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1.
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
2.
Curr Microbiol ; 79(1): 25, 2021 Dec 14.
Artigo em Inglês | MEDLINE | ID: mdl-34905101

RESUMO

Vibrio parahaemolyticus is a moderately halophilic, salt-requiring organism that exhibits optimal growth at approximately 3% salt. Thus, salinity stress is one of the most important stimuli during its lifecycle. The bacterium possesses a strong ability to form biofilms on surfaces, which are thought to be involved in protecting it from adverse environmental conditions. In the present study, salinity-dependent biofilm formation by V. parahaemolyticus was investigated by combining crystal violet staining, colony morphology, intracellular c-di-GMP quantification and quantitative PCR. The results showed that biofilm formation by V. parahaemolyticus was significantly enhanced in low salinity growth conditions and was affected by incubation time. In addition, low salinity reduced intracellular c-di-GMP degradation in V. parahaemolyticus. Transcription of genes encoding ScrABC and ScrG proteins, which are involved in intracellular c-di-GMP metabolism, was inhibited by low salinity growth conditions. Thus, reduced intracellular c-di-GMP degradation in V. parahaemolyticus in low salinity growth conditions may be mediated by repression of scrG and scrABC transcription. Taken together, these results demonstrated for the first time that salinity regulates biofilm formation and c-di-GMP production in V. parahaemolyticus.


Assuntos
Vibrio parahaemolyticus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Biofilmes , GMP Cíclico/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Salinidade , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo
3.
J Fish Dis ; 44(12): 2055-2066, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34496040

RESUMO

The transcriptome and antibiotic resistance of Vibrio parahaemolyticus isolated from Penaeus vannamei cultured in seawater (strain HN1)and freshwater (strain SH1) ponds were studied at different salinity (2‰ and 20‰). At different salinity, 623 differentially expressed genes (DEGs) significantly upregulated and 1,559 DEGs significantly downregulated in SH1. In HN1, 466 DEGs significantly upregulated and 1,930 DEGs significantly downregulated, indicating high salinity can lead to the downregulation of most genes. In KEGG analysis, the expression of DEGs annotated to starch and sucrose metabolism pathway was higher at 2‰ salinity than at 20‰ salinity in HN1 and SH1, implying salinity affected bacterial growth mainly through this pathway. In the enrichment analysis of upregulated DEGs, two pathways (Valine, leucine, and isoleucine degradation, and Butanoate metabolism) were significantly enriched at different salinity. Antibiotic-susceptibility test discovered that SH1 isolated from P. vannamei cultured in freshwater was resistant to multiple drugs, including kanamycin, gentamicin, medemycin, and azithromycin, at a salinity of 2‰, whereas at 20‰ salinity, SH1 was not resistant to the drugs. The HN1 strain isolated from P. vannamei cultured in mariculture was resistant to polymyxin B and clindamycin at 20‰ salinity. Whereas, HN1 was intermediately susceptible to these two antibiotics at 2‰ salinity. These results indicate that the drug resistance of bacteria was affected by salinity. Furthermore, beta-lactam resistance was significantly enriched in SH1 at different salinity, and the inhibition zone of penicillin G was consistent with the results of a beta-lactam resistance pathway.


Assuntos
Resistência Microbiana a Medicamentos/genética , Resistência Microbiana a Medicamentos/fisiologia , Salinidade , Transcriptoma , Vibrio parahaemolyticus/metabolismo , Animais , Antibacterianos/farmacologia , Aquicultura , Penaeidae/microbiologia , Vibrio parahaemolyticus/genética
4.
PLoS One ; 16(6): e0252800, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34143799

RESUMO

Type three secretion is the mechanism of protein secretion found in bacterial flagella and injectisomes. At its centre is the export apparatus (EA), a complex of five membrane proteins through which secretion substrates pass the inner membrane. While the complex formed by four of the EA proteins has been well characterised structurally, little is known about the structure of the membrane domain of the largest subunit, FlhA in flagella, SctV in injectisomes. Furthermore, the biologically relevant nonameric assembly of FlhA/SctV has been infrequently observed and differences in conformation of the cytoplasmic portion of FlhA/SctV between open and closed states have been suggested to reflect secretion system specific differences. FlhA has been shown to bind to chaperone-substrate complexes in an open state, but in previous assembled ring structures, SctV is in a closed state. Here, we identify FlhA and SctV homologues that can be recombinantly produced in the oligomeric state and study them using cryo-electron microscopy. The structures of the cytoplasmic domains from both FlhA and SctV are in the open state and we observe a conserved interaction between a short stretch of residues at the N-terminus of the cytoplasmic domain, known as FlhAL/SctVL, with a groove on the adjacent protomer's cytoplasmic domain, which stabilises the nonameric ring assembly.


