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1.
Nat Commun ; 12(1): 4723, 2021 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-34354064

RESUMO

Translational riboswitches are cis-acting RNA regulators that modulate the expression of genes during translation initiation. Their mechanism is considered as an RNA-only gene-regulatory system inducing a ligand-dependent shift of the population of functional ON- and OFF-states. The interaction of riboswitches with the translation machinery remained unexplored. For the adenine-sensing riboswitch from Vibrio vulnificus we show that ligand binding alone is not sufficient for switching to a translational ON-state but the interaction of the riboswitch with the 30S ribosome is indispensable. Only the synergy of binding of adenine and of 30S ribosome, in particular protein rS1, induces complete opening of the translation initiation region. Our investigation thus unravels the intricate dynamic network involving RNA regulator, ligand inducer and ribosome protein modulator during translation initiation.


Assuntos
Biossíntese de Proteínas , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ribossomos/genética , Ribossomos/metabolismo , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , Subunidades Ribossômicas Menores de Bactérias/química , Subunidades Ribossômicas Menores de Bactérias/genética , Subunidades Ribossômicas Menores de Bactérias/metabolismo , Ribossomos/química , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo
2.
Biochim Biophys Acta Gen Subj ; 1865(1): 129750, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32980502

RESUMO

BACKGROUND: The products of the lysine biosynthesis pathway, meso-diaminopimelate and lysine, are essential for bacterial survival. This paper focuses on the structural and mechanistic characterization of 4-hydroxy-tetrahydrodipicolinate reductase (DapB), which is one of the enzymes from the lysine biosynthesis pathway. DapB catalyzes the conversion of (2S, 4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate (HTPA) to 2,3,4,5-tetrahydrodipicolinate in an NADH/NADPH dependent reaction. Genes coding for DapBs were identified as essential for many pathogenic bacteria, and therefore DapB is an interesting new target for the development of antibiotics. METHODS: We have combined experimental and computational approaches to provide novel insights into mechanism of the DapB catalyzed reaction. RESULTS: Structures of DapBs originating from Mycobacterium tuberculosis and Vibrio vulnificus in complexes with NAD+, NADP+, as well as with inhibitors, were determined and described. The structures determined by us, as well as currently available structures of DapBs from other bacterial species, were compared and used to elucidate a mechanism of reaction catalyzed by this group of enzymes. Several different computational methods were used to provide a detailed description of a plausible reaction mechanism. CONCLUSIONS: This is the first report presenting the detailed mechanism of reaction catalyzed by DapB. GENERAL SIGNIFICANCE: Structural data in combination with information on the reaction mechanism provide a background for development of DapB inhibitors, including transition-state analogues.


Assuntos
Lisina/metabolismo , Mycobacterium tuberculosis/enzimologia , Oxirredutases/metabolismo , Tuberculose/microbiologia , Vibrioses/microbiologia , Vibrio vulnificus/enzimologia , Vias Biossintéticas , Domínio Catalítico , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Oxirredutases/química , Conformação Proteica , Especificidade por Substrato , Vibrio vulnificus/química , Vibrio vulnificus/metabolismo
3.
J Bacteriol ; 202(24)2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-32900828

RESUMO

Pathogenic Vibrio species use many different approaches to subvert, attack, and undermine the host response. The toxins they produce are often responsible for the devastating effects associated with their diseases. These toxins target a variety of host proteins, which leads to deleterious effects, including dissolution of cell organelle integrity and inhibition of protein secretion. Becoming increasingly prevalent as cofactors for Vibrio toxins are proteins of the small GTPase families. ADP-ribosylation factor small GTPases (ARFs) in particular are emerging as a common host cofactor necessary for full activation of Vibrio toxins. While ARFs are not the direct target of Vibrio cholerae cholera toxin (CT), ARF binding is required for its optimal activity as an ADP-ribosyltransferase. The makes caterpillars floppy (MCF)-like and the domain X (DmX) effectors of the Vibrio vulnificus multifunctional autoprocessing repeats-in-toxin (MARTX) toxin also both require ARFs to initiate autoprocessing and activation as independent effectors. ARFs are ubiquitously expressed in eukaryotes and are key regulators of many cellular processes, and as such they are ideal cofactors for Vibrio pathogens that infect many host species. In this review, we cover in detail the known Vibrio toxins that use ARFs as cross-kingdom activators to both stimulate and optimize their activity. We further discuss how these contrast to toxins and effectors from other bacterial species that coactivate, stimulate, or directly modify host ARFs as their mechanisms of action.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Toxinas Bacterianas/metabolismo , Vibrioses/enzimologia , Vibrio vulnificus/metabolismo , Fatores de Ribosilação do ADP/genética , Animais , Toxinas Bacterianas/genética , Interações Hospedeiro-Patógeno , Humanos , Família Multigênica , Vibrioses/genética , Vibrioses/microbiologia , Vibrio vulnificus/classificação , Vibrio vulnificus/genética
4.
Eur J Pharmacol ; 884: 173407, 2020 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-32735984

