Click
here to close Hello! We notice that
you are using Internet Explorer, which is not supported by Echinobase
and may cause the site to display incorrectly. We suggest using a
current version of Chrome,
FireFox,
or Safari.
Front Vet Sci
2023 Jan 01;10:1207831. doi: 10.3389/fvets.2023.1207831.
Show Gene links
Show Anatomy links
Vibrio splendidus Fur regulates virulence gene expression, swarming motility, and biofilm formation, affecting its pathogenicity in Apostichopus japonicus.
Shi Y
,
Liao C
,
Dai F
,
Zhang Y
,
Li C
,
Liang W
.
???displayArticle.abstract???
Vibrio splendidus is an opportunistic pathogen that causes skin ulcer syndrome and results in huge losses to the Apostichopus japonicus breeding industry. Ferric uptake regulator (Fur) is a global transcription factor that affects varieties of virulence-related functions in pathogenic bacteria. However, the role of the V. splendidus fur (Vsfur) gene in the pathogenesis of V. splendidus remains unclear. Hence, we constructed a Vsfur knock-down mutant of the V. splendidus strain (MTVs) to investigate the role of the gene in the effect of biofilm, swarming motility, and virulence on A. japonicus. The result showed that the growth curves of the wild-type V. splendidus strain (WTVs) and MTVs were almost consistent. Compared with WTVs, the significant increases in the transcription of the virulence-related gene Vshppd mRNA were 3.54- and 7.33-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. Similarly, compared with WTVs, the significant increases in the transcription of Vsm mRNA were 2.10- and 15.92-fold in MTVs at the OD600 of 1.0 and 1.5, respectively. On the contrary, the mRNA level of the flagellum assembly gene Vsflic was downregulated 0.56-fold in MTVs at the OD600 of 1.0 compared with the WTVs. MTVs caused delayed disease onset time and reduced A. japonicus mortality. The median lethal doses of WTVs and MTVs were 9.116 × 106 and 1.658 × 1011 CFU·ml-1, respectively. Compared with WTVs, the colonization abilities of MTVs to the muscle, intestine, tentacle, and coelomic fluid of A. japonicus were significantly reduced. Correspondingly, the swarming motility and biofilm formation in normal and iron-replete conditions were remarkably decreased compared with those of WTVs. Overall, these results demonstrate that Vsfur contributes to the pathogenesis of V. splendidus by regulating virulence-related gene expression and affecting its swarming and biofilm formation abilities.
Figure 1. Growth curves of WTVs, MTVs, and furC. WTVs, MTVs, and furC were spread onto 2216E solid plates at 28°C overnight. Three single colonies were inoculated into flasks with 100 ml of fresh 2216E medium and incubated at 28°C with shaking at 180 rpm. Overnight cultures were diluted to the same concentration, and 200 μl aliquots of WTVs, MTVs, and furC were transferred into flasks with 100 ml of fresh 2216E broth (A), 2216E broth supplemented with 50 μM FeCl3
(B), and 2216E broth supplemented with 100 μM iron chelator DP (C). OD600 values were measured at different time points.
Figure 2. (A) Antibiotic resistance test of WTVs, MTVs, and furC. WTVs and MTVs were spread onto 2216E solid plates at 28°C overnight. Single colonies were inoculated into tubes with 5 ml of fresh 2216E medium supplemented with Ap and Kn and incubated at 28°C with shaking at 180 rpm. (a) Negative control group, (b) WTVs group, and (c) MTVs group. furC was spread onto 2216E solid plates at 28°C overnight. Single colonies were inoculated into tubes with 5 ml of fresh 2216E medium supplemented with Ap, Kn and Gm and incubated at 28°C with shaking at 180 rpm. (d)
furC group. (B) Temporal expression analyses of Vsfur in WTVs or MTVs at the OD600 of 0.6, 1.0, and 1.5. Values are presented as mean ± SD (n = 5). Asterisks indicate significant differences: *p < 0.05 and **p < 0.01.
Figure 3. Temporal expression analyses of (A)
Vshppd, (B)
Vsm (The Y-axes were shown as a log scale), and (C)
Vsflic in WTVs or MTVs at the OD600 of 0.6, 1.0, and 1.5. Values are presented as mean ± SD (n = 5). Asterisks indicate significant differences: *p < 0.05 and **p < 0.01.
