies of the ILI strain were used in a challenge test using healthy shrimp. We sequenced the genome of the ILI strain and performed comparative genomics analyses using AHPND and non-AHPND Vibrio isolates. Using available phylogenetic and phylogenomics analyses, we reclassified the ILI strain as Vibrio diabolicus. In summary, this work represents an effort to study the role that probiotics play in the normal gastrointestinal shrimp microbiome and in AHPND-infected shrimp, showing that the ILI probiotic was able to control pathogenic bacterial populations in the host's gastrointestinal tract and stimulate the shrimp's survival. The identification of probiotic bacterial species that are effective in the host's colonization is important to promote animal health and prevent disease. CONCLUSIONS: This study describes probiotic bacteria capable of controlling pathogenic populations of bacteria in the shrimp gastrointestinal tract. Our work provides new insights into the complex dynamics between shrimp and the changes in the microbiota. It also addresses the practical application of probiotics to solve problems with pathogens that cause high mortality-rate in shrimp farming around the world. Video Abstract.


Assuntos
Microbiota , Penaeidae , Probióticos , Vibrio parahaemolyticus , Animais , Humanos , Necrose , Filogenia , RNA Ribossômico 16S/genética , Sobreviventes , Vibrio , Vibrio parahaemolyticus/genética
2.
Nat Commun ; 11(1): 5777, 2020 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-33188170

RESUMO

Vibrio parahaemolyticus is the leading cause of seafood-borne diarrheal diseases. Experimental overproduction of a type 3 secretion system (T3SS1) in this pathogen leads to decreased intestinal colonization, which suggests that T3SS1 repression is required for maximal virulence. However, the mechanisms by which T3SS1 is repressed in vivo are unclear. Here, we show that host-derived nitrite modifies the activity of a bacterial histidine kinase and mediates T3SS1 repression. More specifically, nitrite activates histidine kinase sensor VbrK through S-nitrosylation on cysteine 86, which results in downregulation of the entire T3SS1 operon through repression of its positive regulator exsC. Replacement of cysteine 86 with a serine (VbrK C86S mutant) leads to increased expression of inflammatory cytokines in infected Caco-2 cells. In an infant rabbit model of infection, the VbrK C86S mutant induces a stronger inflammatory response at the early stage of infection, and displays reduced intestinal colonization and virulence at the later stage of infection, in comparison with the parent strain. Our results indicate that the pathogen V. parahaemolyticus perceives nitrite as a host-derived signal and responds by downregulating a proinflammatory factor (T3SS1), thus enhancing intestinal colonization and virulence.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidade , Anaerobiose , Animais , Sequência de Bases , Sítios de Ligação , Células CACO-2 , Citocinas/metabolismo , Regulação para Baixo/genética , Regulação Bacteriana da Expressão Gênica , Interações Hospedeiro-Patógeno/genética , Humanos , Modelos Biológicos , Nitratos/metabolismo , Nitritos/metabolismo , Nitrosação , Fosforilação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Coelhos , Transcrição Genética , Vibrio parahaemolyticus/genética , Virulência/genética
3.
PLoS One ; 15(10): e0240143, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33007026

RESUMO

Vibrio parahaemolyticus is responsible for seafood-borne gastroenteritis worldwide. Isolates of V. parahaemolyticus from clinical samples (n = 54) and environmental samples (n = 38) in Huzhou were analyzed by serological typing, virulence gene detection, antibiotic resistance testing, and pulsed-field gel electrophoresis (PFGE) for molecular typing. O3:K6 was the main serotype and tlh+tdh+trh- was the most frequently detected virulence genotype in clinical strains. O2:Kut was the main serotype and tlh+tdh-trh- was the most frequently detected virulence genotype in environmental strains. Antibiotic resistance testing indicated that the isolates were highly resistant to ampicillin (90.76%), followed by gentamicin and tetracycline. Following the restriction enzyme NotI digestion, the 91 strains yielded 81 PFGE patterns, and 16 clones had similarity values of > 85.00%, indicating a high level of diversity. Finally, there may be cross-contamination between freshwater and seawater products, so it is necessary to strengthen supervision of food processing.


