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1.
Commun Biol ; 5(1): 1352, 2022 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-36494435

RESUMO

Industrial wastewater discharge, agricultural production, marine shipping, oil extraction, and other activities have caused serious marine pollution, including microplastics, petroleum and its products, heavy metals, pesticides, and other organics. Efficiency of bioremediation of marine pollutions may be limited by high salt concentrations (>1%, w/v), which can cause an apparent loss of microbial activities. In this study, functional promoters P1, P2-1, and P2-2 censoring salt stress were isolated and identified from a Vibrio natriegens strain Vmax. Three salt-induced degradation models were constructed to degrade polyethylene terephthalate (PET), chlorpyrifos (CP), and hexabromocyclododecanes (HBCDs) using the marine strain Vmax. The engineered strains are efficient for degradation of the corresponding substrates, with the degradation rates at 15 mg/L PET in 8 d, 50 mg/L CP in 24 h, and 1 mg/L HBCDs in 4 h, respectively. In addition, an immobilization strategy for recycling and reusing of engineered strains was realized by expressing the chitin-binding protein GbpA. This study may help answer the usage of rapidly growing marine bacteria such as V. natriegens Vmax to degrade marine pollution efficiently.


Assuntos
Clorpirifos , Vibrio , Plásticos/metabolismo , Vibrio/genética , Vibrio/metabolismo , Biodegradação Ambiental , Regiões Promotoras Genéticas , Clorpirifos/metabolismo
2.
Mar Drugs ; 20(11)2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36355015

RESUMO

Up until now, the characterizations of GH50 agarases from Vibrio species have rarely been reported compared to GH16 agarases. In this study, a deep-sea strain, WPAGA4, was isolated and identified as Vibrio natriegens due to the maximum similarity of its 16S rRNA gene sequence, the values of its average nucleotide identity, and through digital DNA-DNA hybridization. Two circular chromosomes in V. natriegens WPAGA4 were assembled. A total of 4561 coding genes, 37 rRNA, 131 tRNA, and 59 other non-coding RNA genes were predicted in the genome of V. natriegens WPAGA4. An agarase gene belonging to the GH50 family was annotated in the genome sequence and expressed in E. coli cells. The optimum temperature and pH of the recombinant Aga3420 (rAga3420) were 40 °C and 7.0, respectively. Neoagarobiose (NA2) was the only product during the degradation process of agarose by rAga3420. rAga3420 had a favorable stability following incubation at 10-30 °C for 50 min. The Km, Vmax, and kcat values of rAga3420 were 2.8 mg/mL, 78.1 U/mg, and 376.9 s-1, respectively. rAga3420 displayed cold-adapted properties as 59.7% and 41.2% of the relative activity remained at 10 3 °C and 0 °C, respectively. This property ensured V. natriegens WPAGA4 could degrade and metabolize the agarose in cold deep-sea environments and enables rAga3420 to be an appropriate industrial enzyme for NA2 production, with industrial potential in medical and cosmetic fields.


Assuntos
Alteromonadaceae , Vibrio , Alteromonadaceae/genética , Alteromonadaceae/metabolismo , Sefarose/metabolismo , RNA Ribossômico 16S/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/metabolismo , Vibrio/genética , Vibrio/metabolismo , DNA/metabolismo
3.
World J Microbiol Biotechnol ; 38(12): 241, 2022 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-36271946

RESUMO

Vibrio mimicus is a bacterium that causes gastroenteritis in humans. This pathogen produces an enterotoxic hemolysin called V. mimicus hemolysin (VMH), which is secreted extracellularly as an inactive 80-kDa protoxin and converted to a 66-kDa mature toxin through cleavage between Arg151 and Ser152. The 56-kDa serine protease termed V. mimicus trypsin-like protease (VmtA) is known to mediate this maturating process. However, some strains including strain ES-20 does not possess the vmtA gene. In the present study, the vmtA-negative strains were found to have a replaced gene that encodes a 43-kDa (403 aa) precursor of a serine protease designated by VmtX (V. mimicus trypsin-like protease X). To examine whether VmtX is also involved in the maturation of VMH, VmtX was isolated from the culture supernatant of V. mimicus strain NRE-20, a metalloprotease-negative mutant constructed from strain ES-20. Concretely, the culture supernatant was fractionated with 70% saturated ammonium sulfate and subjected to affinity column chromatography using a HiTrap Benzamidine FF column. The analysis of the N-terminal amino acid sequences of the proteins in the obtained VmtX preparation indicated that the 39-kDa protein was active VmtX consisting of 371 aa (Ile33-Ser403). The VmtX preparation was found to activate pro-VMH through generation of the 66-kDa protein. Additionally, treatment of the VmtX preparation with serine protease inhibitors, such as leupeptin and phenylmethylsulfonyl fluoride, significantly suppressed the activities to hydrolyze the specific peptide substrate and to synthesize the 66-kDa toxin. These findings indicate that VmtX is the second protease that mediats the maturation of VMH.