Assuntos
Proteínas de Bactérias/metabolismo , Flagelos/metabolismo , Proteínas de Membrana/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Microscopia Crioeletrônica/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Fluorescência/métodos , Modelos Moleculares , Conformação Proteica , Sistemas de Secreção Tipo III/genética , Sistemas de Secreção Tipo III/ultraestrutura , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Yersinia enterocolitica/genética , Yersinia enterocolitica/metabolismo
5.
Microbiol Res ; 250: 126791, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34090181

RESUMO

Vibrio parahaemolyticus, a common foodborne pathogen, can form biofilms for survival in various environments and for bacterial transmission. Lux systems in Vibrio species are the typical two-component signal transduction systems, which have been demonstrated to contribute to various phenotypes; however, the functions of each homolog of the Lux system in V. parahaemolyticus in the regulation of biofilm formation remain largely unknown. In this study, we first showed that LuxQ, LuxU, and LuxO are essential for controlling biofilm formation by V. parahaemolyticus, through gene knockout studies. We also found that they acted in the same signaling pathway and their deletion mutants exhibited a similar level of biofilm formation. Furthermore, site-directed mutagenesis revealed that the conserved residues for phosphorylation in LuxQ (D784), LuxU (H56) and LuxO (D47) were critical for their regulatory functions on biofilm formation. Phos-tag™ sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the phosphorylation of LuxU and LuxQ in vivo. Finally, qPCR analysis displayed that the three mutants had a significant decrease in the transcription level of cps loci and cpsQ compared with the wild type strain, which is consistent with the observed phenotype of biofilm formation. Therefore, we propose that LuxQ and its downstream factors LuxU and LuxO function in the same signaling cascade to control biofilm formation by regulating the expression of cpsQ and cps loci. The results of this study provide new data regarding the role of the LuxQ-LuxU-LuxO pathway in biofilm formation by V. parahaemolyticus and help further understand the complex regulatory functions of Lux pathways.


Assuntos
Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Regulação Bacteriana da Expressão Gênica , Fosfoproteínas/genética , Transdução de Sinais/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Mutagênese Sítio-Dirigida , Fenótipo , Fosforilação , Transdução de Sinais/fisiologia , Fatores de Transcrição/genética
6.
J Bacteriol ; 203(13): e0004621, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-33846117

RESUMO

Vibrio parahaemolyticus cells transit from free-swimming to surface adapted lifestyles, such as swarming colonies and three-dimensional biofilms. These transitions are regulated by sensory modules and regulatory networks that involve the second messenger cyclic diguanylate monophosphate (c-di-GMP). In this work, we show that a previously uncharacterized c-di-GMP phosphodiesterase (VP1881) from V. parahaemolyticus plays an important role in modulating the c-di-GMP pool. We found that the product of VP1881 promotes its own expression when the levels of c-di-GMP are low or when the phosphodiesterase (PDE) is catalytically inactive. This behavior has been observed in a class of c-di-GMP receptors called trigger phosphodiesterases, and hence we named the product of VP1881 TpdA, for trigger phosphodiesterase A. The absence of tpdA showed a negative effect on swimming motility while, its overexpression from an isopropyl-ß-d-thiogalactopyranoside (IPTG)-inducible promoter showed a positive effect on both swimming and swarming motility and a negative effect on biofilm formation. Changes in TpdA abundance altered the expression of representative polar and lateral flagellar genes, as well as that of the biofilm-related gene cpsA. Our results also revealed that autoactivation of the native PtpdA promoter is sufficient to alter c-di-GMP signaling responses such as swarming and biofilm formation in V. parahaemolyticus, an observation that could have important implications in the dynamics of these social behaviors. IMPORTANCE c-di-GMP trigger phosphodiesterases (PDEs) could play a key role in controlling the heterogeneity of biofilm matrix composition, a property that endows characteristics that are potentially relevant for sustaining integrity and functionality of biofilms in a variety of natural environments. Trigger PDEs are not always easy to identify based on their sequence, and hence not many examples of these type of signaling proteins have been reported in the literature. Here, we report on the identification of a novel trigger PDE in V. parahaemolyticus and provide evidence suggesting that its autoactivation could play an important role in the progression of swarming motility and biofilm formation, multicellular behaviors that are important for the survival and dissemination of this environmental pathogen.