RESUMO

Vibrio vulnificus (V. vulnificus) infection, frequently resulting in fatal septicemia, has become a growing health concern worldwide. The present study aimed to explore the potential agents that could protect against V. vulnificus cytotoxicity, and to analyze the possible underlying mechanisms. First, we observed that 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid disodium salt hydrate (DIDS) significantly suppressed V. vulnificus cytotoxicity to host cells by using a lactate dehydrogenase (LDH) assay. DIDS did not exhibit any effect on host cell viability, bacterial growth, microbial adhesion and swarming motility. DIDS effectively lowered V. vulnificus RtxA1 toxin-induced calcium influx into host mitochondria and RtxA1 binding to host cells. To further elucidate the underlying mechanism, the synthesis and secretion of RtxA1 toxin were investigated by Western blotting. Intriguingly, DIDS selectively inhibited the secretion of RtxA1 toxin, but did not influence its synthesis. Consequently, the outer membrane portal TolC, a key conduit for RtxA1 export coupled with tripartite efflux pumps, was examined by RT-PCR and Western blotting. We found that DIDS significantly reduced the expression of TolCV1 protein at the transcriptional level. Taken together, these results suggest that DIDS is a promising new paradigm as an antimicrobial drug that targets TolC-mediated toxin.


Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Toxinas Bacterianas/metabolismo , Vibrioses/tratamento farmacológico , Vibrio vulnificus/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Transcrição Genética , Vibrioses/microbiologia , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidade , Fatores de Virulência/metabolismo
5.
Environ Microbiol ; 22(10): 4381-4393, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32515159

RESUMO

With the rapid increase of aquaculture contributing to sustainable food security, comes the need to better understand seafood associated diseases. One of the major aquatic bacterial genera responsible for human infections from seafood is Vibrio, especially from oysters. Currently, in vivo study of bacterial interactions within oysters is limited by the inability to promote high-level uptake of bacteria by oysters. This study has therefore evolved current natural marine snow protocols to generate 'artificial' marine snow, into which bacteria can be incorporated to facilitate extensive uptake by oysters. This presents an adaptable model for bacterial study within filter-feeding shellfish. Using this model, we demonstrate for the first time the antibacterial activity of Vibrio vulnificus Type 6 secretion systems in vivo, revealing an important role for the T6SS in V. vulnificus ecology.


Assuntos
Antibacterianos/farmacologia , Ostreidae/microbiologia , Sistemas de Secreção Tipo VI/metabolismo , Vibrio vulnificus/metabolismo , Animais , Doenças Transmitidas por Alimentos/microbiologia , Humanos , Alimentos Marinhos/microbiologia , Frutos do Mar/microbiologia
6.
Appl Microbiol Biotechnol ; 104(15): 6791-6798, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32533306

RESUMO

Cyclo(Phe-Pro) (cFP), produced by the Vibrio species, plays the dual roles of being a signaling molecule and a virulence factor. Acting modes of this compound have recently been characterized at the molecular level. Nevertheless, the method by which this compound passes across biological membranes remains obscure. Using radiolabeled cFP, we examined the kinetics of transport for this compound across membranes using V. vulnificus, Escherichia coli, and sheep red blood cells. We observed that cFP was taken up by these cells in a concentration-dependent manner and was not affected by the addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that cFP is taken up by passive transport. The kinetics of uptake of cFP by the above three types of cells revealed no significant differences, indicating that no specific protein is involved in this process. When the intracellular accumulation of cFP in the tested cells was measured, the concentrations did not exhibit significant differences between the 1-min and 10-min time points after cFP was added to the culture. In contrast, the intracellular concentration of fumarate, which is well known to be taken up by cells via active transport, was significantly higher at the 10-min than at the 1-min time point after addition. Taken together, this study shows that cFP is a diffusible molecule that does not require energy for transportation across biological membranes, and that cFP does not need membrane machinery in order to cross membranes and consequently act as a virulence factor or signal. KEY POINTS: • Kinetics of cFP uptake into cells of V. vulnificus, E. coli, or RBS was studied. • The uptake was not saturated and required no energy, indicating passive transport. • The lack of cell specificity in cFP uptake means no specific protein is needed. • Therefore, the cFP moves across the biological membrane by simple diffusion.