Assuntos
Proteínas Hemolisinas , Vibrio , Humanos , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Peptídeo Hidrolases/genética , Leupeptinas , Sulfato de Amônio , Tripsina , Fluoreto de Fenilmetilsulfonil , Metaloproteases , Inibidores de Serino Proteinase , Benzamidinas , Vibrio/metabolismo
4.
Int J Mol Sci ; 23(20)2022 Oct 18.
Artigo em Inglês | MEDLINE | ID: mdl-36293339

RESUMO

The increasing prevalence of resistance in carbapenems is an escalating concern as carbapenems are reserved as last-line antibiotics. Although indiscriminate antibiotic usage is considered the primary cause for resistance development, increasing evidence revealed that inconsequential strains without any direct clinical relevance to carbapenem usage are harboring carbapenemase genes. This phenomenon indirectly implies that environmental microbial populations could be the 'hidden vectors' propelling carbapenem resistance. This work aims to explore the carbapenem-resistance profile of Vibrio species across diverse settings. This review then proceeds to identify the different factors contributing to the dissemination of the resistance traits and defines the transmission pathways of carbapenem resistance. Deciphering the mechanisms for carbapenem resistance acquisition could help design better prevention strategies to curb the progression of antimicrobial resistance development. To better understand this vast reservoir selecting for carbapenem resistance in non-clinical settings, Vibrio species is also prospected as one of the potential indicator strains for carbapenem resistance in the environment.


Assuntos
Vibrio , beta-Lactamases , beta-Lactamases/metabolismo , Carbapenêmicos/farmacologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Antibacterianos/farmacologia , Vibrio/genética , Vibrio/metabolismo , Testes de Sensibilidade Microbiana
5.
Elife ; 112022 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-36155655

RESUMO

The type VI secretion system (T6SS) is used by bacteria to deliver toxic effectors directly into target cells. Most T6SSs mediate antibacterial activities, whereas the potential anti-eukaryotic role of T6SS remains understudied. Here, we found a Vibrio T6SS that delivers two novel effectors into mammalian host immune cells. We showed that these effectors induce a pyroptotic cell death in a phagocytosis-dependent manner; we identified the NLRP3 inflammasome as being the underlying mechanism leading to the T6SS-induced pyroptosis. Moreover, we identified a compensatory T6SS-induced pathway that is activated upon inhibition of the canonical pyroptosis pathway. Genetic analyses revealed possible horizontal spread of this T6SS and its anti-eukaryotic effectors into emerging pathogens in the marine environment. Our findings reveal novel T6SS effectors that activate the host inflammasome and possibly contribute to virulence and to the emergence of bacterial pathogens.


Assuntos
Sistemas de Secreção Tipo VI , Vibrio , Animais , Antibacterianos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eucariotos/metabolismo , Inflamassomos/metabolismo , Mamíferos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fagocitose , Sistemas de Secreção Tipo VI/metabolismo , Vibrio/metabolismo
6.
Fish Shellfish Immunol ; 128: 664-675, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35981703

