| 1. |
- Rivera, L., et al.
(författare)
-
Effective qPCR methodology to quantify the expression of virulence genes in Aeromonas salmonicida subsp salmonicida
- 2015
-
Ingår i: Journal of Applied Microbiology. - : Oxford University Press (OUP). - 1364-5072 .- 1365-2672. ; 118:4, s. 792-802
-
Tidskriftsartikel (refereegranskat)abstract
- Aims This study aimed to select and validate different methodological strategies to quantify the expression of the virulence genes ascC and ascV by qPCR in Aeromonas salmonicida subsp. salmonicida (Aer. salmonicida). Methods and Results Using the geNorm, Normfinder and BestKeeper algorithms, reference genes for the qPCR were selected based on their in vitro expression stabilities in three Aer. salmonicida strains. Gene amplification efficiency was calculated by Real-time PCR Miner and LinReg PCR programmes, which have not been used previously in the analysis of bacterial gene expression. The expression of the ascC and ascV virulence genes in a virulent Aer. salmonicida strain was evaluated by three quantification models, including single (least or most stable) or three most stable reference genes, combined with constant or specific gene amplification efficiency. The most stable reference genes were gyrB, proC and rpoC, while rpoD and fabD were the least stable. Quantification models showed different expression patterns. Conclusions The optimal strategy to quantify mRNA expression was to use a combination of the three algorithms and the quantification model including the three most stable reference genes. Real-time PCR Miner or LinReg PCR were valuable tools to estimate amplification efficiency. Significance and Impact of the Study The methods used in this study gave more reliable expression data using qPCR than previously published methods. The quantification and expression dynamics of virulence genes will contribute to a better understanding of how Aer. salmonicida interacts with its host and the environment, and therefore to the prevention of epizootics due to this pathogen.
|
|
| 2. |
- Akram, Neelam, et al.
(författare)
-
Regulation of proteorhodopsin gene expression by nutrient limitation in the marine bacterium Vibrio sp AND4
- 2013
-
Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 15:5, s. 1400-1415
-
Tidskriftsartikel (refereegranskat)abstract
- Proteorhodopsin (PR), a ubiquitous membrane photoprotein in marine environments, acts as a light-driven proton pump and can provide energy for bacterial cellular metabolism. However, knowledge of factors that regulate PR gene expression in different bacteria remains strongly limited. Here, experiments with Vibrio sp. AND4 showed that PR phototrophy promoted survival only in cells from stationary phase and not in actively growing cells. PR gene expression was tightly regulated, with very low values in exponential phase, a pronounced peak at the exponential/stationary phase intersection, and a marked decline in stationary phase. Thus, PR gene expression at the entry into stationary phase preceded, and could therefore largely explain, the stationary phase light-induced survival response in AND4. Further experiments revealed nutrient limitation, not light exposure, regulated this differential PR expression. Screening of available marine vibrios showed that the PR gene, and thus the potential for PR phototrophy, is found in at least three different clusters in the genus Vibrio. In an ecological context, our findings suggest that some PR-containing bacteria adapted to the exploitation of nutrient-rich micro-environments rely on a phase of relatively slowly declining resources to mount a cellular response preparing them for adverse conditions dispersed in the water column.
|
|
| 3. |
- Bobay, Benjamin G, et al.
(författare)
-
Chemical shift assignments and secondary structure prediction of the phosphorelay protein VanU from Vibrio anguillarum
- 2014
-
Ingår i: Biomolecular NMR Assignments. - : Springer Netherlands. - 1874-2718 .- 1874-270X. ; 8:1, s. 177-179
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum is a biofilm forming Gram-negative bacterium that survives prolonged periods in seawater and causes vibriosis in marine life. A quorum-sensing signal transduction pathway initiates biofilm formation in response to environmental stresses. The phosphotransferase protein VanU is the focal point of the quorum-sensing pathway and facilitates the regulation between independent phosphorelay systems that activate or repress biofilm formation. Here we report the (1)H, (13)C, and (15)N backbone and side chain resonance assignments and secondary structure prediction for VanU from V. anguillarum.
|
|
| 4. |
- Buchholtz, Christiane, et al.
(författare)
-
Profiling of acylated homoserine lactones of Vibrio anguillarum in vitro and in vivo : Influence of growth conditions and serotype.