Assuntos
Membrana Celular/metabolismo , Dipeptídeos/metabolismo , Peptídeos Cíclicos/metabolismo , Vibrio vulnificus/metabolismo , Animais , Transporte Biológico , Difusão , Eritrócitos/metabolismo , Escherichia coli/metabolismo , Fumaratos/análise , Fumaratos/metabolismo , Espaço Intracelular/química , Cinética , Ovinos , Fatores de Virulência/metabolismo
7.
J Microbiol Biotechnol ; 30(6): 830-838, 2020 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-32238773

RESUMO

The histone-like nucleoid structuring protein (H-NS) is an abundant global regulator of environmentally controlled gene expression. Herein, we demonstrate that H-NS represses the expression of LeuO, the master regulator of the cyclic(Phe-Pro)-dependent signaling pathway, by directly binding to the upstream region of the gene. H-NS binds to a long stretched region (more than 160-bp long), which overlaps with binding sites for ToxR and LeuO. A high quantity of H-NS outcompetes ToxR for binding to the cis-acting element of leuO. However, our footprinting analyses suggests that the binding of H-NS is relatively weaker than LeuO or ToxR at the same molarity. Considering that the DNA nucleotide sequences of the upstream regions of leuO genes are highly conserved among various Vibrio, such patterns as those found in V. vulnificus would be a common feature in the regulation of leuO gene expression in Vibrionaceae. Taken together, these results suggest that, in species belonging to Vibrionaceae, H-NS regulates the expression of leuO as a basal stopper when cFP-ToxR mediated signaling is absent.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Dipeptídeos/genética , Peptídeos Cíclicos/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Vibrio vulnificus/genética , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Dipeptídeos/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Peptídeos Cíclicos/metabolismo , Fatores de Transcrição/metabolismo , Vibrio vulnificus/metabolismo
8.
BMC Microbiol ; 20(1): 69, 2020 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-32228455

RESUMO

BACKGROUND: Vibrio vulnificus hemolysin (VVH) is a pore-forming toxin secreted by Vibrio vulnificus. Cellular cholesterol was believed to be the receptor for VVH, because cholesterol could bind to VVH and preincubation with cholesterol inhibited cytotoxicity. It has been reported that specific glycans such as N-acetyl-D-galactosamine and N-acetyl-D-lactosamine bind to VVH, however, it has not been known whether these glycans could inhibit the cytotoxicity of VVH without oligomer formation. Thus, to date, binding mechanisms of VVH to cellular membrane, including specific receptors have not been elucidated. RESULTS: We show here that VVH associates with ganglioside GM1a, Fucosyl-GM1, GD1a, GT1c, and GD1b by glycan array. Among them, GM1a could pulldown VVH. Moreover, the GD1a inhibited the cytotoxicity of VVH without the formation of oligomers. CONCLUSION: This is the first report of a molecule able to inhibit the binding of VVH to target cells without oligomerization of VVH.


Assuntos
Membrana Celular/metabolismo , Gangliosídeos/farmacologia , Proteínas Hemolisinas/metabolismo , Vibrio vulnificus/patogenicidade , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Sítios de Ligação/efeitos dos fármacos , Células CHO , Colesterol/metabolismo , Cricetulus , Glicômica/métodos , Proteínas Hemolisinas/química , Análise em Microsséries , Ligação Proteica/efeitos dos fármacos , Conformação Proteica , Multimerização Proteica/efeitos dos fármacos , Vibrio vulnificus/metabolismo
9.
Biochemistry ; 59(10): 1081-1086, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32134253