Assuntos
Microbiologia Ambiental , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificação , China , Análise por Conglomerados , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Eletroforese em Gel de Campo Pulsado , Genes Bacterianos , Humanos , Filogenia , Sorotipagem , Vibrio parahaemolyticus/classificação , Virulência/genética
4.
Int J Food Microbiol ; 335: 108858, 2020 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-33032034

RESUMO

Routine handling of oysters is a common industry practice for off-bottom oyster aquaculture, which aims to produce a high-quality oyster. These practices expose oysters to elevated temperatures and interrupt filter feeding, which can increase Vibrio vulnificus and V. parahaemolyticus levels within the oyster. The resubmersion of oysters after exposure to conditions where the time-temperature controls are exceeded is as an effective mitigation strategy to allow elevated levels of Vibrio spp. to "recover", or return to ambient levels, prior to harvest. Previous work examined the effect of desiccation on recovery times; the objective of this study was to evaluate the effect of additional handling treatments [tumbled and refrigerated (TR), tumbled and not refrigerated (TNR), not tumbled and refrigerated (NTR), and not tumbled and not refrigerated (NTNR)] on the time needed for V. vulnificus, total V. parahaemolyticus, and pathogenic V. parahaemolyticus (tdh+/trh+) to recover in oysters. A set of non-treated (control) oysters remained submerged throughout the study to determine the ambient Vibrio spp. (inclusive of genotypes) levels within oysters. Vibrio spp. levels were measured immediately before (pre) and after (post) the treatments, and 1, 2, 4, 7, 10, and 14 days after resubmersion using a three-tube MPN real-time PCR method. The non-refrigerated oysters (TNR, NTNR) had Vibrio spp. levels 1.54 to 2.10 log MPN/g higher than the pre-treatment levels, while the Vibrio spp. levels in refrigerated oysters were not significantly higher than pre-treatment levels. After resubmersion, Vibrio spp. levels increased by 0.84 to 1.78 log MPN/g in the refrigerated oysters (TR, NTR). Vibrio spp. levels in oysters returned to ambient after 1-7 days of resubmersion, depending on the handling treatment and the Vibrio spp. These results provide data on handling treatments not previously reported and further support the seven-day resubmersion requirement for farmers in Alabama using the adjustable longline system.


Assuntos
Crassostrea/microbiologia , Manipulação de Alimentos , Refrigeração , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio vulnificus/crescimento & desenvolvimento , Alabama , Animais , Aquicultura , Contaminação de Alimentos , Reação em Cadeia da Polimerase em Tempo Real , Alimentos Marinhos/microbiologia , Temperatura , Fatores de Tempo , Vibrio parahaemolyticus/genética , Vibrio vulnificus/genética
5.
Mar Pollut Bull ; 160: 111551, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32810670

RESUMO

In characterization of food borne pathogens from the environment, assessment of virulence, genetic diversity and AMR are essential preludes to formulate preventive strategies and to combat the spread. This study aimed to identify and characterize pathogenic Vibrio parahaemolyticus in the coastal aquaculture farms of Kerala, India. Twenty-seven ß-haemolytic V. parahaemolyticus were isolated from 7 out of 40 farms studied. Among the 27 isolates, 15 possessed the tdh gene and 4 had trh. ERIC PCR and PFGE illustrated the presence of pathogenic isolates that shared genetic similarity with clinical strains. One pathogenic isolate was identified to be multidrug resistant (MDR) and 59% exhibited a MAR index of 0.2 or above. Seventy four percent of the pathogenic isolates were ESBL producers and 3.7% of them were carbapenemase producers phenotypically. This asks for adoption of control measures during farming to prevent the transmission of pathogenic V. parahaemolyticus to the environment and food chain.


Assuntos
Vibrioses , Vibrio parahaemolyticus , Aquicultura , Variação Genética , Humanos , Índia , Alimentos Marinhos , Vibrio parahaemolyticus/genética
6.
PLoS One ; 15(8): e0237181, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32813697

RESUMO

Multidrug-resistant Vibrio parahaemolyticus has become a significant public health concern. The development of effective drugs and vaccines against Vibrio parahaemolyticus is the current research priority. Thus, we aimed to find out effective drug and vaccine targets using a comprehensive genome-based analysis. A total of 4822 proteins were screened from V. parahaemolyticus proteome. Among 16 novel cytoplasmic proteins, 'VIBPA Type II secretion system protein L' and 'VIBPA Putative fimbrial protein Z' were subjected to molecular docking with 350 human metabolites, which revealed that Eliglustat, Simvastatin and Hydroxocobalamin were the top drug molecules considering free binding energy. On the contrary, 'Sensor histidine protein kinase UhpB' and 'Flagellar hook-associated protein of 25 novel membrane proteins were subjected to T-cell and B-cell epitope prediction, antigenicity testing, transmembrane topology screening, allergenicity and toxicity assessment, population coverage analysis and molecular docking analysis to generate the most immunogenic epitopes. Three subunit vaccines were constructed by the combination of highly antigenic epitopes along with suitable adjuvant, PADRE sequence and linkers. The designed vaccine constructs (V1, V2, V3) were analyzed by their physiochemical properties and molecular docking with MHC molecules- results suggested that the V1 is superior. Besides, the binding affinity of human TLR-1/2 heterodimer and construct V1 could be biologically significant in the development of the vaccine repertoire. The vaccine-receptor complex exhibited deformability at a minimum level that also strengthened our prediction. The optimized codons of the designed construct was cloned into pET28a(+) vector of E. coli strain K12. However, the predicted drug molecules and vaccine constructs could be further studied using model animals to combat V. parahaemolyticus associated infections.