- 2006
-
Ingår i: Syst Appl Microbiol. - 0723-2020. ; 29:6, s. 433-45
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum produces several interlinked acylated homoserine lactone (AHL) signal molecules which may influence expression of its virulence factors such as exoprotease production and biofilm formation. Using both thin layer chromatography and HPLC-high resolution mass spectrometry (HPLC-HRMS), we demonstrate in this study that the same types of AHLs are produced by many serotypes of V. anguillarum and that altering in vitro growth conditions (salinity, temperature and iron concentration) has little influence on the AHL-profile. Most strains produced N-(3-oxodecanoyl)-l-homoserine lactone (3-oxo-C10-HSL) and N-(3-hydroxy-hexanoyl)-l-homoserine lactone (3-hydroxy-C6-HSL) as the dominant molecules. Also, two spots with AHL activity appeared on TLC plates, which could not be identified as AHL structures. Trace amounts of N-(3-hydroxy-octanoyl)-l-homoserine lactone, N-(3-hydroxy-decanoyl)-l-homoserine lactone and N-(3-hydroxy-dodecanoyl)-l-homoserine lactone (3-hydroxy-C8-HSL, 3-hydroxy-C10-HSL and 3-oxo-C12-HSL, respectively) were also detected by HPLC-HRMS analysis from in vitro cultures. Most studies of quorum sensing (QS) systems have been conducted in vitro, the purpose of our study was to determine if the same acylated homoserine lactones were produced in vivo during infection. Extracts from infected fish were purified using several solid phase extraction strategies to allow chromatographic detection and separation by both TLC and HLPC-HRMS. 3-oxo-C10-HSL and 3-hydroxy-C6-HSL were detected in organs from fish dying from vibriosis, however, compared to in vitro culturing where 3-oxo-C10-HSL is the dominant molecule, 3-hydroxy-C6-HSL was prominent in the infected fish tissues. Hence, the balance between the QS systems may be different during infection compared to in vitro cultures. For future studies of QS systems and the possible specific interference with expression of virulence factors, in vitro cultures should be optimised to reflect the in vivo situation.
|
|
| 5. |
- Cámara, Miguel, et al.
(författare)
-
Quorum sensing in Vibrio cholerae.
- 2002
-
Ingår i: Nature Genetics. - : Springer Science and Business Media LLC. - 1061-4036 .- 1546-1718.
-
Annan publikation (refereegranskat)
|
|
| 6. |
- Croxatto, Antony, et al.
(författare)
-
A distinctive dual-channel quorum-sensing system operates in Vibrio anguillarum
- 2004
-
Ingår i: Molecular Microbiology. - : Wiley-Blackwell. - 0950-382X .- 1365-2958. ; 52:6, s. 1677-1689
-
Tidskriftsartikel (refereegranskat)abstract
- Many bacterial cells communicate using diffusible signal molecules to monitor cell population density via a process termed quorum sensing. In marine Vibrio species, the Vibrio harveyi-type LuxR protein is a key player in a quorum-sensing phosphorelay cascade, which controls the expression of virulence, symbiotic and survival genes. Previously, we characterized Vibrio anguillarum homologues of LuxR (VanT) and LuxMN (VanMN) and, in this study, we have identified homologues of LuxPQ (VanPQ) and LuxOU (VanOU). In contrast to other Vibrio species, vanT was expressed at low cell density and showed no significant induction as the cell number increased. In addition, although the loss of VanO increased vanT expression, the loss of VanU, unexpectedly, decreased it. Both VanN and VanQ were required for repression of vanT even in a vanU mutant, suggesting an alternative route for VanNQ signal transduction other than via VanU. VanT negatively regulated its own expression by binding and repressing the vanT promoter and by binding and activating the vanOU promoter. The signal relay results in a cellular response as expression of the metalloprotease, empA, was altered similar to that of vanT in all the mutants. Consequently, the V. anguillarum quorum-sensing phosphorelay systems work differently from those of V. harveyi and may be used to limit rather than induce vanT expression.
|
|
| 7. |
|
|
| 8. |
- Croxatto, Antony, et al.
(författare)
-
VanT, a Homologue of Vibrio harveyi LuxR, Regulates Serine, Metalloprotease, Pigment, and Biofilm Production in Vibrio anguillarum
- 2002
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 184:6, s. 1617-1629
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum possesses at least two N-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the luxMN system of Vibrio harveyi. In this study, we have cloned an additional gene of this circuit, vanT, encoding a V. harveyi LuxR-like transcriptional regulator. A V. anguillarum ΔvanT null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless lacZ. Three lacZ fusions (serA::lacZ, hpdA-hgdA::lacZ, and sat-vps73::lacZ) were identified which exhibited decreased expression in the ΔvanT strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production. V. anguillarum strains require an active VanT to produce high levels of an l-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to Vibrio cholerae proteins encoded within a DNA locus required for biofilm formation. A V. anguillarum ΔvanT mutant and a mutant carrying a polar mutation in the sat-vps73 DNA locus were shown to produce defective biofilms. Hence, a new member of the V. harveyi LuxR transcriptional activator family has been characterized in V. anguillarum that positively regulates serine, metalloprotease, pigment, and biofilm production.
|
|
| 9. |
- Croxatto, Antony, et al.
(författare)
-
Vibrio anguillarum colonization of rainbow trout integument requires a DNA locus involved in exopolysaccharide transport and biosynthesis.