RESUMO

The adenine-sensing riboswitch from the Gram-negative bacterium Vibrio vulnificus is an RNA-based gene regulatory element that acts in response to both its cognate low-molecular weight ligand and temperature. The combined sensitivity to environmental temperature and ligand concentration is maintained by an equilibrium of three distinct conformations involving two ligand-free states and one ligand-bound state. The key structural element that undergoes refolding in the ligand-free states comprises a 35-nucleotide temperature response module. Here, we present the structural characterization of this temperature response module. We employ high-resolution NMR spectroscopy and photocaged RNAs as molecular probes to decipher the kinetic and thermodynamic framework of the secondary structure transition in the apo state of the riboswitch. We propose a model for the transition state adopted during the thermal refolding of the temperature response module that connects two mutually exclusive long-lived and stable conformational states. This transition state is characterized by a comparatively low free activation enthalpy. A pseudoknot conformation in the transition state, as commonly seen in RNA refolding, is therefore unlikely. More likely, the transition state of the adenine-sensing riboswitch temperature response module features a linear conformation.


Assuntos
Riboswitch/genética , Riboswitch/fisiologia , Vibrio vulnificus/química , Aclimatação , Aptâmeros de Nucleotídeos/metabolismo , Cinética , Ligantes , Espectroscopia de Ressonância Magnética/métodos , Modelos Moleculares , Conformação de Ácido Nucleico , Dobramento de RNA/fisiologia , RNA Bacteriano/química , Temperatura , Termodinâmica , Vibrio vulnificus/metabolismo
10.
Cells ; 9(3)2020 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-32151068

RESUMO

Curcumin, a hydrophobic polyphenol of turmeric, has a variety of biological functions as a herbal supplement, but its poor gastric absorption rate is one of the major factors limiting its oral bioavailability. In the present study, we have investigated the functional role of a nanosphere loaded with curcumin (CN) during host cell death elicited by the Gram-negative bacterium V. vulnificus in human gastrointestinal epithelial HT-29 cells and an ileal-ligated mouse model. The recombinant protein (r) VvhA produced by V. vulnificus significantly reduced the viability of HT-29 cells. The cytotoxic effect of rVvhA was restored upon a treatment with CN (100 ng/mL), which had shown 1000-fold higher anti-apoptotic efficacy than curcumin. CN inhibited the phosphorylation of c-Src and PKC mediated by intracellular ROS responsible for the distinctive activation of the MAPKs in rVvhA-treated HT-29 cells. Interestingly, CN significantly restored the expression of Bax, Bcl-2, and cleaved caspase-3 as regulated by the phosphorylation of NF-κB. In mouse models of V. vulnificus infection, treatment with CN had a blocking effect that elevated the levels of TUNEL-positive DNA fragmentation and apoptosis-related proteins. These results indicate that CN is a functional agent that manipulates the V. vulnificus VvhA signaling pathway responsible for gastrointestinal cell death.


Assuntos
Morte Celular/efeitos dos fármacos , Curcumina/farmacologia , Nanosferas/uso terapêutico , Vibrio vulnificus/metabolismo , Apoptose/efeitos dos fármacos , Proteínas de Bactérias/genética , Curcumina/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , NF-kappa B/metabolismo , Proteínas Recombinantes/metabolismo , Vibrio vulnificus/genética
11.
J Biol Chem ; 295(16): 5350-5361, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32169898

RESUMO

For successful infection of their hosts, pathogenic bacteria recognize host-derived signals that induce the expression of virulence factors in a spatiotemporal manner. The fulminating food-borne pathogen Vibrio vulnificus produces a cytolysin/hemolysin protein encoded by the vvhBA operon, which is a virulence factor preferentially expressed upon exposure to murine blood and macrophages. The Fe-S cluster containing transcriptional regulator IscR activates the vvhBA operon in response to nitrosative stress and iron starvation, during which the cellular IscR protein level increases. Here, electrophoretic mobility shift and DNase I protection assays revealed that IscR directly binds downstream of the vvhBA promoter P vvhBA , which is unusual for a positive regulator. We found that in addition to IscR, the transcriptional regulator HlyU activates vvhBA transcription by directly binding upstream of P vvhBA , whereas the histone-like nucleoid-structuring protein (H-NS) represses vvhBA by extensively binding to both downstream and upstream regions of its promoter. Of note, the binding sites of IscR and HlyU overlapped with those of H-NS. We further substantiated that IscR and HlyU outcompete H-NS for binding to the P vvhBA regulatory region, resulting in the release of H-NS repression and vvhBA induction. We conclude that concurrent antirepression by IscR and HlyU at regions both downstream and upstream of P vvhBA provides V. vulnificus with the means of integrating host-derived signal(s) such as nitrosative stress and iron starvation for precise regulation of vvhBA transcription, thereby enabling successful host infection.