Assuntos
Vacinas Bacterianas/imunologia , Descoberta de Drogas/métodos , Genoma Bacteriano , Vibrioses/tratamento farmacológico , Vibrioses/prevenção & controle , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/imunologia , Biologia Computacional/métodos , Farmacorresistência Bacteriana Múltipla/genética , Epitopos de Linfócito B/imunologia , Epitopos de Linfócito T/imunologia , Escherichia coli K12/genética , Humanos , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mapas de Interação de Proteínas , Proteoma/genética , Proteômica/métodos , Vacinas de Subunidades/imunologia , Vibrioses/microbiologia
7.
J Food Sci ; 85(6): 1834-1844, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32449955

RESUMO

Vibrio parahaemolyticus is an important pathogenic bacterium in both food safety management and mariculture. Rapid and accurate detection technologies are critical for effective control of its outbreak and spreading. Conventional technologies and polymerase chain reaction (PCR)-based approaches have limited usage because of the requirement of laboratory instruments and trained personnel. Using the isothermal recombinase polymerase amplification (RPA) technology, several detection assays have been developed with added convenience. Combining the lateral flow strip (LFS) test with RPA can further simplify the detection. In this study, an improved RPA assay using LFS for visual detection of V. parahaemolyticus was developed. Primers were designed targeting the virulence genes and screened for amplification efficiency, nonspecific amplification, and primer-dimer formation. Probes were designed for the best primer pairs, and the weakness of LFS tests, being easily affected by primer-dependent artifacts, was overcome by sequence modifications on primers and probe. The RPA-LFS assay took 25 min at 35 to 45 °C, and showed excellent specificity. It detected as low as one colony forming unit (CFU) of V. parahaemolyticus per reaction without DNA purification, or 10 CFU/10 g spiked food samples with 2 hr of enrichment. The detection limit was better than the currently available RPA-based detection methods. Application of the RPA-LFS assay for simulated samples or real clinical samples showed accurate and consistent detection results compared to bioassay and quantitative PCR. The RPA-LFS assay provided a rapid, accurate, and convenient V. parahaemolyticus detection method suitable for on-site detection in resource-limited conditions. PRACTICAL APPLICATION: This research developed a rapid and visual detection technology for Vibrio parahaemolyticus that is not dependent on complicated equipment. The detection process takes 25 min and the result is read with the naked eye. A detection kit can be developed based on this technology for on-site detection of V. parahaemolyticus in resource-limited regions for food safety management and mariculture.


Assuntos
Microbiologia de Alimentos/métodos , Reação em Cadeia da Polimerase/métodos , Vibrio parahaemolyticus/isolamento & purificação , Primers do DNA/genética , DNA Bacteriano/genética , Contaminação de Alimentos/análise , Limite de Detecção , Sensibilidade e Especificidade , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/crescimento & desenvolvimento
8.
Emerg Microbes Infect ; 9(1): 855-867, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32306848

RESUMO

The adsorption of phages to hosts is the first step of phage infection. Studies have shown that tailed phages use tail fibres or spikes to recognize bacterial receptors and mediate adsorption. However, whether other phage tail components can also recognize host receptors is unknown. To identify potential receptors, we screened a transposon mutagenesis library of the marine pathogen Vibrio parahaemolyticus and discovered that a vp0980 mutant (vp0980 encodes a predicted transmembrane protein) could not be lysed by phage OWB. Complementation of this mutant with wild-type vp0980 in trans restored phage-mediated lysis. Phage adsorption and confocal microscopy assays demonstrated that phage OWB had dramatically reduced adsorption to the vp0980 mutant compared to that to the wild type. Pulldown assays showed that phage tail tubular proteins A and B (TTPA and TTPB) interact with Vp0980, suggesting that Vp0980 is a TTPA and TTPB receptor. Vp0980 lacking the outer membrane region (aa 114-127) could not bind to TTPA and TTPB, resulting in reduced phage adsorption. These results strongly indicated that TTPA and TTPB binding with their receptor Vp0980 mediates phage adsorption and subsequent bacterial lysis. To the best of our knowledge, this study is the first report of a bacterial receptor for phage tail tubular proteins.