- 2007
-
Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 9:2, s. 370-382
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum, part of the normal flora of the aquatic milieu, causes a fatal haemorrhagic septicaemia in marine fish. In this study, a rainbow trout model was used to characterize the colonization of fish skin by V. anguillarum. Within 5 h after infection, the bacterium penetrated the skin mucosal layer, attached to the scales within 12 h, and formed a biofilm by 24-48 h. Two divergently transcribed putative operons, orf1-wbfD-wbfC-wbfB and wza-wzb-wzc, were shown to play a role in skin colonization and virulence. The first operon encodes proteins of unknown function. The wza-wzb-wzc genes encode a secretin, tyrosine kinase and tyrosine phosphatase, respectively, which are similar to proteins in polysaccharide transport complexes. Compared with the wild type, polar mutations in wza, orf1 and wbfD caused a decrease in exopolysaccharide biosynthesis but not lipopolysaccharide biosynthesis. The wza and orf1 mutants did not attach to fish scales; whereas, the wbfD mutant had a wild-type phenotype. Moreover, the wza and orf1 mutants had decreased exoprotease activity, in particular the extracellular metalloprotease EmpA, as well as mucinase activity suggesting that these mutations also affect exoenzyme secretion. Thus, the exopolysaccharide transport system in V. anguillarum is required for attachment to fish skin, possibly preventing mechanical removal of bacteria via natural sloughing of mucus.
|
|
| 10. |
- Farto, R, et al.
(författare)
-
Colonization of turbot tissues by virulent and avirulent Aeromonas salmonicida subsp. salmonicida strains during infection
- 2011
-
Ingår i: Diseases of Aquatic Organisms. - : Inter Research. - 0177-5103 .- 1616-1580. ; 95, s. 167-173
-
Tidskriftsartikel (refereegranskat)abstract
- Preventing disease outbreaks in cultured turbot Psetta maxima L. caused by Aeromonas salmonicida subsp. salmonicida (ASS) requires a better understanding of how this pathogen colonizes its host. Distribution of 1 virulent and 2 avirulent ASS strains in turbot tissues was investigated during early and late stages of infection following an immersion challenge. To track bacteria within the turbot, the ASS strains were tagged with green fluorescent protein (GFP). Both virulent and avirulent strains colonized the epidermal mucus, gills, and intestine within the first 12 h post challenge, suggesting that these sites may serve as points of entry into turbot. Although the avirulent strains colonized these initial sites in the turbot tissues, they were rarely found in the internal organs and were cleared from the host 4 d post challenge. In contrast, the virulent ASS strain was found in the liver and kidney as early as 12 h post challenge and was found in the muscle tissue at very late stages of infection. The virulent strain persisted in all tested host tissues until death occurred 7 d post challenge, suggesting that ASS must colonize and survive within the turbot tissues for an infection to result in death of the fish. Comparisons of the distribution profiles of both virulent and avirulent strains during early and late stages of an infection in turbot has provided important information on the route and persistence of an ASS infection in this host.
|
|
| 11. |
- Garcia-Aljaro, Cristina, et al.
(författare)
-
Quorum-sensing regulates biofilm formation in Vibrio scophthalmi
- 2012
-
Ingår i: BMC Microbiology. - : BioMed Central. - 1471-2180. ; 12, s. 287-
-
Tidskriftsartikel (refereegranskat)abstract
- Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits.Results: The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorum-sensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study.Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.
|
|
| 12. |
- Gómez-Consarnau, Laura, et al.
(författare)
-
Proteorhodopsin phototrophy promotes survival of marine bacteria during starvation
- 2010
-
Ingår i: PLoS biology. - : Public Library Science. - 1544-9173 .- 1545-7885. ; 8
-
Tidskriftsartikel (refereegranskat)abstract
- Proteorhodopsins are globally abundant photoproteins found in bacteria in the photic zone of the ocean. Although their function as proton pumps with energy-yielding potential has been demonstrated, the ecological role of proteorhodopsins remains largely unexplored. Here, we report the presence and function of proteorhodopsin in a member of the widespread genus Vibrio, uncovered through whole-genome analysis. Phylogenetic analysis suggests that the Vibrio strain AND4 obtained proteorhodopsin through lateral gene transfer, which could have modified the ecology of this marine bacterium. We demonstrate an increased long-term survival of AND4 when starved in seawater exposed to light rather than held in darkness. Furthermore, mutational analysis provides the first direct evidence, to our knowledge, linking the proteorhodopsin gene and its biological function in marine bacteria. Thus, proteorhodopsin phototrophy confers a fitness advantage to marine bacteria, representing a novel mechanism for bacterioplankton to endure frequent periods of resource deprivation at the ocean’s surface.
|
|
| 13. |
- Holm, Kare Olav, et al.
(författare)
-
Complete genome sequence of Vibrio anguillarum strain NB10, a virulent isolate from the Gulf of Bothnia
- 2015
-
Ingår i: Standards in Genomic Sciences. - : Springer Science and Business Media LLC. - 1944-3277. ; 10
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum causes a fatal hemorrhagic septicemia in marine fish that leads to great economical losses in aquaculture world-wide. Vibrio anguillarum strain NB10 serotype O1 is a Gram-negative, motile, curved rod-shaped bacterium, isolated from a diseased fish on the Swedish coast of the Gulf of Bothnia, and is slightly halophilic. Strain NB10 is a virulent isolate that readily colonizes fish skin and intestinal tissues. Here, the features of this bacterium are described and the annotation and analysis of its complete genome sequence is presented. The genome is 4,373,835 bp in size, consists of two circular chromosomes and one plasmid, and contains 3,783 protein-coding genes and 129 RNA genes.
|
|
| 14. |
- Ishikawa, Takahiko, et al.