RESUMO

The New Zealand Greenshell™ mussel (Perna canaliculus) is an endemic bivalve species with cultural importance, that is harvested recreationally and commercially. However, production is currently hampered by increasing incidences of summer mortality in farmed and wild populations. While the causative factors for these mortality events are still unknown, it is believed that increasing seawater temperatures and pathogen loads are potentially at play. To improve our understanding of these processes, challenge experiments were conducted to investigate the combined effects of increased seawater temperature and Vibrio infection on the immune and metabolic responses of adult mussels. Biomarkers that measure the physiological response of mussels to multiple-stressors can be utilised to study resilience in a changing environment, and support efforts to strengthen biosecurity management. Mussels acclimated to two temperatures (16 °C and 24 °C) were injected with either autoclaved, filtered seawater (control) or Vibriosp. DO1 (infected). Then, haemolymph was sampled 24 h post-injection and analysed to quantify haemocyte immune responses (via flow-cytometry), antioxidant capacity (measured electrochemically) and metabolic responses (via gas chromatography-mass spectrometry) to bacterial infection. Both seawater temperature and injection type significantly influenced the immune and metabolite status of mussels. A lack of interaction effects between temperature and injection type indicated that the effects of Vibrio sp. 24 h post-infection were similar between seawater temperatures. Infected mussels had a higher proportion of dead haemocytes and lower overall haemocyte counts than uninfected controls. The proportion of haemocytes showing evidence of apoptosis was higher in mussels held at 24 °C compared with those held at 16 °C. The proportion of haemocytes producing reactive oxygen species did not differ between temperatures or injection treatments. Mussels held at 24 °C exhibited elevated levels of metabolites linked to the glycolysis pathway to support energy production. The saccharopin-lysine pathway metabolites were also increased in these mussels, indicating the role of lysine metabolism. A decrease in metabolic activity (decreases in BCAAs, GABA, urea cycle metabolites, oxidative stress metabolites) was largely seen in mussels injected with Vibrio sp. Itaconate increased as seen in previous studies, suggesting that antimicrobial activity may have been activated in infected mussels. This study highlights the complex nature of immune and metabolic responses in mussels exposed to multiple stressors and gives an insight into Vibrio sp. infection mechanisms at different seawater temperatures.


Assuntos
Anti-Infecciosos , Perna (Organismo) , Vibrioses , Vibrio , Animais , Anti-Infecciosos/farmacologia , Antioxidantes/metabolismo , Biomarcadores/metabolismo , Lisina/farmacologia , Perna (Organismo)/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Água do Mar , Temperatura , Ureia/metabolismo , Vibrio/metabolismo , Ácido gama-Aminobutírico/farmacologia
7.
Int J Mol Sci ; 23(16)2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-36012135

RESUMO

Piscibactin is a widespread siderophore system present in many different bacteria, especially within the Vibrionaceae family. Previous works showed that most functions required for biosynthesis and transport of this siderophore are encoded by the high-pathogenicity island irp-HPI. In the present work, using Vibrio anguillarum as a model, we could identify additional key functions encoded by irp-HPI that are necessary for piscibactin production and transport and that have remained unknown. Allelic exchange mutagenesis, combined with cross-feeding bioassays and LC-MS analysis, were used to demonstrate that Irp4 protein is an essential component for piscibactin synthesis since it is the thioesterase required for nascent piscibactin be released from the NRPS Irp1. We also show that Irp8 is a MFS-type protein essential for piscibactin secretion. In addition, after passage through the outer membrane transporter FrpA, the completion of ferri-piscibactin internalization through the inner membrane would be achieved by the ABC-type transporter FrpBC. The expression of this transporter is coordinated with the expression of FrpA and with the genes encoding biosynthetic functions. Since piscibactin is a major virulence factor of some pathogenic vibrios, the elements of biosynthesis and transport described here could be additional interesting targets for the design of novel antimicrobials against these bacterial pathogens.


Assuntos
Vibrio , Vibrionaceae , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Ilhas Genômicas , Sideróforos/metabolismo , Vibrio/genética , Vibrio/metabolismo , Vibrionaceae/genética , Vibrionaceae/metabolismo , Fatores de Virulência/metabolismo
8.
Mar Drugs ; 20(8)2022 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-35892947

RESUMO

The applications of alginate lyase are diverse, but efficient commercial enzymes are still unavailable. In this study, a novel alginate lyase with high activity was obtained from the marine bacteria Vibrio sp. Ni1. The ORF of the algB gene has 1824 bp, encoding 607 amino acids. Homology analysis shows that AlgB belongs to the PL7 family. There are two catalytic domains with the typical region of QIH found in AlgB. The purified recombinant enzyme of AlgB shows highest activity at 35 °C, pH 8.0, and 50 mmol/L Tris-HCl without any metal ions. Only K+ slightly enhances the activity, while Fe2+ and Cu2+ strongly inhibit the activity. The AlgB preferred polyM as substrate. The end products of enzymatic mixture are DP2 and DP3, without any metal ion to assist them. This enzyme has good industrial application prospects.