Assuntos
Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , Diester Fosfórico Hidrolases/metabolismo , Vibrio parahaemolyticus/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , GMP Cíclico/química , GMP Cíclico/genética , GMP Cíclico/metabolismo , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/genética , Sistemas do Segundo Mensageiro , Vibrio parahaemolyticus/genética
7.
Biochem Biophys Res Commun ; 555: 102-108, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-33813268

RESUMO

VbrK and VbrR from the gastroenteritis-causing Vibrio parahaemolyticus are a histidine kinase and response regulator, respectively, that constitute a two-component regulatory system. VbrK responds to ß-lactam antibiotics or nitrate and activates VbrR via phosphorylation. Consequently, VbrR transcriptionally regulates the expression of ß-lactamase and ExsC and contributes to the survival or virulence of V. parahaemolyticus. Due to the unavailability of the VbrR structure, it remains unclear how VbrR is activated via its N-terminal receiver domain (RD) and recognizes dsDNA via its C-terminal DNA-binding domain (DBD). To reveal the mechanism underlying VbrR-mediated activation, we generated the phosphomimetic protein (VbrRRD-D51E) of the VbrR RD by replacing the D51 residue at the phosphorylation site with glutamate. VbrRRD-D51E exhibits a ß7α5 structure rather than the typical ß5α5 structure because it contains a unique two-stranded ß-sheet. The VbrRRD-D51E structure represents an active state in which the D51E residue interacts with the T78 residue. As a result, the Y97 residue adopts an inward conformation, allowing VbrRRD-D51E to dimerize using the α4-ß5-α5 face. These activation events are facilitated by a VbrR-specific residue, R52. Further structural study demonstrated that the VbrR DBD adopts a ß-strand-decorated three-helix structure. Based on a comparative structural study, we propose that VbrR recognizes dsDNA by inserting the α8 helix into the major groove of dsDNA and interacting with the minor groove of dsDNA via the ß11-ß12 region. Our findings will provide a new avenue for development of new antibacterial drugs for treating V. parahaemolyticus infections.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Vibrio parahaemolyticus/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , DNA/química , DNA/metabolismo , Genes Reguladores , Modelos Moleculares , Fosforilação , Domínios Proteicos , Vibrio parahaemolyticus/metabolismo
8.
Front Immunol ; 12: 634497, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33868255

RESUMO

Programmed cell death (PCD) is an essential process for the immune system's development and homeostasis, enabling the remotion of infected or unnecessary cells. There are several PCD's types, depending on the molecular mechanisms, such as non-inflammatory or pro-inflammatory. Hemocytes are the main component of cellular immunity in bivalve mollusks. Numerous infectious microorganisms produce toxins that impair hemocytes functions, but there is little knowledge on the role of PCD in these cells. This study aims to evaluate in vitro whether marine toxins induce a particular type of PCD in hemocytes of the bivalve mollusk Crassostrea gigas during 4 h at 25°C. Hemocytes were incubated with two types of marine toxins: non-proteinaceous toxins from microalgae (saxitoxin, STX; gonyautoxins 2 and 3, GTX2/3; okadaic acid/dynophysistoxin-1, OA/DTX-1; brevetoxins 2 and 3, PbTx-2,-3; brevetoxin 2, PbTx-2), and proteinaceous extracts from bacteria (Vibrio parahaemolyticus, Vp; V. campbellii, Vc). Also, we used the apoptosis inducers, staurosporine (STP), and camptothecin (CPT). STP, CPT, STX, and GTX 2/3, provoked high hemocyte mortality characterized by apoptosis hallmarks such as phosphatidylserine translocation into the outer leaflet of the cell membrane, exacerbated chromatin condensation, DNA oligonucleosomal fragments, and variation in gene expression levels of apoptotic caspases 2, 3, 7, and 8. The mixture of PbTx-2,-3 also showed many apoptosis features; however, they did not show apoptotic DNA oligonucleosomal fragments. Likewise, PbTx-2, OA/DTX-1, and proteinaceous extracts from bacteria Vp, and Vc, induced a minor degree of cell death with high gene expression of the pro-inflammatory initiator caspase-1, which could indicate a process of pyroptosis-like PCD. Hemocytes could carry out both PCD types simultaneously. Therefore, marine toxins trigger PCD's signaling pathways in C. gigas hemocytes, depending on the toxin's nature, which appears to be highly conserved both structurally and functionally.