(författare)
-
Pathoadaptive conditional regulation of the type VI secretion system in Vibrio cholerae O1 strains
- 2012
-
Ingår i: Infection and Immunity. - : American Society for Microbiology. - 0019-9567 .- 1098-5522. ; 80:2, s. 575-584
-
Tidskriftsartikel (refereegranskat)abstract
- The most recently discovered secretion pathway in gram-negative bacteria, the type VI secretion system (T6SS), is present in many species and is considered important for the survival of non-O1 non-O139 Vibrio cholerae in aquatic environments. Until now, it was not known whether there is a functionally active T6SS in wild-type V. cholerae O1 strains, the cause of cholera disease in humans. Here, we demonstrate the presence of a functionally active T6SS in wild-type V. cholerae O1 strains, as evidenced by the secretion of the T6SS substrate Hcp, which required several gene products encoded within the putative vas gene cluster. Our analyses showed that the T6SS of wild-type V. cholerae O1 strain A1552 was functionally activated when the bacteria were grown under high-osmolarity conditions. The T6SS was also active when the bacteria were grown under low temperature (23°C), suggesting that the system may be important for the survival of the bacterium in the environment. A test of the interbacterial virulence of V. cholerae strain A1552 against an Escherichia coli K-12 strain showed that it was strongly enhanced under high osmolarity and that it depended on the hcp genes. Interestingly, we found that the newly recognized osmoregulatory protein OscR plays a role in the regulation of T6SS gene expression and secretion of Hcp from V. cholerae O1 strains.
|
|
| 15. |
- Ishikawa, Takahiko, et al.
(författare)
-
Quorum sensing regulation of the two hcp alleles in Vibrio cholerae O1 strains
- 2009
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 4:8
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The type VI secretion system (T6SS) has emerged as a protein secretion system important to several gram-negative bacterial species. One of the common components of the system is Hcp, initially described as a hemolysin co-regulated protein in a serotype O17 strain of Vibrio cholerae. Homologs to V. cholerae hcp genes have been found in all characterized type VI secretion systems and they are present also in the serotype O1 strains of V. cholerae that are the cause of cholera diseases but seemed to have non-functional T6SS.METHODOLOGY/PRINCIPAL FINDINGS: The serotype O1 V. cholerae strain A1552 was shown to express detectable levels of Hcp as determined by immunoblot analyses using polyclonal anti-Hcp antiserum. We found that the expression of Hcp was growth phase dependent. The levels of Hcp in quorum sensing deficient mutants of V. cholerae were compared with the levels in wild type V. cholerae O1 strain A1552. The expression of Hcp was positively and negatively regulated by the quorum sensing regulators HapR and LuxO, respectively. In addition, we observed that expression of Hcp was dependent on the cAMP-CRP global transcriptional regulatory complex and required the RpoN sigma factor.CONCLUSION/SIGNIFICANCE: Our results show that serotype O1 strains of V. cholerae do express Hcp which is regarded as one of the important T6SS components and is one of the secreted substrates in non-O1 non-O139 V. cholerae isolates. We found that expression of Hcp was strictly regulated by the quorum sensing system in the V. cholerae O1 strain. In addition, the expression of Hcp required the alternative sigma factor RpoN and the cAMP-CRP global regulatory complex. Interestingly, the environmental isolates of V. cholerae O1 strains that showed higher levels of the HapR quorum sensing regulator in comparison with our laboratory standard serotype O1 strain A1552 where also expressing higher levels of Hcp.
|
|
| 16. |
|
|
| 17. |
- Li, Xuan, et al.
(författare)
-
RpoS and Indole Signaling Control the Virulence of Vibrio anguillarum towards Gnotobiotic Sea Bass (Dicentrarchus labrax) Larvae
- 2014
-
Ingår i: PLOS ONE. - : PLOS one. - 1932-6203. ; 9:10, s. e111801-
-
Tidskriftsartikel (refereegranskat)abstract
- Quorum sensing, bacterial cell-to-cell communication with small signal molecules, controls the virulence of many pathogens. In contrast to other vibrios, neither the VanI/VanR acylhomoserine lactone quorum sensing system, nor the three-channel quorum sensing system affects virulence of the economically important aquatic pathogen Vibrio anguillarum. Indole is another molecule that recently gained attention as a putative signal molecule. The data presented in this study indicate that indole signaling and the alternative sigma factor RpoS have a significant impact on the virulence of V. anguillarum. Deletion of rpoS resulted in increased expression of the indole biosynthesis gene tnaA and in increased production of indole. Both rpoS deletion and the addition of exogenous indole (50-100 mu M) resulted in decreased biofilm formation, exopolysaccharide production (a phenotype that is required for pathogenicity) and expression of the exopolysaccharide synthesis gene wbfD. Further, indole inhibitors increased the virulence of the rpoS deletion mutant, suggesting that indole acts downstream of RpoS. Finally, in addition to the phenotypes found to be affected by indole, the rpoS deletion mutant also showed increased motility and decreased sensitivity to oxidative stress.
|
|
| 18. |
- McGee, Karen, et al.