Assuntos
Regulação Bacteriana da Expressão Gênica , Ferro/deficiência , Nitrogênio/metabolismo , Óperon , Estresse Fisiológico , Vibrio vulnificus/genética , Animais , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Células Cultivadas , Ferro/metabolismo , Camundongos , Regiões Promotoras Genéticas , Células RAW 264.7 , Fatores de Transcrição/metabolismo , Vibrio vulnificus/metabolismo
12.
J Biomed Sci ; 27(1): 21, 2020 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-31906951

RESUMO

BACKGROUND: Melatonin (5-methoxy-N-acetyltryptamine), a hormone produced in the pineal gland, has a variety of biological functions as an antioxidant, but a functional role of melatonin in the regulation of intestinal mucin (Muc) production during bacterial infection has yet to be described in detail. In this study, we investigate the effects of melatonin during Muc2 repression elicited by the Gram-negative bacterium V. vulnificus. METHODS: Mucus-secreting human HT29-MTX cells were used to study the functional role of melatonin during Muc2 depletion induced by the recombinant protein (r) VvpM produced by V. vulnificus. The regulatory effects of melatonin coupling with melatonin receptor 2 (MT2) on the production of reactive oxygen species (ROS), the activation of PKCδ and ERK, and the hypermethylation of the Muc2 promoter as induced by rVvpM were examined. Experimental mouse models of V. vulnificus infection were used to study the role of melatonin and how it neutralizes the bacterial toxin activity related to Muc2 repression. RESULTS: Recombinant protein (r) VvpM significantly reduced the level of Muc2 in HT29-MTX cells. The repression of Muc2 induced by rVvpM was significantly restored upon a treatment with melatonin (1 µM), which had been inhibited by the knockdown of MT2 coupling with Gαq and the NADPH oxidase subunit p47 phox. Melatonin inhibited the ROS-mediated phosphorylation of PKCδ and ERK responsible for region-specific hypermethylation in the Muc2 promoter in rVvpM-treated HT29-MTX cells. In the mouse models of V. vulnificus infection, treatment with melatonin maintained the level of Muc2 expression in the intestine. In addition, the mutation of the VvpM gene from V. vulnificus exhibited an effect similar to that of melatonin. CONCLUSIONS: These results demonstrate that melatonin acting on MT2 inhibits the hypermethylation of the Muc2 promoter to restore the level of Muc2 production in intestinal epithelial cells infected with V. vulnificus.


Assuntos
Toxinas Bacterianas/metabolismo , Metilação de DNA , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Melatonina/farmacologia , Mucina-2/biossíntese , Receptor MT2 de Melatonina/metabolismo , Vibrioses/metabolismo , Vibrio vulnificus/metabolismo , Animais , Toxinas Bacterianas/farmacologia , Células HT29 , Humanos , Camundongos , Vibrioses/patologia
13.
Microbiologyopen ; 9(1): e00947, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31595707

RESUMO

In gram-negative bacteria, energy-dependent active transport of iron-bound substrates across the outer membrane is achieved through the TonB systems of proteins. Three TonB systems have been identified in the human pathogen Vibrio vulnificus. The TonB1 system contains three proteins: TonB1, ExbB1, and ExbD1. Both the TonB2 and TonB3 systems have been shown to also contain a fourth protein, TtpC2 and TtpC3, respectively. Here, we report and begin to characterize two additional proteins in the TonB2 and TonB3 systems: TtpB and TtpD. Both TtpB2 and TtpD2 are absolutely required for the function of the TonB2 system in V. vulnificus. However, although both TtpB3 and TtpD3 in the TonB3 system are related to the proteins in the TonB2 system, neither are active in iron transport. All six protein components of the TonB2 system-TonB2, ExbB2, ExbD2, TtpB2, TtpC2, and TtpD2-are essential for the uptake of both endogenously produced iron-bound siderophores and exogenous siderophores produced from other organisms. Through complementation, we have shown that V. vulnificus is capable of using different TtpD2 proteins from other Vibrio species to bring in multiple siderophores. In contrast, we also demonstrate that TtpB2 must come from V. vulnificus, and not other species within the genus, to complement mutations in the TonB2 system.