Assuntos
Apoptose/efeitos dos fármacos , Toxinas Bacterianas/toxicidade , Crassostrea/efeitos dos fármacos , Hemócitos/efeitos dos fármacos , Toxinas Marinhas/toxicidade , Animais , Toxinas Bacterianas/isolamento & purificação , Caspases/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Crassostrea/imunologia , Crassostrea/metabolismo , Quebras de DNA de Cadeia Dupla , Hemócitos/imunologia , Hemócitos/metabolismo , Hemócitos/patologia , Fosfatidilserinas/metabolismo , Vibrio/metabolismo , Vibrio parahaemolyticus/metabolismo
9.
PLoS Pathog ; 17(1): e1009194, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439894

RESUMO

The viable but non culturable (VBNC) state is a condition in which bacterial cells are viable and metabolically active, but resistant to cultivation using a routine growth medium. We investigated the ability of V. parahaemolyticus to form VBNC cells, and to subsequently become resuscitated. The ability to control VBNC cell formation in the laboratory allowed us to selectively isolate VBNC cells using fluorescence activated cell sorting, and to differentiate subpopulations based on their metabolic activity, cell shape and the ability to cause disease in Galleria mellonella. Our results showed that two subpopulations (P1 and P2) of V. parahaemolyticus VBNC cells exist and can remain dormant in the VBNC state for long periods. VBNC subpopulation P2, had a better fitness for survival under stressful conditions and showed 100% revival under favourable conditions. Proteomic analysis of these subpopulations (at two different time points: 12 days (T12) and 50 days (T50) post VBNC) revealed that the proteome of P2 was more similar to that of the starting microcosm culture (T0) than the proteome of P1. Proteins that were significantly up or down-regulated between the different VBNC populations were identified and differentially regulated proteins were assigned into 23 functional groups, the majority being assigned to metabolism functional categories. A lactate dehydrogenase (lldD) protein, responsible for converting lactate to pyruvate, was significantly upregulated in all subpopulations of VBNC cells. Deletion of the lactate dehydrogenase (RIMD2210633:ΔlldD) gene caused cells to enter the VBNC state significantly more quickly compared to the wild-type, and adding lactate to VBNC cells aided their resuscitation and extended the resuscitation window. Addition of pyruvate to the RIMD2210633:ΔlldD strain restored the wild-type VBNC formation profile. This study suggests that lactate dehydrogenase may play a role in regulating the VBNC state.


Assuntos
Fenômenos Fisiológicos Bacterianos , Proteínas de Bactérias/metabolismo , Viabilidade Microbiana , Proteoma/metabolismo , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio parahaemolyticus/patogenicidade , Virulência , Células Cultivadas , Meios de Cultura , Regulação Bacteriana da Expressão Gênica , Proteoma/análise , Vibrioses/metabolismo , Vibrioses/microbiologia , Vibrio parahaemolyticus/metabolismo
10.
J Bacteriol ; 203(4)2021 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-33199284