(författare)
-
Identification and characterization of additional flagellin genes from Vibrio anguillarum
- 1996
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 178:17, s. 5188-5198
-
Tidskriftsartikel (refereegranskat)abstract
- Previously, the flagellar filament of Vibrio anguillarum was suggested to consist of flagellin A and three additional flagellin proteins, FlaB, -C, and -D. This study identifies the genes encoding FlaB, -C, and -D and a possible fifth flagellin gene that may encode FlaE. The flagellin genes map at two separate DNA loci and are most similar to the four polar flagellin genes of Vibrio parahaemolyticus, also located at two DNA loci. The genetic organization of these two loci is conserved between both organisms. For each gene, in-frame deletions of the entire gene, the 5' end, and the 3' end were made. Mutant analysis showed that each mutation, except those in flaE, caused a loss of flagellin from the filament. However, no obvious structural loss in the filament, as determined by electron microscopy, and only slight decreases in motility were seen. Virulence analysis indicated that all but two of the mutations gave a wild-type phenotype. The 5'-end deletions of flaD and flaE decreased virulence significantly (>10(4)-fold) of infections via both the intraperitoneal and immersion routes. These results indicate that, like FlaA, FlaD and FlaE may also be involved in virulence.
|
|
| 19. |
- Milton, Debra L., et al.
(författare)
-
Cloning of a metalloprotease gene involved in the virulence mechanism of Vibrio anguillarum
- 1992
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 174:22, s. 7235-7244
-
Tidskriftsartikel (refereegranskat)abstract
- Genetic evidence has previously suggested that a zinc metalloprotease is involved in the invasive mechanism of the fish pathogen Vibrio anguillarum NB10. In this study, the metalloprotease gene was cloned and sequenced. The sequence encodes a polypeptide (611 amino acids) that contains a putative signal sequence followed by a large leader sequence and the mature protein (44.6 kDa). Since the purified protein has a molecular mass of 36 kDa instead of the predicted 44.6 kDa, the mature protein is most likely processed a third time. Comparative analyses of the protein sequence showed high homologies to other bacterial metalloproteases within the zinc-binding and active-site regions. The Vibrio cholerae hemagglutinin/protease and the Pseudomonas aeruginosa elastase were exceptions in that the homology extended throughout the entire putative preproprotein. A chromosomal metalloprotease mutant was made via the integration of foreign DNA into the protease gene. This mutant did not secrete the metalloprotease, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide protein analysis and by growth on gelatin agar. Transcomplementation of the chromosomal mutation revived the secretion of the metalloprotease and its activity on gelatin agar. Interestingly, when supernatant proteins were analyzed by gelatin-SDS-polyacrylamide electrophoresis, two different proteases (75 and 30 kDa) were detected in the mutant strain but not in the transcomplemented strain or the wild-type strain. Moreover, fish infection studies were done, and implications for the role of the metalloprotease in the virulence mechanism of V. anguillarum are discussed.
|
|
| 20. |
- Milton, Debra L., et al.
(författare)
-
Flagellin A is essential for the virulence of Vibrio anguillarum
- 1996
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 178:5, s. 1310-1319
-
Tidskriftsartikel (refereegranskat)abstract
- A flagellin gene from the fish pathogen Vibrio anguillarum was cloned, sequenced, and mutagenized. The DNA sequence suggests that the flaA gene encodes a 40.1-kDa protein and is a single transcriptional unit. A polar mutation and four in-frame deletion mutations (180 bp deleted from the 5' end of the gene, 153 bp deleted from the 3' end of the gene, a double deletion of both the 180- and 153-bp deletions, and 942 bp deleted from the entire gene) were made. Compared with the wild type, all mutants were partially motile, and a shortening of the flagellum was seen by electron microscopy. Wild-type phenotypes were regained when the mutations were transcomplemented with the flaA gene. Protein analysis indicated that the flaA gene corresponds to a 40-kDa protein and that the flagellum consists of three additional flagellin proteins with molecular masses of 41, 42, and 45 kDa. N-terminal sequence analysis confirmed that the additional proteins were flagellins with N termini that are 82 to 88% identical to the N terminus of FlaA. Virulence studies showed that the N terminal deletion, the double deletion, and the 942-bp deletion increased the 50% lethal dose between 70- and 700-fold via immersion infection, whereas infection via intraperitoneal injection showed no loss in virulence. In contrast, the polar mutant and the carboxy-terminal deletion mutant showed approximately a 10(4)-fold increase in the 50% lethal dose by both immersion and intraperitoneal infection. In summary, FlaA is needed for crossing the fish integument and may play a role in virulence after invasion of the host.
|
|
| 21. |
- Milton, Debra L., 1956-, et al.