Assuntos
Bacteriófagos/fisiologia , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/virologia , Proteínas da Cauda Viral/metabolismo , Ligação Viral , Biblioteca Gênica , Mutação , Proteínas da Cauda Viral/genética , Vírion/fisiologia
9.
Talanta ; 214: 120818, 2020 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-32278427

RESUMO

Vibrio parahaemolyticus is a major cause of seafood-associated food poisoning. It is of great significance to develop an accurate, simple and cost-effective method to identify infected seafood, especially for on-site application. Polymerase chain reaction (PCR) remains the golden standard for nucleic acid detection. But traditional methods heavily reply on sophisticated instrument and specialized operators, which limits the application for on-site detections. Here we developed a novel, specific and visualized detection method for PCR based on CRISPR/Cas12a system. On a low-cost thermal cycler, amplification reaction can be conducted easily. The CRISPR/Cas12a system was specifically designed to evaluate amplicons, eliminating false positive results. Besides the negative samples remained colorless, the positive samples generated obvious green fluorescence, which could be easily distinguished by the naked eye using a homemade UV device. The presented detection method was verified by detecting shrimp samples. The limit of detection is 1.02 × 102 copies/µL. This presented method provided a new strategy for specific endpoint detection of PCR and advanced its application in field for food safety assurance.


Assuntos
Sistemas CRISPR-Cas/genética , Penaeidae/microbiologia , Reação em Cadeia da Polimerase , RNA Bacteriano/genética , Temperatura , Vibrio parahaemolyticus/isolamento & purificação , Animais , Técnicas Biossensoriais , Técnicas Eletroquímicas , Contaminação de Alimentos/análise , Tamanho da Partícula , Propriedades de Superfície , Vibrio parahaemolyticus/genética
10.
Microbiol Res ; 235: 126448, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32114363

RESUMO

Vibrio parahaemolyticus is a common foodborne pathogen in seafood and represents a major threat to human health worldwide. In this study, we identified that PhoR, a histidine kinase, is involved in the regulation of swarming and flagella assembly. RNA sequencing analysis showed that 1122 genes were differentially expressed in PhoR mutant, including 394 upregulated and 728 downregulated genes. KEGG enrichment and heatmap analysis demonstrated that the bacterial secretion system, flagella assembly and chemotaxis pathways were significantly downregulated in PhoR mutant, while the microbial metabolism in diverse environments and carbon metabolism pathways were upregulated in PhoR mutant. qRT-PCR further confirmed that genes responsible for the type III secretion system (T3SS), swarming and the thermostable direct hemolysin were positively regulated by PhoR. Phosphorylation assays suggested that PhoR was highly activated in BHI medium compared to LB medium. Taken together, these data suggested that activated PhoR contributes to the expression of swarming motility and secretion system genes in Vibrio parahaemolyticus.


Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Transcriptoma , Sistemas de Secreção Tipo III/genética , Vibrio parahaemolyticus/genética , Toxinas Bacterianas/genética , Biofilmes/crescimento & desenvolvimento , Regulação para Baixo , Perfilação da Expressão Gênica , Proteínas Hemolisinas/genética , Movimento , Regulação para Cima , Vibrio parahaemolyticus/enzimologia
11.
Artigo em Inglês | MEDLINE | ID: mdl-32178325

RESUMO

Expression of the regulatory stress rpoS gene controls the transcription of cspA genes, which are involved in survival and adaptation to low temperatures. The purpose of this study was to assess the growth kinetics of naturally occurring V. parahaemolyticus in shellstock oysters and in vitro and the cold-shock-induced expression of the rpoS and cspA gene response in vitro during postharvest refrigeration. Naturally contaminated eastern oysters (Crassostrea virginica) and pathogenic (Vp-tdh) and nonpathogenic (Vp-tlh) isolates were stored at 7 ± 1 °C for 168 h and 216 h, respectively. The regulatory stress (rpos) and cold-shock (cspA) gene expressions were determined by reverse transcription PCR. At 24 h, the (Vp-tdh) strain grew faster (p < 0.05) than the (Vp-tlh) strain in oysters (λ = 0.33, 0.39, respectively) and in vitro (λ = 0.89, 37.65, respectively), indicating a better adaptation to cold shock for the (Vp-tdh) strain in live oysters and in vitro. At 24 h, the (Vp-tdh) strain rpoS and cspA gene expressions were upregulated by 1.9 and 2.3-fold, respectively, but the (Vp-tlh) strain rpoS and cspA gene expressions were repressed and upregulated by -0.024 and 1.9-fold, respectively. The V. parahaemolyticus strains that were isolated from tropical oysters have adaptive expression changes to survive and grow at 7 °C, according to their virulence.