Assuntos
Polissacarídeo-Liases , Vibrio , Alginatos/metabolismo , Proteínas de Bactérias/metabolismo , Clonagem Molecular , Concentração de Íons de Hidrogênio , Íons , Metais/farmacologia , Polissacarídeo-Liases/metabolismo , Especificidade por Substrato , Vibrio/metabolismo
9.
J Biol Inorg Chem ; 27(6): 565-572, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35834122

RESUMO

Amphi-enterobactin is an amphiphilic siderophore isolated from a variety of microbial Vibrio species. Like enterobactin, amphi-enterobactin is a triscatecholate siderophore; however, it is framed on an expanded tetralactone core comprised of four L-Ser residues, of which one L-Ser is appended by a fatty acid and the remaining L-Ser residues are appended by 2,3-dihydroxybenzoate (DHB). Fragments of amphi-enterobactin composed of 2-Ser-1-DHB-FA and 3-Ser-2-DHB-FA have been identified in the supernatant of Vibrio campbellii species. The origin of these fragments has not been determined, although two distinct isomers could exist for 2-Ser-1-DHB-FA and three distinct isomers could exist for 3-Ser-2-DHB-FA. The fragments of amphi-enterobactin could originate from hydrolysis of the amphi-enterobactin macrolactone, or from premature release due to an inefficient biosynthetic pathway. Unique masses in the tandem MS analysis establish that certain fragments isolated from the culture supernatant must originate from hydrolysis of the amphi-enterobactin macrolactone, while others cannot be distinguished from premature release during biosynthesis or hydrolysis of amphi-enterobactin.


Assuntos
Enterobactina , Vibrio , Hidroxibenzoatos/metabolismo , Sideróforos/química , Vibrio/metabolismo
10.
Gene ; 839: 146726, 2022 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-35835408

RESUMO

Bacteria adhesion to fish mucus is a crucial virulence mechanism. As the initial step of bacterial infection, adhesion is impacted by bacterial motility and environmental conditions. However, its molecular mechanism is yet unclear. In this study, a significant decrease in gene expression of adhesion-deficient Vibrio harveyi was observed when the bacteria were subjected by Cu2+(50 mg/L), Pb2+(100 mg/L), Hg2+(25 mg/L), and Zn2+(50 mg/L). The genes fliA, fliR, and flrB were responsible for flagellation; being crucial for adhesion, these genes were identified and silenced via RNAi. After silencing of these genes by RNAi technology, the ability of adhesion, biofilm formation, motility, and flagella synthesis of V. harveyi were considerably reduced. Compared with the control group, it was observed that the expression levels of fliS, fliD, flgH, and flrC were significant down-regulated in fliR-RNAi, flrB-RNAi, and fliA-RNAi. This data indicates that the expression levels of most virulence genes are affected by fliA, fliR, and flrB. Also, the expression of fliA, fliR, and flrB can be influenced by the salinity, temperature, and pH. The results show that: (1) fliA, fliR, and flrB have important roles in the adhesion of V. harveyi; (2) fliA, fliR, and flrB can regulate bacterial adhesion by affecting its motility, and biofilm formation; (3) fliA, fliR, and flrB can regulate adhesion ability of V. harveyi in different environments.


Assuntos
Aderência Bacteriana , Vibrio , Animais , Aderência Bacteriana/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Vibrio/genética , Vibrio/metabolismo , Virulência/genética
11.
Curr Microbiol ; 79(6): 181, 2022 May 05.
Artigo em Inglês | MEDLINE | ID: mdl-35508788