RESUMO

Vibrio parahaemolyticus rapidly colonizes surfaces using swarming motility. Surface contact induces the surface-sensing regulon, including lateral flagellar genes, spurring dramatic shifts in physiology and behavior. The bacterium can also adopt a sessile, surface-associated lifestyle and form robust biofilms. These alternate colonization strategies are influenced reciprocally by the second messenger c-di-GMP. Although V. parahaemolyticus possesses 43 predicted proteins with the c-di-GMP-forming GGDEF domain, none have been previously been identified as contributors to surface colonization. We sought to explore this knowledge gap by using a suppressor transposon screen to restore the swarming motility of a nonswarming, high-c-di-GMP strain. Two diguanylate cyclases, ScrJ and ScrL, each containing tetratricopeptide repeat-coupled GGDEF domains, were demonstrated to contribute additively to swarming gene repression. Both proteins required an intact catalytic motif to regulate. Another suppressor mapped in lafV, the last gene in a lateral flagellar operon. Containing a degenerate phosphodiesterase (EAL) domain, LafV repressed transcription of multiple genes in the surface sensing regulon; its repressive activity required LafK, the primary swarming regulator. Mutation of the signature EAL motif had little effect on LafV's repressive activity, suggesting that LafV belongs to the subclass of EAL-type proteins that are regulatory but not enzymatic. Consistent with these activities and their predicted effects on c-di-GMP, scrJ and scrL but not lafV, mutants affected the transcription of the c-di-GMP-responsive biofilm reporter cpsA::lacZ Our results expand the knowledge of the V. parahaemolyticus GGDEF/EAL repertoire and its roles in this surface colonization regulatory network.IMPORTANCE A key survival decision, in the environment or the host, is whether to emigrate or aggregate. In bacteria, c-di-GMP signaling almost universally influences solutions to this dilemma. In V. parahaemolyticus, c-di-GMP reciprocally regulates swarming and sticking (i.e., biofilm formation) programs of surface colonization. Key c-di-GMP-degrading phosphodiesterases responsive to quorum and nutritional signals have been previously identified. c-di-GMP binding transcription factors programming biofilm development have been studied. Here, we further develop the blueprint of the c-di-GMP network by identifying new participants involved in dictating the complex decision of whether to swarm or stay. These include diguanylate cyclases with tetratricopeptide domains and a degenerate EAL protein that, analogously to the negative flagellar regulator RflP/YdiV of enteric bacteria, serves to regulate swarming.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Vibrio parahaemolyticus/metabolismo , Sequência de Aminoácidos , Aderência Bacteriana/fisiologia , Proteínas de Bactérias/genética , Biofilmes , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Mutação , Fósforo-Oxigênio Liases/genética , Fósforo-Oxigênio Liases/metabolismo , Domínios Proteicos , Vibrio parahaemolyticus/genética
11.
J Appl Microbiol ; 131(1): 50-67, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33151560

RESUMO

AIMS: Acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus has emerged as a severe bacterial disease of cultured shrimp. To identify the key virulence factors, two AHPND-causing V. parahaemolyticus (VpAHPND ) strains (123 and 137) and two non-VpAHPND strains (HZ56 and ATCC 17082) were selected. METHODS AND RESULTS: Challenge tests showed that the four strains exhibited different virulence towards shrimp with cumulative mortalities at 48 h postinfection (hpi) ranging from 10 to 92%. The expression of pirABVP in strain 123 and 137 was not significantly different. Genomic analysis revealed that the two VpAHPND strains contain a plasmid with the PirABVP toxins (pirABVP ) flanked by the insertion sequence (ISVal1) that has been identified in various locations of chromosomes in VpAHPND strains. The two VpAHPND strains possessed almost identical virulence factors, while ISVal1 disrupted three genes related to flagellar motility in strain 137. Phenotype assay showed that strain 123 possessed the highest growth rate and swimming motility, followed by strain 137, suggesting that the disruption of essential genes mediated by ISVal1 significantly affected the virulence level. Transcriptome analysis of two VpAHPND strains (123 and 137) further suggested that virulence genes related to the capsule, flagella and primary metabolism were highly expressed in strain 123. CONCLUSIONS: Here for the first time, it is demonstrated that the virulence of VpAHPND is not only determined by the expression of pirABVP , but also is mediated by ISVal1 which affects the genes involved in flagellar motility and primary metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: The genomic and transcriptomic analysis of VpAHPND strains provides valuable information on the virulence factors affecting the pathogenicity of VpAHPND.


Assuntos
Penaeidae/microbiologia , Vibrioses/veterinária , Vibrio parahaemolyticus/patogenicidade , Fatores de Virulência/metabolismo , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Elementos de DNA Transponíveis/genética , Plasmídeos/genética , Alimentos Marinhos/microbiologia , Vibrioses/microbiologia , Vibrioses/mortalidade , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo , Virulência/genética , Fatores de Virulência/genética
12.
EMBO J ; 40(2): e105699, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33347626

RESUMO

Pathogen type 3 secretion systems (T3SS) manipulate host cell pathways by directly delivering effector proteins into host cells. In Vibrio parahaemolyticus, the leading cause of bacterial seafood-borne diarrheal disease, we showed that a T3SS effector, VgpA, localizes to the host cell nucleolus where it binds Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2). An amino acid substitution in VgpA (VgpAL10A ) did not alter its translocation to the nucleus but abolished the effector's capacity to interact with EBP2. VgpA-EBP2 interaction led to the re-localization of c-Myc to the nucleolus and increased cellular rRNA expression and proliferation of cultured cells. The VgpA-EBP2 interaction elevated EBP2's affinity for c-Myc and prolonged the oncoprotein's half-life. Studies in infant rabbits demonstrated that VgpA is translocated into intestinal epithelial cells, where it interacts with EBP2 and leads to nucleolar re-localization of c-Myc. Moreover, the in vivo VgpA-EBP2 interaction during infection led to proliferation of intestinal cells and heightened V. parahaemolyticus' colonization and virulence. These observations suggest that direct effector stimulation of a c-Myc controlled host cell growth program can contribute to pathogenesis.