Assuntos
Temperatura Baixa , Crassostrea , Regulação da Expressão Gênica , Ostreidae , Vibrio parahaemolyticus , Animais , Refrigeração , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidade
12.
Microbes Environ ; 35(2)2020.
Artigo em Inglês | MEDLINE | ID: mdl-32201414

RESUMO

Vibrio parahaemolyticus is the leading cause of bacteria-associated foodborne diarrheal diseases and specifically causes early mortality syndrome (EMS), which is technically known as acute hepatopancreatic necrosis disease (AHPND), a serious threat to shrimp aquaculture. To investigate the genetic and evolutionary relationships of V. parahaemolyticus in China, 184 isolates from clinical samples (VPC, n=40), AHPND-infected shrimp (VPE, n=10), and various aquatic production sources (VPF, n=134) were collected and evaluated by a multilocus sequence analysis (MLST). Furthermore, the presence of potential virulence factors (tlh, tdh, and trh) and single nucleotide polymorphisms (SNPs) in V. parahaemolyticus isolates was assessed using genomic sequencing. Analyses of virulence factors revealed that the majority of VPC isolates (97.5%) possessed the tdh and/or trh genes, while most of the VPF isolates (83.58%) did not encode hemolysin genes. Therefore, we hypothesized that the environment is a potential reservoir that promotes horizontal DNA transfer, which drives evolutionary change that, in turn, leads to the emergence of novel, potentially pathogenic strains. Phylogenetic analyses identified VPF-112 as a non-pathogenic maternal strain isolated from aquatic products and showed that it had a relatively high evolutionary status. All VPE strains and some VPC strains were grouped into several small subgroups and evenly distributed on phylogenetic trees. Anthropogenic activities and environmental selective pressure may be important factors influencing the process of transforming strains from non-pathogenic to pathogenic bacteria.


Assuntos
Evolução Molecular , Penaeidae/microbiologia , Vibrio parahaemolyticus/genética , Animais , Aquicultura , Tipagem de Sequências Multilocus , Filogenia , Vibrioses/veterinária , Fatores de Virulência/genética
13.
Appl Environ Microbiol ; 86(10)2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32169942

RESUMO

Bacteria accumulate small, organic compounds called compatible solutes via uptake from the environment or biosynthesis from available precursors to maintain the turgor pressure of the cell in response to osmotic stress. The halophile Vibrio parahaemolyticus has biosynthesis pathways for the compatible solutes ectoine (encoded by ectABC-asp_ect) and glycine betaine (encoded by betIBA-proXWV), four betaine-carnitine-choline transporters (encoded by bccT1 to bccT4), and a second ProU transporter (encoded by proVWX). All of these systems are osmotically inducible with the exception of bccT2 Previously, it was shown that CosR, a MarR-type regulator, was a direct repressor of ectABC-asp_ect in Vibrio species. In this study, we investigated whether CosR has a broader role in the osmotic stress response. Expression analyses demonstrated that betIBA-proXWV, bccT1, bccT3, bccT4, and proVWX are repressed in low salinity. Examination of an in-frame cosR deletion mutant showed that expression of these systems is derepressed in the mutant at low salinity compared with the wild type. DNA binding assays demonstrated that purified CosR binds directly to the regulatory region of both biosynthesis systems and four transporters. In Escherichia coli green fluorescent protein (GFP) reporter assays, we demonstrated that CosR directly represses transcription of betIBA-proXWV, bccT3, and proVWX Similar to Vibrio harveyi, we showed betIBA-proXWV was directly activated by the quorum-sensing LuxR homolog OpaR, suggesting a conserved mechanism of regulation among Vibrio species. Phylogenetic analysis demonstrated that CosR is ancestral to the Vibrionaceae family, and bioinformatics analysis showed widespread distribution among Gammaproteobacteria in general. Incidentally, in Aliivibrio fischeri, Aliivibrio finisterrensis, Aliivibrio sifiae, and Aliivibrio wodanis, an unrelated MarR-type regulator gene named ectR was clustered with ectABC-asp, which suggests the presence of another novel ectoine biosynthesis regulator. Overall, these data show that CosR is a global regulator of osmotic stress response that is widespread among bacteria.IMPORTANCE Vibrio parahaemolyticus can accumulate compatible solutes via biosynthesis and transport, which allow the cell to survive in high salinity conditions. There is little need for compatible solutes under low salinity conditions, and biosynthesis and transporter systems need to be repressed. However, the mechanism(s) of this repression is not known. In this study, we showed that CosR played a major role in the regulation of multiple compatible solute systems. Phylogenetic analysis showed that CosR is present in all members of the Vibrionaceae family as well as numerous Gammaproteobacteria Collectively, these data establish CosR as a global regulator of the osmotic stress response that is widespread in bacteria, controlling many more systems than previously demonstrated.