RESUMO

Vibrio fluvialis is a marine opportunistic pathogen that frequently causes diseases in aquatic animals and humans. V. fluvialis can produce quorum sensing signaling molecules to coordinate cell density-dependent behavioral changes, including N-acyl homoserine lactone (AHL), which acts as a vital mediator of virulence-associated gene expression. Currently, several AHL molecules in V. fluvialis have been detected via biological and physicochemical methods, although different detection approaches have generated diverse AHL profiles. Here, we describe the AHL-producing bacterium, V. fluvialis BJ-1, which was isolated from marine sediments from the East China Sea. V. fluvialis BJ-1 could stimulate AHL-mediated ß-galactosidase synthesis of the biosensor Agrobacterium tumefaciens NTL4 (pZLR4) but could not induce violacein production in the AHL reporter strain, Chromobacterium violaceum CV026. This bacterial isolate exhibited strong AHL-producing activity at low cell density; however, the AHL activity declined when population density remained at high levels. Analysis of the AHLs by Ultra-High-Performance Liquid Chromatography tandem Mass Spectrometry demonstrated that V. fluvialis BJ-1 produced five different AHL signaling molecules, including two linear chain AHL products (C8- and C10-HSL), and three ß-carbon-oxidative AHL products (3-O-C8-, 3-O-C10- and 3-O-C12-HSL). Significantly, the present study is the first to accurately define the AHL profile of marine V. fluvialis. In future, the coupling of UHPLC to ESI-MS/MS is expected to be utilized for the accurate determination of AHL profiles in marine Vibrio.


Assuntos
Acil-Butirolactonas , Vibrio , 4-Butirolactona/metabolismo , Animais , Cromatografia Líquida de Alta Pressão , Homosserina/metabolismo , Lactonas/metabolismo , Percepção de Quorum , Espectrometria de Massas em Tandem , Vibrio/genética , Vibrio/metabolismo
12.
Appl Environ Microbiol ; 88(11): e0051922, 2022 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-35612301

RESUMO

Bacteria coping with oxygen deficiency use alternative terminal electron acceptors for NADH regeneration, particularly fumarate. Fumarate is reduced by the FAD_binding_2 domain of cytoplasmic fumarate reductase in many bacteria. The variability of the primary structure of this domain in homologous proteins suggests the existence of reducing activities with different specificities. Here, we produced and characterized one such protein encoded in the Vibrio harveyi genome (GenBank ID: AIV07243) and found it to be a specific NADH:acrylate oxidoreductase (ARD). This previously unknown enzyme is formed by the OYE-like, FMN_bind, and FAD_binding_2 domains and contains covalently bound flavin mononucleotide (FMN) and noncovalently bound flavin adenine dinucleotide (FAD) and FMN in a ratio of 1:1:1. The covalently bound FMN is absolutely required for activity and is attached by the specific flavin transferase, ApbE, to the FMN_bind domain. Quantitative reverse transcription PCR (RT-qPCR) and activity measurements indicated dramatic stimulation of ARD biosynthesis by acrylate in the V. harveyi cells grown aerobically. In contrast, the ard gene expression in the cells grown anaerobically without acrylate was higher than that in aerobic cultures and increased only 2-fold in the presence of acrylate. These findings suggest that the principal role of ARD in Vibrio is energy-saving detoxification of acrylate coming from the environment. IMPORTANCE The benefits of the massive genomic information accumulated in recent years for biological sciences have been limited by the lack of data on the function of most gene products. Approximately half of the known prokaryotic genes are annotated as "proteins with unknown functions," and many other genes are annotated incorrectly. Thus, the functional and structural characterization of the products of such genes, including identification of all existing enzymatic activities, is a pressing issue in modern biochemistry. In this work, we have shown that the product of the V. harveyi ard gene exhibits a yet-undescribed NADH:acrylate oxidoreductase activity. This activity may allow acrylate detoxification and its use as a terminal electron acceptor in anaerobic or substrate in aerobic respiration of marine and other bacteria.


Assuntos
Mononucleotídeo de Flavina , Vibrio , Acrilatos , Sequência de Aminoácidos , FMN Redutase/metabolismo , Mononucleotídeo de Flavina/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Fumaratos , NAD/metabolismo , NADH Desidrogenase/metabolismo , NADH NADPH Oxirredutases/metabolismo , Vibrio/metabolismo
13.
Appl Microbiol Biotechnol ; 106(9-10): 3721-3734, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35488933