Assuntos
Proteínas de Bactérias/metabolismo , Nucléolo Celular/metabolismo , Proliferação de Células/fisiologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Sistemas de Secreção Tipo III/metabolismo , Vibrio parahaemolyticus/metabolismo , Virulência/fisiologia , Animais , Células CACO-2 , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/metabolismo , Infecções por Vírus Epstein-Barr/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Herpesvirus Humano 4/patogenicidade , Humanos , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Coelhos , Vibrioses/metabolismo
13.
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
14.
Microb Pathog ; 149: 104579, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-33091577

RESUMO

The type VI secretion system 2 (T6SS2) gene locus of Vibrio parahaemolyticus is comprised of three operons, VPA1027-1024, VPA1043-1028, and VPA1044-1046. QsvR is a virulence regulator of V. parahaemolyticus. In this study, the regulation of VPA1027, VPA1043 and VPA1044 by QsvR was investigated by primer extension, quantitative real-time PCR, LacZ fusion, electrophoretic mobility shift assay and DNase I footprinting. The results demonstrated that QsvR binds to the promoter-proximal DNA regions of each of these three operons, activating their transcription. T6SS2 was shown to predominately contribute to V. parahaemolyticus adhesion, with qsvR deletion significantly decreasing V. parahaemolyticus adhesion to HeLa cells. Thus, QsvR is not only a positive regulator of T6SS2 gene transcription but also a mediator of V. parahaemolyticus adhesion to host cells.


Assuntos
Sistemas de Secreção Tipo VI , Vibrio parahaemolyticus , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Células HeLa , Humanos , Sistemas de Secreção Tipo VI/genética , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/metabolismo
15.
Biochem Biophys Res Commun ; 533(1): 155-161, 2020 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-32943185

RESUMO

Bacteria express ß-lactamase to counteract the bactericidal effects of ß-lactam antibiotics, which are the most widely employed antibacterial drugs. In gram-negative bacteria, the expression of ß-lactamase is generally regulated in response to the muropeptide that is generated from the peptidoglycan of the cell wall during ß-lactam antibiotic challenge. The direct regulation of ß-lactamase expression by ß-lactams was recently reported in Vibrio parahaemolyticus, and this regulation is mediated by a two-component regulatory system that consists of the histidine kinase VbrK and the response regulator VbrR. VbrK directly recognizes ß-lactam antibiotics using the periplasmic sensor domain (VbrKSD), a PF11884 Pfam family member, and it delivers the ß-lactam signal to VbrR to induce the transcription of the ß-lactamase gene. To determine the structural features of VbrKSD as the prototype of the PF11884 family and provide insights into the ß-lactam antibiotic-binding mode of VbrKSD, we determined the crystal structure of VbrKSD at 1.65 Å resolution. VbrKSD folds into a unique curved rod-like structure that has not been previously reported in other families. VbrKSD consists of two domains (D1 and D2). The D1 domain contains two helix-decorated ß-sheets, and the D2 domain adopts a helix-rich structure. VbrKSD features two terminal disulfide bonds, which would be the canonical property of the PF11884 family. In the VbrKSD structure, the L82 residue, which was previously shown to play a key role in ß-lactam antibiotic recognition, forms a pocket along with its neighboring hydrophobic or positively charged residues.