Assuntos
Proteínas de Bactérias/genética , Transporte Biológico/genética , Ferro/metabolismo , Proteínas de Membrana/genética , Sideróforos/genética , Vibrio vulnificus/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Transporte Biológico/fisiologia , Proteínas de Membrana/metabolismo , Alinhamento de Sequência , Sideróforos/metabolismo , Vibrio vulnificus/metabolismo , Virulência
14.
Folia Microbiol (Praha) ; 65(2): 265-274, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31840198

RESUMO

Due to the development of Marine aquaculture, infections caused by Vibrio vulnificus are common all over the world. Symptoms of V. vulnificus infection vary from gastrointestinal illness to septicemia. After infection with V. vulnificus, some patients showed gastrointestinal symptoms, including vomiting, fever, diarrhea, and so on. Others appeared wound infection at the site of contact with bacteria, and even developed sepsis. Once it develops into sepsis, the prognosis of patients is very poor. However, its underlying pathogenic mechanism remains largely undetermined. Growing evidence shows that it can induce primary septicemia mainly via essential virulence factors and regulators. Therefore, it is important to identify the factors that play roles in sepsis. In this review, we systematically expounded the role of V. vulnificus virulence factors and regulators in its infection-induced sepsis in order to provide useful information for the treatment and prevention of V. vulnificus.


Assuntos
Proteínas de Bactérias/metabolismo , Sepse/microbiologia , Vibrioses/microbiologia , Vibrio vulnificus/metabolismo , Fatores de Virulência/metabolismo , Animais , Proteínas de Bactérias/genética , Humanos , Vibrio vulnificus/genética , Fatores de Virulência/genética
15.
Cell Microbiol ; 22(2): e13133, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31658406

RESUMO

Studies have successfully elucidated the mechanism of action of several effector domains that comprise the multifunctional-autoprocessing repeats-in-toxins (MARTX) toxins of Vibrio vulnificus. However, the biochemical linkage between the cysteine proteolytic activity of Makes Caterpillars Floppy (MCF)-like effector and its cellular effects remains unknown. In this study, we identify the host cell factors that activate in vivo and in vitro MCF autoprocessing as adenosine diphosphate (ADP)-Ribosylation Factor 1 (ARF1) and ADP-Ribosylation Factor 3 (ARF3). Autoprocessing activity is enhanced when ARF1 is in its active [guanosine triphosphate (GTP)-bound] form compared to the inactive [guanosine diphosphate (GDP)-bound] form. Subsequent to auto-cleavage, MCF is acetylated on its exposed N-terminal glycine residue. Acetylation apparently does not dictate subcellular localization as MCF is found localized throughout the cell. However, the cleaved form of MCF gains the ability to bind to the specialized lipid phosphatidylinositol 5-phosphate enriched in Golgi and other membranes necessary for endocytic trafficking, suggesting that a fraction of MCF may be subcellularly localized. Traditional thin-section electron microscopy, high-resolution cryoAPEX localization, and fluorescent microscopy show that MCF causes Golgi dispersal resulting in extensive vesiculation. In addition, host mitochondria are disrupted and fragmented. Mass spectrometry analysis found no reproducible modifications of ARF1 suggesting that ARF1 is not post-translationally modified by MCF. Further, catalytically active MCF does not stably associate with ARF1. Our data indicate not only that ARF1 is a cross-kingdom activator of MCF, but also that MCF may mediate cytotoxicity by directly targeting another yet to be identified protein. This study begins to elucidate the biochemical activity of this important domain and gives insight into how it may promote disease progression.


Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Toxinas Bacterianas/metabolismo , Complexo de Golgi/metabolismo , Vibrio vulnificus/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Processamento de Proteína Pós-Traducional , Transporte Proteico
16.
Proc Natl Acad Sci U S A ; 116(36): 18031-18040, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31427506

RESUMO

Upon invading target cells, multifunctional autoprocessing repeats-in-toxin (MARTX) toxins secreted by bacterial pathogens release their disease-related modularly structured effector domains. However, it is unclear how a diverse repertoire of effector domains within these toxins are processed and activated. Here, we report that Makes caterpillars floppy-like effector (MCF)-containing MARTX toxins require ubiquitous ADP-ribosylation factor (ARF) proteins for processing and activation of intermediate effector modules, which localize in different subcellular compartments following limited processing of holo effector modules by the internal cysteine protease. Effector domains structured tandemly with MCF in intermediate modules become disengaged and fully activated by MCF, which aggressively interacts with ARF proteins present at the same location as intermediate modules and is converted allosterically into a catalytically competent protease. MCF-mediated effector processing leads ultimately to severe virulence in mice via an MCF-mediated ARF switching mechanism across subcellular compartments. This work provides insight into how bacteria take advantage of host systems to induce systemic pathogenicity.


Assuntos
Fatores de Ribosilação do ADP , ADP-Ribosilação , Toxinas Bacterianas , Vibrio vulnificus , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Células HEK293 , Células HeLa , Humanos , Camundongos , Domínios Proteicos , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Vibrio vulnificus/patogenicidade
17.
Emerg Microbes Infect ; 8(1): 934-945, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31237474

RESUMO

Cytoskeletal rearrangement and acute cytotoxicity occur in Vibrio vulnificus-infected host cells. RtxA1 toxin, a multifunctional autoprocessing repeats-in-toxin (MARTX), is essential for the pathogenesis of V. vulnificus and the programmed necrotic cell death. In this study, HeLa cells expressing RtxA1 amino acids 1491-1971 fused to GFP were observed to be rounded. Through yeast two-hybrid screening and subsequent immunoprecipitation validation assays, we confirmed the specific binding of a RtxA11491-1971 fragment with host-cell filamin A, an actin cross-linking scaffold protein. Downregulation of filamin A expression decreased the cytotoxicity of RtxA1 toward host cells. Furthermore, the phosphorylation of JNK and p38 MAPKs was induced by the RtxA1-filamin A interaction during the toxin-mediated cell death. However, the phosphorylation of these MAPKs was not observed during the RtxA1 intoxication of filamin A-deficient M2 cells. In addition, the depletion of pak1, which appeared to be activated by the RtxA1-filamin A interaction, inhibited RtxA1-induced phosphorylation of JNK and p38, and the cells treated with a pak1 inhibitor exhibited decreased RtxA1-mediated cytoskeletal rearrangement and cytotoxicity. Thus, the binding of filamin A by the RtxA11491-1971 domain appears to be a requisite to pak1-mediated MAPK activation, which contributes to the cytoskeletal reorganization and host cell death.


Assuntos
Toxinas Bacterianas/metabolismo , Citoesqueleto/metabolismo , Filaminas/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Vibrioses/metabolismo , Vibrio vulnificus/metabolismo , Quinases Ativadas por p21/metabolismo , Motivos de Aminoácidos , Toxinas Bacterianas/toxicidade , Morte Celular , Citoesqueleto/genética , Filaminas/genética , Células HeLa , Interações Hospedeiro-Patógeno , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Ligação Proteica , Vibrioses/genética , Vibrioses/microbiologia , Vibrioses/fisiopatologia , Vibrio vulnificus/química , Vibrio vulnificus/genética , Quinases Ativadas por p21/genética
18.
PLoS Pathog ; 15(5): e1007731, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31083688