RESUMO

Autoinducer-2 (AI-2), a quorum-sensing signal molecule from the human pathogen Vibrio vulnificus, was assessed for its effect on the gut microbiome of mice. For this, we employed 16S rRNA sequencing to compare the gut microbiome of mice infected with either wild-type V. vulnificus or with the isotype ΔluxS that has a deletion in luxS which encodes the biosynthetic function of AI-2. The relative ratio of wild-type Vibrio species in the jejunum and ileum of mice infected with the wild type was significantly higher than that in mice infected with ΔluxS, suggesting that AI-2 plays an important role in the colonization of V. vulnificus in the small intestine. The bacterial composition in the gut of mice infected with ΔluxS comprises a higher proportion of Firmicutes, composed mainly of Lactobacillus, compared to the mice infected with wild-type cells. In the large intestine, Vibrio species were barely detected regardless of genetic background. Three Lactobacillus spp. isolated from fecal samples from mice infected with ΔluxS manifested significant antibacterial activities against V. vulnificus. Culture supernatants from these three species were dissolved by HPLC, and a substance in fractions showing inhibitory activity against V. vulnificus was determined to be lactic acid. Our results suggest that luxS in V. vulnificus affects not only the ability of the species to colonize the host gut but also its susceptibility to the growth-inhibiting activity of commensal bacteria including Lactobacillus. KEY POINTS: • Gut microbiomes of ΔluxS-infected and WT Vibrio-infected mice differed greatly. • Difference was most prominent in the jejunum and ileum compared to the duodenum or large intestine. • In the small and large intestines of mice, the relative proportions of Vibrio and Lactobacillus species showed a negative relationship. • Effector molecules produced by Lactobacillus in mouse gut inhibit Vibrio growth.


Assuntos
Microbioma Gastrointestinal , Vibrio vulnificus , Vibrio , Animais , Proteínas de Bactérias/genética , Liases de Carbono-Enxofre/genética , Liases de Carbono-Enxofre/metabolismo , Regulação Bacteriana da Expressão Gênica , Lactobacillus/metabolismo , Camundongos , Percepção de Quorum , RNA Ribossômico 16S/genética , Vibrio/genética , Vibrio/metabolismo , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
14.
Appl Environ Microbiol ; 88(7): e0167721, 2022 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-35285716

RESUMO

Vibrio collagenases of the M9A subfamily are closely related to Vibrio pathogenesis for their role in collagen degradation during host invasion. Although some Vibrio collagenases have been characterized, the collagen degradation mechanism of Vibrio collagenase is still largely unknown. Here, an M9A collagenase, VP397, from marine Vibrio pomeroyi strain 12613 was characterized, and its fragmentation pattern on insoluble type I collagen fibers was studied. VP397 is a typical Vibrio collagenase composed of a catalytic module featuring a peptidase M9N domain and a peptidase M9 domain and two accessory bacterial prepeptidase C-terminal domains (PPC domains). It can hydrolyze various collagenous substrates, including fish collagen, mammalian collagens of types I to V, triple-helical peptide [(POG)10]3, gelatin, and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-o-Arg (Pz-peptide). Atomic force microscopy (AFM) observation and biochemical analyses revealed that VP397 first assaults the C-telopeptide region to dismantle the compact structure of collagen and dissociate tropocollagen fragments, which are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets. In addition, domain deletion mutagenesis showed that the catalytic module of VP397 alone is capable of hydrolyzing type I collagen fibers and that its C-terminal PPC2 domain functions as a collagen-binding domain during collagenolysis. Based on our results, a model for the collagenolytic mechanism of VP397 is proposed. This study sheds light on the mechanism of collagen degradation by Vibrio collagenase, offering a better understanding of the pathogenesis of Vibrio and helping in developing the potential applications of Vibrio collagenase in industrial and medical areas. IMPORTANCE Many Vibrio species are pathogens and cause serious diseases in humans and aquatic animals. The collagenases produced by pathogenic Vibrio species have been regarded as important virulence factors, which occasionally exhibit direct pathogenicity to the infected host or facilitate other toxins' diffusion through the digestion of host collagen. However, our knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited. This study reveals the degradation strategy of Vibrio collagenase VP397 on type I collagen. VP397 binds on collagen fibrils via its C-terminal PPC2 domain, and its catalytic module first assaults the C-telopeptide region and then attacks the Y-Gly bonds in the dissociated tropocollagen fragments to release peptides and amino acids. This study offers new knowledge regarding the collagenolytic mechanism of Vibrio collagenase, which is helpful for better understanding the role of collagenase in Vibrio pathogenesis and for developing its industrial and medical applications.