Assuntos
Antibacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Vibrio parahaemolyticus/metabolismo , beta-Lactamas/metabolismo , Proteínas de Bactérias/química , Cristalografia por Raios X , Histidina Quinase/química , Humanos , Modelos Moleculares , Conformação Proteica , Domínios Proteicos , Vibrioses/microbiologia , Vibrio parahaemolyticus/química
16.
Elife ; 92020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32808593

RESUMO

Pathogens find diverse niches for survival including inside a host cell where replication occurs in a relatively protective environment. Vibrio parahaemolyticus is a facultative intracellular pathogen that uses its type 3 secretion system 2 (T3SS2) to invade and replicate inside host cells. Analysis of the T3SS2 pathogenicity island encoding the T3SS2 appeared to lack a mechanism for egress of this bacterium from the invaded host cell. Using a combination of molecular tools, we found that VPA0226, a constitutively secreted lipase, is required for escape of V. parahaemolyticus from the host cells. This lipase must be delivered into the host cytoplasm where it preferentially uses fatty acids associated with innate immune response to esterify cholesterol, weakening the plasma membrane and allowing egress of the bacteria. This study reveals the resourcefulness of microbes and the interplay between virulence systems and host cell resources to evolve an ingenious scheme for survival and escape.


Assuntos
Proteínas de Bactérias/metabolismo , Colesterol/metabolismo , Ácidos Graxos/metabolismo , Lipase/metabolismo , Vibrio parahaemolyticus/metabolismo , Esterificação , Ilhas Genômicas , Sistemas de Secreção Tipo III , Vibrio parahaemolyticus/enzimologia
17.
J Bacteriol ; 202(24)2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-32817090

RESUMO

Fluctuations in osmolarity are one of the most prevalent stresses to which bacteria must adapt, both hypo- and hyperosmotic conditions. Most bacteria cope with high osmolarity by accumulating compatible solutes (osmolytes) in the cytoplasm to maintain the turgor pressure of the cell. Vibrio parahaemolyticus, a halophile, utilizes at least six compatible solute transporters for the uptake of osmolytes: two ABC family ProU transporters and four betaine-carnitine-choline transporter (BCCT) family transporters. The full range of compatible solutes transported by this species has yet to be determined. Using an osmolyte phenotypic microarray plate for growth analyses, we expanded the known osmolytes used by V. parahaemolyticus to include N,N-dimethylglycine (DMG), among others. Growth pattern analysis of four triple-bccT mutants, possessing only one functional BCCT, indicated that BccT1 (VP1456), BccT2 (VP1723), and BccT3 (VP1905) transported DMG. BccT1 was unusual in that it could take up both compounds with methylated head groups (glycine betaine [GB], choline, and DMG) and cyclic compounds (ectoine and proline). Bioinformatics analysis identified the four coordinating amino acid residues for GB in the BccT1 protein. In silico modeling analysis demonstrated that GB, DMG, and ectoine docked in the same binding pocket in BccT1. Using site-directed mutagenesis, we showed that a strain with all four residues mutated resulted in the loss of uptake of GB, DMG, and ectoine. We showed that three of the four residues were essential for ectoine uptake, whereas only one of the residues was important for GB uptake. Overall, we have demonstrated that DMG is a highly effective compatible solute for Vibrio species and have elucidated the amino acid residues in BccT1 that are important for the coordination of GB, DMG, and ectoine transport.IMPORTANCE Vibrio parahaemolyticus possesses at least six osmolyte transporters, which allow the bacterium to adapt to high-salinity conditions. In this study, we identified several additional osmolytes that were utilized by V. parahaemolyticus We demonstrated that the compound DMG, which is present in the marine environment, was a highly effective osmolyte for Vibrio species. We determined that DMG is transported via BCCT family carriers, which have not been shown previously to take up this compound. BccT1 was a carrier for GB, DMG, and ectoine, and we identified the amino acid residues essential for the coordination of these compounds. The data suggest that for BccT1, GB is more easily accommodated than ectoine in the transporter binding pocket.


Assuntos
Diamino Aminoácidos/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sarcosina/análogos & derivados , Vibrio parahaemolyticus/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Betaína/metabolismo , Transporte Biológico , Carnitina/metabolismo , Colina/metabolismo , Regulação Bacteriana da Expressão Gênica , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Família Multigênica , Sarcosina/metabolismo , Vibrio parahaemolyticus/química , Vibrio parahaemolyticus/genética
18.
Food Microbiol ; 91: 103500, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32539983