(författare)
-
Low risk for microbial contamination of syringe and tube feeding bag surfaces after multiple reuses with home blenderized tube feeding
- 2022
-
Ingår i: Nutrition in clinical practice. - : John Wiley & Sons. - 0884-5336 .- 1941-2452. ; 37:4, s. 907-912
-
Tidskriftsartikel (refereegranskat)abstract
- Background: Guidelines for the reuse of enteral tube feeding (ETF) equipment guidelines are limited to manufacturer recommendations. ETF equipment reuse studies are needed as the enteral population has increased, along with blenderized tube feeding (BTF).Methods: This experiment tested microbial contamination of a reusable gravity feeding bag and syringe after 15 BTF reuses and cleanings. Eight bags and syringes were filled with the BTF, held at room temperature for 20 min, and then emptied, washed, and air dried. After the last air drying, the inner surfaces of the bag and syringe were swabbed, and aerobic microbial counts were performed using serial dilutions and plate counts.Results: The microbial counts for all syringes and six bags were <1 colony-forming unit (CFU)/cm2; one bag was <5 CFU/cm2 and one bag was 12.5 CFU/cm2. No legal guidelines for surface cleanliness exist for the food sector. Several studies propose a safe microbial level to be <2.5 CFU/cm2, and the European Commission recommended <10 CFU/cm2. Based on these proposed guidelines, microbial counts of all syringes and seven bags were within the proposed guidelines, except for one bag just above 10 CFU/cm2.Conclusion: The feeding bag used in this study may be used multiple times for BTF with a reduced risk of microbial contamination when manufacturer's cleaning guidelines are followed. Although bolus tube feeding is an off-label use for syringes, they are frequently used for BTF, and in this study the cleaning after 15 uses over 5 days was effective to reduce microbial counts.
|
|
| 22. |
- Milton, Debra L., et al.
(författare)
-
Quorum sensing in Vibrio anguillarum : characterization of the vanI/vanR locus and identification of the autoinducer N-(3-oxodecanoyl)-L-homoserine lactone
- 1997
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 179:9, s. 3004-3012
-
Tidskriftsartikel (refereegranskat)abstract
- Certain gram-negative pathogens are known to control virulence gene expression through cell-cell communication via small diffusible signal molecules termed autoinducers. This intercellular signal transduction mechanism termed quorum sensing depends on the interaction of an N-acylhomoserine lactone (AHL) auto-inducer molecule with a receptor protein belonging to the LuxR family of positive transcriptional activators. Vibrio anguillarum is a gram-negative pathogen capable of causing a terminal hemorrhagic septicemia known as vibriosis in fish such as rainbow trout. In this study, we sought to determine whether V. anguillarum employs AHLs to regulate virulence gene expression. Spent V. anguillarum culture supernatants stimulated bioluminescence in a recombinant lux-based Escherichia coli AHL biosensor strain, whereas they both stimulated and inhibited AHL-mediated violacein pigment production in Chromobacterium violaceum. This finding suggested that V. anguillarum may produce multiple AHL signal molecules. Using high-performance liquid chromatography and high-resolution tandem mass spectrometry, we identified the major V. anguillarum AHL as N-(3-oxodecanoyl)-L-homoserine lactone (ODHL), a structure which was unequivocally confirmed by chemical synthesis. The gene (vanI) responsible for ODHL synthesis was cloned and sequenced and shown to belong to the LuxI family of putative AHL synthases. Further sequencing downstream of vanI revealed a second gene (vanR) related to the LuxR family of transcriptional activators. Although deletion of vanI abolished ODHL synthesis, no reduction of either metalloprotease production or virulence in a fish infection model was observed. However, the vanI mutant remained capable of weakly activating both bioluminescence and violacein in the E. coli and C. violaceum biosensors, respectively, indicating the existence of additional layers of AHL-mediated regulatory complexity.
|
|
| 23. |
- Milton, Debra L
(författare)
-
Quorum sensing in vibrios : complexity for diversification.
- 2006
-
Ingår i: Int J Med Microbiol. - 1438-4221. ; 296:2-3, s. 61-71
-
Tidskriftsartikel (refereegranskat)abstract
- N-acylhomoserine lactone-dependent quorum sensing was first discovered in two luminescent marine bacteria, Vibrio fischeri and Vibrio harveyi. The LuxI/R system of V. fischeri is the paradigm of Gram-negative quorum-sensing systems; however, it is not found in all vibrios. A more complex quorum-sensing regulation is found in V. harveyi. Three parallel systems transmit signals via phosphorelays that converge onto one regulatory protein LuxO. Components of the three systems are found only in vibrios. Of the five Vibrio strains analysed, the number and types of signal circuits found in each strain are diverse. The signalling systems have different regulatory responses depending on the type of association the Vibrio strains have with an animal host, which may reflect the diverse roles the vibrios have in structuring and maintaining microniches within the aquatic milieu. Further studies are likely to show that the diversity and complexity of the Vibrio quorum-sensing systems coordinate intraspecies behaviour, niche occupation, and possibly evolution.
|
|
| 24. |
- Milton, Debra L., et al.