Assuntos
Colágeno Tipo I , Vibrio , Sequência de Aminoácidos , Aminoácidos , Animais , Colágeno/metabolismo , Colágeno Tipo I/genética , Colagenases/genética , Colagenases/metabolismo , Mamíferos , Peptídeos/metabolismo , Tropocolágeno , Vibrio/metabolismo
15.
J Biol Chem ; 298(3): 101667, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35120925

RESUMO

In marine environments, organisms are confronted with numerous microbial challenges, although the differential regulation of xenophagy in response to different pathogenic bacterial species remains relatively unknown. Here, we addressed this issue using Apostichopus japonicus as a model. We identified 39 conserved autophagy-related genes by genome-wide screening, which provided a molecular basis for autophagy regulation in sea cucumbers. Furthermore, xenophagy of two Gram-negative bacteria, Vibrio splendidus and Escherichia coli, but not a Gram-positive bacteria, Micrococcus luteus, was observed in different autophagy assays. Surprisingly, a significantly higher autophagy capacity was found in the E. coli-challenged group than in the V. splendidus-challenged group. To confirm these findings, two different lipopolysaccharides, LPSV. splendidus and LPSE. coli, were isolated; we found that these LPS species differentially activated coelomocyte xenophagy. To explore the molecular mechanism mediating differential levels of xenophagy, we used an siRNA knockdown assay and confirmed that LPSV. splendidus-mediated xenophagy was dependent on an AjTLR3-mediated pathway, whereas LPSE. coli-mediated xenophagy was dependent on AjToll. Moreover, the activation of different AjTLRs resulted in AjTRAF6 ubiquitination and subsequent activation of K63-linked ubiquitination of AjBeclin1. Inversely, the LPSV. splendidus-induced AjTLR3 pathway simultaneously activated the expression of AjA20, which reduced the extent of K63-linked ubiquitination of AjBeclin1 and impaired the induction of autophagy; however, this finding was no t evident with LPSE. coli. Our present results provide the first evidence showing that xenophagy could be differentially induced by different bacterial species to yield differential autophagy levels in echinoderms.


Assuntos
Proteína Beclina-1 , Equinodermos , Fator 6 Associado a Receptor de TNF , Receptores Toll-Like , Vibrio , Animais , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Equinodermos/metabolismo , Equinodermos/microbiologia , Escherichia coli/genética , Escherichia coli/metabolismo , Lipopolissacarídeos/farmacologia , Macroautofagia , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Receptores Toll-Like/metabolismo , Vibrio/metabolismo
16.
Int J Mol Sci ; 23(2)2022 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-35054801

RESUMO

Osmotic changes are common challenges for marine microorganisms. Bacteria have developed numerous ways of dealing with this stress, including reprogramming of global cellular processes. However, specific molecular adaptation mechanisms to osmotic stress have mainly been investigated in terrestrial model bacteria. In this work, we aimed to elucidate the basis of adjustment to prolonged salinity challenges at the proteome level in marine bacteria. The objects of our studies were three representatives of bacteria inhabiting various marine environments, Shewanella baltica, Vibrio harveyi and Aliivibrio fischeri. The proteomic studies were performed with bacteria cultivated in increased and decreased salinity, followed by proteolytic digestion of samples which were then subjected to liquid chromatography with tandem mass spectrometry analysis. We show that bacteria adjust at all levels of their biological processes, from DNA topology through gene expression regulation and proteasome assembly, to transport and cellular metabolism. The finding that many similar adaptation strategies were observed for both low- and high-salinity conditions is particularly striking. The results show that adaptation to salinity challenge involves the accumulation of DNA-binding proteins and increased polyamine uptake. We hypothesize that their function is to coat and protect the nucleoid to counteract adverse changes in DNA topology due to ionic shifts.


Assuntos
Adaptação Fisiológica , Aliivibrio fischeri/fisiologia , Oceanos e Mares , Proteômica , Salinidade , Shewanella/fisiologia , Vibrio/fisiologia , Adaptação Fisiológica/genética , Aliivibrio fischeri/genética , Aliivibrio fischeri/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Ontologia Genética , Chaperonas Moleculares/metabolismo , Ácidos Nucleicos/metabolismo , Concentração Osmolar , Osmose , Pressão Osmótica , Ligação Proteica , Proteoma/metabolismo , Shewanella/genética , Shewanella/metabolismo , Transcrição Genética , Vibrio/genética , Vibrio/metabolismo
17.
Nat Commun ; 13(1): 566, 2022 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-35091565

RESUMO

The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase.