Figure 4. The analysis for Fur binding box prediction in the (A)
Vshppd, (B)
Vsm, and (C)
Vsflic promoter region. The putative Fur binding boxes are indicated as gray boxes. The transcriptional initiation site, the corresponding −10 and −35 boxes, and the translational start site are indicated in red letters. (D) Sequence alignment of these putative Fur binding boxes with the Fur box consensus sequence. Bases identical to the consensus are shown in red.
Figure 5. (A) The lethality of WTVs and MTVs. Apostichopus japonicus was randomly divided into seven tanks with 15 individuals each. The WTVs and MTVs strains used for infection were cultured in 2216E medium (24 h, 28°C) until OD600 was approximately 1.0. The strains were then washed and re-suspended in PBS (28°C). For survival assays, weight-matched A. japonicus individuals were infected with 1 × 107, 1 × 106, and 1 × 105 CFU·ml−1
V. splendidus (WTVs or MTVs). Apostichopus japonicus infected with PBS was used as the negative control. The water temperature during infection was 16°C. The daily mortality of infected A. japonicus was recorded. (B) The observed symptoms of (a) MTVs and (b) WTVs. Dead A. japonicus were removed in a timely manner and photographed to observe symptoms.
Figure 6. The colonization abilities of WTVs and MTVs to different A. japonicus tissues (A) and coelomic fluids (B) were demonstrated by colony counting. A. japonicus was soaked in WTVs and MTVs (1.0 × 107 CFU·ml−1) for 48 h infection. The A. japonicus tissues were weighed and homogenized. The coelomic fluids were filtered through a 200-mesh. The homogenized solution was diluted in gradients and then coated on 2216E plates.
Figure 7. (A) Swarming motility of WTVs, MTVs, and furC in normal conditions. (B) Bar graph of the swimming motility of WTVs, MTVs, and furC. (C) The same concentration of WTVs (a) and MTVs (b) (2 μl) were dropped on low-agar 2216E plates supplemented with 100 μM FeCl3 and cultured for 48 h at 28°C. (D) The same concentration of WTVs (c) and MTVs (d) (2 μl) was dropped on low-agar 2216E plates supplemented with 200 μM DP and cultured for 48 h at 28°C. The error line represents the SD (n = 5). Asterisks represent the significant difference (*p<0.05, **p<0.01).
Figure 8. (A) Biofilm formation of WTVs, MTVs, and furC in normal conditions. (B) Biofilm formation of WTVs and MTVs in iron-replete conditions and iron-starved conditions. The error line represents the SD (n = 5). Asterisks represent the significant difference (*p < 0.05, **p < 0.01).
Carpenter,
This is not your mother's repressor: the complex role of fur in pathogenesis.
2009, Pubmed
Carpenter,
This is not your mother's repressor: the complex role of fur in pathogenesis.
2009,
Pubmed
Cha,
Functional analysis of the role of Fur in the virulence of Pseudomonas syringae pv. tabaci 11528: Fur controls expression of genes involved in quorum-sensing.
2008,
Pubmed
Chen,
Iron redox pathway revealed in ferritin via electron transfer analysis.
2020,
Pubmed
Chen,
A unique NLRC4 receptor from echinoderms mediates Vibrio phagocytosis via rearrangement of the cytoskeleton and polymerization of F-actin.
2021,
Pubmed
,
Echinobase
Crosa,
Signal transduction and transcriptional and posttranscriptional control of iron-regulated genes in bacteria.
1997,
Pubmed
Dai,
Vibrio splendidus flagellin C binds tropomodulin to induce p38 MAPK-mediated p53-dependent coelomocyte apoptosis in Echinodermata.
2022,
Pubmed
,
Echinobase
Dai,
FliC of Vibrio splendidus-related strain involved in adhesion to Apostichopus japonicus.
2020,
Pubmed
,
Echinobase
Di Lorenzo,
Plasmid-Encoded Iron Uptake Systems.
2014,
Pubmed
Gao,
Direct Binding and Regulation by Fur and HapR of the Intermediate Regulator and Virulence Factor Genes Within the ToxR Virulence Regulon in Vibrio cholerae.
2020,
Pubmed
Gao,
Fur Represses Vibrio cholerae Biofilm Formation via Direct Regulation of vieSAB, cdgD, vpsU, and vpsA-K Transcription.
2020,
Pubmed
Guo,
secA, secD, secF, yajC, and yidC contribute to the adhesion regulation of Vibrio alginolyticus.
2018,
Pubmed
Hahn,
Regulation of the furA and catC operon, encoding a ferric uptake regulator homologue and catalase-peroxidase, respectively, in Streptomyces coelicolor A3(2).