RESUMO

The objective of this study was to investigate the antibacterial and antibiofilm activity of eugenol against V. parahaemolyticus planktonic and biofilm cells and the involved mechanisms as well. Atime-kill assay, a biofilm formation assay on the surface of crab shells, an assay to determine the reduction of virulence using eugenol at different concentrations, energy-filtered transmission electron microscope (EF-TEM), field emission scanning electron microscopy (FE-SEM), confocal laser scanning microscope (CLSM) and high-performance liquid chromatography (HPLC) were performed to evaluate the antibacterial and antibiofilm activity of eugenol. The results indicated that different concentrations of eugenol (0.1-0.6%) significantly reduced biofilm formation, metabolic activities, and secretion of extracellular polysaccharide (EPS), with effective antibacterial effect. Eugenol at 0.4% effectively eradicated the biofilms formed by clinical and environmental V. parahaemolyticus on crab surface by more than 4.5 and 4 log CFU/cm2, respectively. At 0.6% concentration, the reduction rates of metabolic activities for ATCC27969 and NIFS29 were 79% and 68%, respectively. Whereas, the reduction rates of EPS for ATCC27969 and NIFS29 were 78% and 71%, respectively. On visual evaluation, significant results were observed for biofilm reduction, live/dead cell detection, and quorum sensing (QS). This study demonstrated that eugenol can be used to control V. parahaemolyticus biofilms and biofilm-related infections and can be employed for the protection of seafood.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Eugenol/farmacologia , Vibrio parahaemolyticus/efeitos dos fármacos , Animais , Biofilmes/crescimento & desenvolvimento , Braquiúros/microbiologia , Microbiologia de Alimentos , Conservantes de Alimentos/farmacologia , Testes de Sensibilidade Microbiana , Polissacarídeos Bacterianos/metabolismo , Percepção de Quorum/efeitos dos fármacos , Frutos do Mar/microbiologia , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio parahaemolyticus/metabolismo , Vibrio parahaemolyticus/patogenicidade , Virulência/efeitos dos fármacos
19.
Nat Struct Mol Biol ; 27(6): 589-597, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32424347

RESUMO

The Vibrio parahaemolyticus T3SS effector VopQ targets host-cell V-ATPase, resulting in blockage of autophagic flux and neutralization of acidic compartments. Here, we report the cryo-EM structure of VopQ bound to the Vo subcomplex of the V-ATPase. VopQ inserts into membranes and forms an unconventional pore while binding directly to subunit c of the V-ATPase membrane-embedded subcomplex Vo. We show that VopQ arrests yeast growth in vivo by targeting the immature Vo subcomplex in the endoplasmic reticulum (ER), thus providing insight into the observation that VopQ kills cells in the absence of a functional V-ATPase. VopQ is a bacterial effector that has been discovered to inhibit a host-membrane megadalton complex by coincidentally binding its target, inserting into a membrane and disrupting membrane potential. Collectively, our results reveal a mechanism by which bacterial effectors modulate host cell biology and provide an invaluable tool for future studies on V-ATPase-mediated membrane fusion and autophagy.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vibrio parahaemolyticus/metabolismo , Proteínas de Bactérias/genética , Membrana Celular , Microscopia Crioeletrônica , Interações Hospedeiro-Patógeno , Modelos Moleculares , Conformação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , ATPases Vacuolares Próton-Translocadoras/genética
20.
Environ Microbiol ; 22(10): 4279-4294, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32219943

RESUMO

Bacteria constantly experience changes to their external milieu and need to adapt accordingly to ensure their survival. Certain bacteria adapt by means of cellular differentiation, resulting in the development of a specific cell type that is specialized for life in a distinct environment. Furthermore, to understand how bacteria adapt, it is essential to appreciate the significant changes that occur at the proteomic level. By analysing the proteome of our model organism Vibrio parahaemolyticus from distinct environmental conditions and cellular differential states, we demonstrate that the proteomic expression profile is highly flexible, which likely allows it to adapt to life in different environmental conditions and habitats. We show that, even within the same swarm colony, there are specific zones of cells with distinct expression profiles. Furthermore, our data indicate that cell surface attachment and swarmer cell differentiation are distinct programmes that require specific proteomic expression profiles. This likely allows V. parahaemolyticus to adapt to life in different environmental conditions and habitats. Finally, our analyses reveal that the expression profile of the essential protein pool is highly fluid, with significant fluctuations that dependent on the specific life-style, environment and differentiation state of the bacterium.


Assuntos
Aclimatação/fisiologia , Proteínas de Bactérias/metabolismo , Transcriptoma/genética , Vibrio parahaemolyticus/metabolismo , Adaptação Fisiológica/fisiologia , Proteínas de Bactérias/genética , Proteoma/análise , Proteômica , Vibrio parahaemolyticus/citologia , Vibrio parahaemolyticus/crescimento & desenvolvimento
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