Assuntos
Proteínas de Bactérias/química , Colágeno/metabolismo , Colagenases/química , Conformação Proteica , Vibrio/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação/genética , Biocatálise , Cromatografia Líquida , Colagenases/genética , Colagenases/metabolismo , Cristalografia por Raios X , Espectrometria de Massas , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Vibrio/enzimologia , Vibrio/genética
18.
Gene ; 807: 145961, 2022 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-34530088

RESUMO

Vibrio parahaemolyticus produces two types of IV pili: mannose-sensitive haemagglutinin type IV pili (MSHA) and chitin-regulated pili (ChiRP). Both of them are required for biofilm formation and the pathogen persistence in hosts. However, there are few reports on the regulation of their expression. In the present study, we showed that the master quorum sensing (QS) regulators AphA and OpaR oppositely regulated the transcription of mshA1 encoding the pilin of MSHA pilus in V. parahaemolyticus. At low cell density (LCD), AphA indirectly repressed mshA1 transcription. In contrast, at high cell density (HCD), OpaR bound to the regulatory DNA region of mshA1 to activate its transcription. Oppositely regulation of mshA1 by AphA and OpaR led to a gradual increase in the expression level of mshA1 from LCD to HCD. Thus, regulation of type IV pili production was one of the mechanisms that V. parahaemolyticus adopted to control biofilm formation.


Assuntos
Proteínas de Fímbrias/genética , Percepção de Quorum/genética , Vibrio/genética , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Contagem de Células , Proteínas de Fímbrias/metabolismo , Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/genética , Lectina de Ligação a Manose/genética , Lectina de Ligação a Manose/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Vibrio/metabolismo , Vibrio cholerae/genética , Vibrio cholerae/metabolismo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo
19.
J Proteomics ; 251: 104412, 2022 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-34737109

RESUMO

The gram-negative bacterium Vibrio (Listonella) anguillarum (VA) is the causative agent of vibriosis, a terminal hemorrhagic septicemia affecting the aquacultural industry across the globe. In the current study we used label-free quantitative proteomics to investigate how VA adapts to conditions that mimic defined aspects of vibriosis-related stress such as exposure to oxidative stress (H2O2), exposure to humoral factors of innate immunity through incubation with Atlantic salmon serum, and iron deprivation upon supplementation of 2,2'-dipyridyl (DIP) to the growth medium. We also investigated how regulation of virulence factors may be governed by the VA growth phase and availability of nutrients. All experimental conditions explored revealed stress-specific proteomic adaption of VA and only nine proteins were found to be commonly regulated in all conditions. A general observation made for all stress-related conditions was regulation of multiple metabolic pathways. Notably, iron deprivation and exposure to Atlantic salmon serum evoked upregulation of iron acquisition mechanisms. The findings made in the present study represent a source of potential virulence determinants that can be of use in the search for means to understand vibriosis. SIGNIFICANCE: Vibriosis in fish and shellfish caused by V. anguillarum (VA) is responsible for large economic losses in the aquaculture sector across the globe. However, not much is known about the defense mechanism of this pathogen to percept and adapt to the imposed stresses during infection. Analyzing the response of VA to multiple host-related physiochemical stresses, the quantitative proteomic analysis of the present study indicates modulation of several virulence determinants and key defense networks of this pathogen. Our findings provide a theoretical basis to enhance our understanding of VA pathogenesis and can be employed to improve current intervention strategies to control vibriosis in aquaculture.


Assuntos
Doenças dos Peixes , Vibrio , Animais , Doenças dos Peixes/microbiologia , Peróxido de Hidrogênio/metabolismo , Imunidade Inata , Ferro/metabolismo , Estresse Oxidativo , Proteômica , Vibrio/metabolismo
20.
Trends Biotechnol ; 40(4): 381-384, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-34794836

RESUMO

Vibrio natriegens is a Gram-negative marine bacterium with an exceptionally fast growth rate and a doubling time of less than 10 min. Its high substrate uptake rates and metabolic prowess make it a promising next-generation workhorse for rapid molecular biology, protein expression, and metabolic engineering.


Assuntos
Vibrio , Biotecnologia , Engenharia Metabólica , Biologia Molecular , Vibrio/genética , Vibrio/metabolismo
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