RESUMO

The type II secretion system (T2SS) is a cell envelope-spanning macromolecular complex that is prevalent in Gram-negative bacterial species. It serves as the predominant virulence mechanism of many bacteria including those of the emerging human pathogens Vibrio vulnificus and Aeromonas hydrophila. The system is composed of a core set of highly conserved proteins that assemble an inner membrane platform, a periplasmic pseudopilus and an outer membrane complex termed the secretin. Localization and assembly of secretins in the outer membrane requires recognition of secretin monomers by two different partner systems: an inner membrane accessory complex or a highly sequence-diverse outer membrane lipoprotein, termed the pilotin. In this study, we addressed the question of differential secretin assembly mechanisms by using cryo-electron microscopy to determine the structures of the secretins from A. hydrophila (pilotin-independent ExeD) and V. vulnificus (pilotin-dependent EpsD). These structures, at approximately 3.5 Å resolution, reveal pentadecameric stoichiometries and C-terminal regions that carry a signature motif in the case of a pilotin-dependent assembly mechanism. We solved the crystal structure of the V. vulnificus EpsS pilotin and confirmed the importance of the signature motif for pilotin-dependent secretin assembly by performing modelling with the C-terminus of EpsD. We also show that secretin assembly is essential for membrane integrity and toxin secretion in V. vulnificus and establish that EpsD requires the coordinated activity of both the accessory complex EpsAB and the pilotin EpsS for full assembly and T2SS function. In contrast, mutation of the region of the S-domain that is normally the site of pilotin interactions has little effect on assembly or function of the ExeD secretin. Since secretins are essential outer membrane channels present in a variety of secretion systems, these results provide a structural and functional basis for understanding the key assembly steps for different members of this vast pore-forming family of proteins.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Lipoproteínas/metabolismo , Secretina/química , Sistemas de Secreção Tipo II/química , Vibrio vulnificus/metabolismo , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/química , Microscopia Crioeletrônica , Cristalografia por Raios X , Lipoproteínas/química , Modelos Moleculares , Conformação Proteica , Secretina/metabolismo , Homologia de Sequência , Sistemas de Secreção Tipo II/metabolismo , Vibrio vulnificus/crescimento & desenvolvimento
19.
Mol Microbiol ; 112(1): 266-279, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31058375

RESUMO

How motile bacteria recognize their environment and decide whether to stay or navigate toward more favorable location is a fundamental issue in survival. The flagellum is an elaborate molecular device responsible for bacterial locomotion, and the flagellum-driven motility allows bacteria to move themselves to the appropriate location at the right time. Here, we identify the polar landmark protein HubP as a modulator of polar flagellation that recruits the flagellar assembly protein FapA to the old cell pole, thereby controlling its activity for the early events of flagellar assembly in Vibrio vulnificus. We show that dephosphorylated EIIAGlc of the PEP-dependent sugar transporting phosphotransferase system sequesters FapA from HubP in response to glucose and hence inhibits FapA-mediated flagellation. Thus, flagellar assembly and motility is governed by spatiotemporal control of FapA, which is orchestrated by the competition between dephosphorylated EIIAGlc and HubP, in the human pathogen V. vulnificus.


Assuntos
Quimiotaxia/fisiologia , Flagelos/metabolismo , Vibrio vulnificus/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Polaridade Celular/genética , Polaridade Celular/fisiologia , Quimiotaxia/genética , Flagelos/fisiologia , Regulação Bacteriana da Expressão Gênica/genética , Glucose/metabolismo , Vibrio vulnificus/genética
20.
Environ Microbiol Rep ; 11(4): 581-588, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31102321

RESUMO

The Roseobacter group is a widespread marine bacterial group, of which some species produce the broad-spectrum antibiotic tropodithietic acid (TDA). A mode of action for TDA has previously been proposed in Escherichia coli, but little is known about its effect on non-producing marine bacteria at in situ concentrations. The purpose of this study was to investigate how a sub-lethal level of TDA affects Vibrio vulnificus at different time points (30 and 60 min) using a transcriptomic approach. Exposure to TDA for as little as 30 min resulted in the differential expression of genes associated with cell regeneration, including the up-regulation of those involved in biogenesis of the cell envelope. Defence mechanisms including oxidative stress defence proteins and iron uptake systems were also up-regulated in response to TDA, while motility-related genes were down-regulated. Gene expression data and scanning electron microscopy imaging revealed a switch to a biofilm phenotype in the presence of TDA. Our study shows that a low concentration of this antibiotic triggers a defence response to reactive oxygen species and iron depletion in V. vulnificus, which indicates that the mode of action of TDA is likely more complex in this bacterium than what is known for E. coli.


Assuntos
Antibacterianos/farmacologia , Expressão Gênica/efeitos dos fármacos , Tropolona/análogos & derivados , Vibrio vulnificus/efeitos dos fármacos , Proteínas de Bactérias/genética , Biofilmes/crescimento & desenvolvimento , Transporte Biológico/genética , Membrana Celular/metabolismo , Parede Celular/metabolismo , Perfilação da Expressão Gênica , Ferro/metabolismo , Estresse Oxidativo/genética , Tropolona/farmacologia , Vibrio vulnificus/genética , Vibrio vulnificus/metabolismo , Vibrio vulnificus/ultraestrutura
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