Assuntos
Proteínas de Bactérias/genética , Pressão Osmótica , Proteínas Repressoras/genética , Vibrio parahaemolyticus/fisiologia , Proteínas de Bactérias/metabolismo , Sequência de Bases , Regulação Bacteriana da Expressão Gênica , Filogenia , Proteínas Repressoras/metabolismo , Alinhamento de Sequência , Vibrio parahaemolyticus/genética
14.
Nat Commun ; 11(1): 1085, 2020 02 27.
Artigo em Inglês | MEDLINE | ID: mdl-32109231

RESUMO

Gram-negative bacteria deliver effectors via the type VI secretion system (T6SS) to outcompete their rivals. Each bacterial strain carries a different arsenal of effectors; the identities of many remain unknown. Here, we present an approach to identify T6SS effectors encoded in bacterial genomes of interest, without prior knowledge of the effectors' domain content or genetic neighborhood. Our pipeline comprises a comparative genomics analysis followed by screening using a surrogate T6SS+ strain. Using this approach, we identify an antibacterial effector belonging to the T6SS1 of Vibrio parahaemolyticus, representing a widespread family of T6SS effectors sharing a C-terminal domain that we name Tme (Type VI membrane-disrupting effector). Tme effectors function in the periplasm where they intoxicate bacteria by disrupting membrane integrity. We believe our approach can be scaled up to identify additional T6SS effectors in various bacterial genera.


Assuntos
Membrana Externa Bacteriana/metabolismo , Proteínas de Bactérias/genética , Toxinas Bacterianas/genética , Sistemas de Secreção Tipo VI/genética , Vibrio parahaemolyticus/genética , Antibacterianos/farmacologia , Membrana Externa Bacteriana/efeitos dos fármacos , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Descoberta de Drogas , Genoma Bacteriano , Genômica , Periplasma/metabolismo , Vibrio parahaemolyticus/citologia , Vibrio parahaemolyticus/metabolismo
15.
Curr Microbiol ; 77(5): 710-715, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-31897665

RESUMO

Phosphatidylserine synthase (Pss) is involved in the metabolic pathway in phospholipid synthesis in different organisms. In this study, Pss expression in Vibrio parahaemolyticus was verified through liquid chromatography tandem-mass spectrometry. To analyze the characteristics of Pss, the recombinant Pss was overexpressed and purified from Escherichia coli. The optimum temperature and pH of Pss were 40 °C and 8, respectively. When reacting with divalent metal, Pss activity decreased. In addition, Pss could not only use cytidine diphosphate diacylglycerol (CDP-DAG, 16:0), but also CDP-DAG (18:1) as a substrate to produce cytidine 5'-monophosphate. Furthermore, the 3D structure of Pss was predicted, and the results revealed that histidine and lysine of the two HKD motifs were present in the catalytic site. Moreover, CDP-DAG (16:0) was docked with the Pss model. To investigate whether the two HKD motifs in Pss are important to its activity, site-directed mutagenesis of histidine was performed. The results revealed that the activities of both H131A and H352A were diminished. Little is known regarding the catalytic site of type I Pss. This is the first report on the biochemical characterization of Pss in V. parahaemolyticus.