(författare)
-
Sequence of a novel virulence-mediating gene, virC, from Vibrio anguillarum
- 1995
-
Ingår i: Gene. - 0378-1119 .- 1879-0038. ; 164:1, s. 95-100
-
Tidskriftsartikel (refereegranskat)abstract
- Previously, the double-transposon (Tn) mutant VAN20 of Vibrio anguillarum (Va) 775.17B was isolated. This mutant lacked a major surface antigen (MSA) suggested to be a lipopolysaccharide (LPS) and showed a 10(5)-fold increase in the 50% lethal dose (LD50) when fish were infected intraperitoneally. In this study, the two Tn insertion sites within the chromosome were identified, a plasmid insertion mutation was made at each locus in a more virulent strain of Va, NB10, and the virulence was analyzed. One mutant displayed a 10(4)-fold increase in LD50, whereas the second mutant showed the wild-type (wt) phenotype. However, both mutants still expressed the MSA, suggesting that there may be more than two Tn insertions in VAN20 or that a double mutation is required to prevent production of the MSA. The DNA locus for the virulent phenotype was cloned and sequenced. A potential transcriptional unit consisting of three putative open reading frames (ORFs) was identified. The Tn was located in the second ORF, virC (virulence). The first ORF (34.8 kDa) showed 30% homology to the Escherichia coli and Salmonella typhimurium cysG (cysteine) genes. The virC gene (51.4 kDa) and the third ORF (24 kDa) showed no homology to other proteins in GenBank. Plasmid insertion mutants were made within each of these ORFs and the virulence was assayed. Only the virC mutant showed a loss in virulence, indicating that virC is a novel gene that is essential for the virulence of Va.
|
|
| 25. |
- Milton, Debra L., et al.
(författare)
-
The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and N-hexanoylhomoserine lactone :
- 2001
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 183:12, s. 3537-3547
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum, which causes terminal hemorrhagic septicemia in fish, was previously shown to possess a LuxRI-type quorum-sensing system (vanRI) and to produce N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL). However, a vanI null mutant still activated N-acylhomoserine lactone (AHL) biosensors, indicating the presence of an additional quorum-sensing circuit in V. anguillarum. In this study, we have characterized this second system. Using high-pressure liquid chromatography in conjunction with mass spectrometry and chemical analysis, we identified two additional AHLs as N-hexanoylhomoserine lactone (C6-HSL) and N-(3-hydroxyhexanoyl)homoserine lactone (3-hydroxy-C6-HSL). Quantification of each AHL present in stationary-phase V. anguillarum spent culture supernatants indicated that 3-oxo-C10-HSL, 3-hydroxy-C6-HSL, and C6-HSL are present at approximately 8.5, 9.5, and 0.3 nM, respectively. Furthermore, vanM, the gene responsible for the synthesis of these AHLs, was characterized and shown to be homologous to the luxL and luxM genes, which are required for the production of N-(3-hydroxybutanoyl)homoserine lactone in Vibrio harveyi. However, resequencing of the V. harveyi luxL/luxM junction revealed a sequencing error present in the published sequence, which when corrected resulted in a single open reading frame (termed luxM). Downstream of vanM, we identified a homologue of luxN (vanN) that encodes a hybrid sensor kinase which forms part of a phosphorelay cascade involved in the regulation of bioluminescence in V. harveyi. A mutation in vanM abolished the production of C6-HSL and 3-hydroxy-C6-HSL. In addition, production of 3-oxo-C10-HSL was abolished in the vanM mutant, suggesting that 3-hydroxy-C6-HSL and C6-HSL regulate the production of 3-oxo-C10-HSL via vanRI. However, a vanN mutant displayed a wild-type AHL profile. Neither mutation affected either the production of proteases or virulence in a fish infection model. These data indicate that V. anguillarum possesses a hierarchical quorum sensing system consisting of regulatory elements homologous to those found in both V. fischeri (the LuxRI homologues VanRI) and V. harveyi (the LuxMN homologues, VanMN).
|
|
| 26. |
- Ormonde, P., et al.
(författare)
-
Role of motility in adherence to and invasion of a fish cell line by Vibrio anguillarum
- 2000
-
Ingår i: Journal of Bacteriology. - 0021-9193 .- 1098-5530. ; 182:8, s. 2326-2328
-
Tidskriftsartikel (refereegranskat)abstract
- To understand further the role of the flagellum of Vibrio anguillarum in virulence, invasive and adhesive properties of isogenic motility mutants were analyzed by using a chinook salmon embryo cell line. Adhesion was unaffected but invasion of the cell line was significantly decreased in nonmotile or partially motile mutants, and the chemotactic mutant was hyperinvasive. These results suggest that active motility aids invasion by V. anguillarum, both in vivo and in vitro.
|
|
| 27. |
- O'Toole, R, et al.
(författare)
-
Chemotactic motility is required for invasion of the host by the fish pathogen Vibrio anguillarum.