2000,
Pubmed
Hantke,
Iron and metal regulation in bacteria.
2001,
Pubmed
Hoe,
Bacterial sRNAs: regulation in stress.
2013,
Pubmed
Hu,
Identification, characterization, and molecular application of a virulence-associated autotransporter from a pathogenic Pseudomonas fluorescens strain.
2009,
Pubmed
Kobayashi,
Pathological Roles of Iron in Cardiovascular Disease.
2018,
Pubmed
Lacoste,
A Vibrio splendidus strain is associated with summer mortality of juvenile oysters Crassostrea gigas in the Bay of Morlaix (North Brittany, France).
2001,
Pubmed
Liang,
Vibrio splendidus virulence to Apostichopus japonicus is mediated by hppD through glutamate metabolism and flagellum assembly.
2022,
Pubmed
,
Echinobase
Liu,
Ferric Uptake Regulator (FurA) is Required for Acidovorax citrulli Virulence on Watermelon.
2019,
Pubmed
Liu,
Identification of the pathogens associated with skin ulceration and peristome tumescence in cultured sea cucumbers Apostichopus japonicus (Selenka).
2010,
Pubmed
,
Echinobase
Livak,
Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.
2001,
Pubmed
Luo,
Time-resolved dual RNA-seq of tissue uncovers Pseudomonas plecoglossicida key virulence genes in host-pathogen interaction with Epinephelus coioides.
2020,
Pubmed
Luo,
flrA, flrB and flrC regulate adhesion by controlling the expression of critical virulence genes in Vibrio alginolyticus.
2016,
Pubmed
Mey,
Identification of the Vibrio cholerae enterobactin receptors VctA and IrgA: IrgA is not required for virulence.
2002,
Pubmed
Morales,
Targeting iron metabolism in cancer therapy.
2021,
Pubmed
Pajuelo,
Iron and Fur in the life cycle of the zoonotic pathogen Vibrio vulnificus.
2016,
Pubmed
Pasqua,
Ferric Uptake Regulator Fur Is Conditionally Essential in Pseudomonas aeruginosa.
2017,
Pubmed
Pohl,
Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator.
2003,
Pubmed
Porcheron,
Interplay between iron homeostasis and virulence: Fur and RyhB as major regulators of bacterial pathogenicity.
2015,
Pubmed
Sawabe,
Simple conjugation and outgrowth procedures for tagging vibrios with GFP, and factors affecting the stable expression of the gfp tag.
2006,
Pubmed
Shao,
Interplay between ferric uptake regulator Fur and horizontally acquired virulence regulator EsrB coordinates virulence gene expression in Edwardsiella piscicida.
2021,
Pubmed
Snoussi,
High potential of adhesion to abiotic and biotic materials in fish aquaculture facility by Vibrio alginolyticus strains.
2009,
Pubmed
Song,
Characteristics of the iron uptake-related process of a pathogenic Vibrio splendidus strain associated with massive mortalities of the sea cucumber Apostichopus japonicus.
2018,
Pubmed
,
Echinobase
Stoebner,
Iron-regulated hemolysin production and utilization of heme and hemoglobin by Vibrio cholerae.
1988,
Pubmed
Sun,
Sedoheptulose kinase bridges the pentose phosphate pathway and immune responses in pathogen-challenged sea cucumber Apostichopus japonicus.
2020,
Pubmed
,
Echinobase
Thomson,
Vibrio splendidus biotype 1 as a cause of mortalities in hatchery-reared larval turbot, Scophthalmus maximus (L.).
2005,
Pubmed
Troxell,
Transcriptional regulation by Ferric Uptake Regulator (Fur) in pathogenic bacteria.
2013,
Pubmed
Ushijima,
Influence of Chemotaxis and Swimming Patterns on the Virulence of the Coral Pathogen Vibrio coralliilyticus.
2018,
Pubmed
Wang,
Vibrio campbellii hmgA-mediated pyomelanization impairs quorum sensing, virulence, and cellular fitness.
2013,
Pubmed
Wing,
Regulation of IcsP, the outer membrane protease of the Shigella actin tail assembly protein IcsA, by virulence plasmid regulators VirF and VirB.
2004,
Pubmed
Yu,
Fur-mediated global regulatory circuits in pathogenic Neisseria species.
2012,
Pubmed
Zhang,
Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain.
2008,
Pubmed