Assuntos
Proteínas de Bactérias/metabolismo , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/metabolismo , Vibrio parahaemolyticus/enzimologia , Proteínas de Bactérias/genética , CDPdiacilglicerol-Serina O-Fosfatidiltransferase/genética , Cromatografia Líquida , Escherichia coli/genética , Histidina/genética , Concentração de Íons de Hidrogênio , Mutagênese Sítio-Dirigida , Fosfolipídeos/metabolismo , Espectrometria de Massas em Tandem , Temperatura , Vibrio parahaemolyticus/genética
16.
Nat Commun ; 11(1): 547, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992706

RESUMO

TrkH is a bacterial ion channel implicated in K+ uptake and pH regulation. TrkH assembles with its regulatory protein, TrkA, which closes the channel when bound to ADP and opens it when bound to ATP. However, it is unknown how nucleotides control the gating of TrkH through TrkA. Here we report the structures of the TrkH-TrkA complex in the presence of ADP or ATP. TrkA forms a tetrameric ring when bound to ADP and constrains TrkH to a closed conformation. The TrkA ring splits into two TrkA dimers in the presence of ATP and releases the constraints on TrkH, resulting in an open channel conformation. Functional studies show that both the tetramer-to-dimer conversion of TrkA and the loss of constraints on TrkH are required for channel gating. In addition, deletion of TrkA in Escherichia coli depolarizes the cell, suggesting that the TrkH-TrkA complex couples changes in intracellular nucleotides to membrane potential.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Potenciais da Membrana/fisiologia , Canais de Potássio/química , Canais de Potássio/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Difosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Modelos Moleculares , Mutagênese , Potássio/metabolismo , Canais de Potássio/genética , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Vibrio parahaemolyticus/genética , Difração de Raios X
17.
J Food Sci ; 85(3): 744-754, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31999364

RESUMO

Salmonella enterica, Listeria monocytogenes, Shigella flexneri, Escherichia coli O157:H7, Vibrio parahaemolyticus, Staphylococcus aureus, Vibrio cholerae, Clostridium botulinum type A, Bacillus cereus, Clostridium perfringens Alpha toxin, and Yersinia enterocolitica are 11 common foodborne pathogens. Traditional bacterial culture methods for detecting pathogens are time-consuming and labor-intensive. Multiplex PCR technology, which can detect multiple targets in a single tube, has been increasingly applied to microbial detection due to its high specificity, sensitivity, and fast response. This paper is to establish a multiplex PCR technology mediated by a common primer for the detection of these 11 common foodborne pathogens in order to achieve the goal of nondirectional screening for these 11 common foodborne pathogens. The specificity of the established CP-MPCR detection system was first verified by 100 clinical isolates. The sensitivity of the CP-MPCR detection system was then detected by using cultured bacteria preparations and has been confirmed with a high sensitivity of 103 to 104 CFU/mL, among them, the sensitivity of the CP-MPCR for Vibrio cholerae and S. flexneri can even achieve 102 CFU/mL. Sixty anal swab samples collected from Suzhou CDC and 16 enrichment cultured solutions of food samples collected from the Suzhou Food Inspection and Testing Center were tested using the CP-MPCR system. A total of 32 positive results were detected. PRACTICAL APPLICATION: Food poisoning incidents occur frequently around the world, mainly because of the contamination of food by pathogenic bacteria and serious harm to human health. The method provided in this study can detect 11 foodborne pathogens in food, which can effectively prevent the spread of pathogenic microorganisms. At the same time, for the food poisoning incident that has already occurred, this method can be used for diagnosis to find out the cause.


Assuntos
Bactérias/isolamento & purificação , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Reação em Cadeia da Polimerase Multiplex/métodos , Bacillus cereus/genética , Bacillus cereus/isolamento & purificação , Bactérias/classificação , Bactérias/genética , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Salmonella enterica/genética , Salmonella enterica/isolamento & purificação , Sensibilidade e Especificidade , Staphylococcus aureus/isolamento & purificação , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/isolamento & purificação
18.
Nat Microbiol ; 5(3): 395-406, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31988380

RESUMO

A major form of transcriptional regulation in bacteria occurs through the exchange of the primary σ factor of RNA polymerase (RNAP) with an alternative extracytoplasmic function (ECF) σ factor1. ECF σ factors are generally intrinsically active and are retained in an inactive state via the sequestration into σ factor-anti-σ factor complexes until their action is warranted2-20. Here, we report a previously uncharacterized mechanism of transcriptional regulation that relies on intrinsically inactive ECF σ factors, the activation of which and interaction with the ß'-subunit of RNAP depends on σ factor phosphorylation. In Vibrio parahaemolyticus, the threonine kinase PknT phosphorylates the σ factor EcfP, which results in EcfP activation and expression of an essential polymyxin-resistant regulon. EcfP phosphorylation occurs at a highly conserved threonine residue, Thr63, positioned within a divergent region in the σ2.2 helix. Our data indicate that EcfP is intrinsically inactive and unable to bind the ß'-subunit of RNAP due to the absence of a negatively charged DAED motif in this region. Furthermore, our results indicate that phosphorylation at residue Thr63 mimics this negative charge and licenses EcfP to interact with the ß'-subunit in the formation of the RNAP holoenzyme, which in turn results in target gene expression. This regulatory mechanism is a previously unrecognized paradigm in bacterial signal transduction and transcriptional regulation, and our data suggest that it is widespread in bacteria.


Assuntos
Bactérias/genética , Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Fator sigma/farmacologia , Transcrição Genética/efeitos dos fármacos , DNA Bacteriano/genética , RNA Polimerases Dirigidas por DNA , Genes Bacterianos/genética , Modelos Moleculares , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteômica , Transcriptoma , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo
19.
Arch Microbiol ; 202(5): 1025-1033, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31938849

RESUMO

Nanogold enhanced surface plasmon resonance (SPR), colloidal gold immunochromatographic test strips (ICTS), and polymerase chain reaction (PCR), combined with immunomagnetic separation (IMS) were established in this study for the rapid detection of Vibrio parahaemolyticus (VP). The sensitivities of SPR, ICTS, and PCR was determined to be 101, 103, and 103 CFU/mL for VP, respectively. After separation and enrichment by IMS, the sensitivities of SPR, ICTS, and PCR were 100, 101, and 102 CFU/mL for VP, respectively, which were improved by 10-, 100-, and 10-fold compared to the direct detection by SPR, ICTS, and PCR, respectively. When the VP-polluted water samples were directly assessed by SPR, ICTS, and PCR, the results were negative. By contrast, after separation and enrichment for 45 min by IMS, the results were all positive. The IMS-SPR, IMS-ICTS, and IMS-PCR detection methods were able to yield results in approximately 1.5 h, 55 min, and 3.5 h, respectively. These combined detection methods have advantages in being high-throughput and easy to operate without the need for sophisticated equipment or specialized skills. These methods might aid in the development of SPR, ICTS, and PCR technologies for simultaneously examining multiple food-borne pathogens in food products.


Assuntos
Cromatografia de Afinidade/métodos , Separação Imunomagnética/métodos , Ressonância de Plasmônio de Superfície/métodos , Vibrio parahaemolyticus/isolamento & purificação , Animais , Cromatografia de Afinidade/instrumentação , Doenças Transmitidas por Alimentos/microbiologia , Coloide de Ouro/química , Ensaios de Triagem em Larga Escala/métodos , Reação em Cadeia da Polimerase/métodos , Sensibilidade e Especificidade , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/imunologia
20.
Microbiol Immunol ; 64(3): 167-181, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31850542

RESUMO

Vibrio parahaemolyticus is a leading cause of seafood-borne bacterial gastroenteritis in humans. Since its discovery in 1950, this bacterium has been isolated in widespread outbreaks and in sporadic cases of gastroenteritis worldwide. Although the exotoxin, thermostable direct hemolysin, had been the focus of extensive research on the pathogenicity of V. parahaemolyticus, the whole-genome sequencing of a clinical isolate, RIMD2210633 strain, was a breakthrough in this field. The possession of two sets of gene clusters for type III secretion systems (T3SS1 and T3SS2) was unveiled by that genome project. T3SS is a protein export apparatus that delivers bacterial proteins, called effectors, directly into the host's cytosol, to disrupt host cell function. The subsequent studies have established that T3SS2, which is encoded in an 80 kb pathogenicity island called V. parahaemolyticus pathogenicity island (Vp-PAI), is closely related to enteropathogenicity. Recent functional analyses of Vp-PAI-encoded genes revealed the sophisticated mechanisms in V. parahaemolyticus for sensing the intestinal environment and host cell contact, and a dozen T3SS2-exported proteins encoded in Vp-PAI. In this review, we summarize recent advances in V. parahaemolyticus research regarding the control of the expression of Vp-PAI-encoded genes, structural components and the secretory regulation of T3SS2, and the biological activities of T3SS2-exported effectors. Thus, Vp-PAI-encoded T3SS2 becomes an important key in the postgenomic era to shed light on the enteropathogenic mechanism of V. parahaemolyticus.


Assuntos
Ilhas Genômicas/genética , Sistemas de Secreção Tipo III , Vibrioses/microbiologia , Vibrio parahaemolyticus , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Interações entre Hospedeiro e Microrganismos , Humanos , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidade
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