- 1996
-
Ingår i: Molecular Microbiology. - 0950-382X .- 1365-2958. ; 19:3, s. 625-637
-
Tidskriftsartikel (refereegranskat)abstract
- The role of the flagellum and motility in the virulence of the marine fish pathogen Vibrio anguillarum was examined. Non-motile mutants were generated by transposon mutagenesis. Infectivity studies revealed that disruption of the flagellum and subsequent loss of motility correlated with an approximate 500-fold decrease in virulence when fish were inoculated by immersion in bacteria-containing water. However, the flagellar filament and motility were not required for pathogenicity following intraperitoneal injection of fish. The transposon-insertion site for six mutants was determined by cloning and sequencing of the Vibrio DNA flanking the transposon. V. anguillarum genes whose products showed strong homology to proteins with an established role in flagellum biosynthesis were identified. One of the aflagellate mutants had a transposon insertion in the rpoN gene of V. anguillarum. This rpoN mutant failed to grow at low concentrations of available iron and was avirulent by both the immersion and intraperitoneal modes of inoculation. A chemotaxis gene, cheR, was located upstream of one transposon insertion and an in-frame deletion was constructed in the coding region of this gene. The resulting non-chemotactic mutant exhibited wild-type pathogenicity when injected intra-peritoneally into fish but showed a decrease in virulence similar to that seen for the non-motile aflagellate mutants following immersion infection. Hence, chemotactic motility is a required function of the flagellum for the virulence of V. anguillarum.
|
|
| 28. |
- O'Toole, R, et al.
(författare)
-
RpoN of the fish pathogen Vibrio (Listonella) anguillarum is essential for flagellum production and virulence by the water-borne but not intraperitoneal route of inoculation.
- 1997
-
Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 143 ( Pt 12)
-
Tidskriftsartikel (refereegranskat)abstract
- To investigate the involvement of RpoN in flagellum production and pathogenicity of Vibrio (Listonella) anguillarum, the rpoN gene was cloned and sequenced. The deduced product of the rpoN gene displayed strong homology to the alternative sigma 54 factor (RpoN) of numerous species of bacteria. In addition, partial sequencing of rpoN-linked ORFs revealed a marked resemblance to similarly located ORFs in other bacterial species. A polar insertion or an in-frame deletion in the coding region of rpoN abolished expression of the flagellin subunits and resulted in loss of motility. Introduction of the rpoN gene of V. anguillarum or Pseudomonas putida into the rpoN mutants restored flagellation and motility. The rpoN mutants were proficient in the expression of other proposed virulence determinants of V. anguillarum, such as ability to grow under low available iron conditions, and expression of the LPS O-antigen and of haemolytic and proteolytic extracellular products. The infectivity of the rpoN mutants with respect to the wild-type strain was unaffected following intraperitoneal injection of fish but was reduced significantly when fish were immersed in bacteria-containing water. Thus, RpoN does not appear to regulate any factors required for virulence subsequent to penetration of the fish epithelium, but is important in the infection of fish by water-borne V. anguillarum.
|
|
| 29. |
- Schwenteit, J. M., et al.
(författare)
-
Construction of Aeromonas salmonicida subsp. achromogenes AsaP1-toxoid strains and study of their ability to induce immunity in Arctic char, Salvelinus alpinus L.
- 2015
-
Ingår i: Journal of Fish Diseases. - : Wiley. - 0140-7775 .- 1365-2761. ; 38:10, s. 891-900
-
Tidskriftsartikel (refereegranskat)abstract
- The metalloendopeptidase AsaP1 is one of the major extracellular virulence factors of A. salmonicida subsp. achromogenes, expressed as a 37-kDa pre-pro-peptide and processed to a 19-kDa active peptide. The aim of this study was to construct mutant strains secreting an AsaP1-toxoid instead of AsaP1-wt, to study virulence of these strains and to test the potency of the AsaP1-toxoid bacterin and the recombinant AsaP1-toxoids to induce protective immunity in Arctic char. Two A. salmonicida mutants were constructed that secrete either AsaP1(E294A) or AsaP1(Y309F). The secreted AsaP1(Y309F)-toxoid had weak caseinolytic activity and was processed to the 19-kDa peptide, whereas the AsaP1(E294A)-toxoid was found as a 37-kDa pre-pro-peptide suggesting that AsaP1 is auto-catalytically processed. The LD50 of the AsaP1(Y309F)-toxoid mutant in Arctic char was significantly higher than that of the corresponding wt strain, and LD50 of the AsaP1(E294A)-toxoid mutant was comparable with that of an AsaP1-deficient strain. Bacterin based on AsaP1(Y309F)-toxoid mutant provided significant protection, comparable with that induced by a commercial polyvalent furunculosis vaccine. Detoxification of AsaP1 is very hard, expensive and time consuming. Therefore, an AsaP1-toxoid-secreting mutant is more suitable than the respective wt strain for production of fish bacterins aimed to protect against atypical furunculosis.
|
|
| 30. |
- Tait, Karen, et al.
(författare)
-
Disruption of quorum sensing in seawater abolishes attraction of zoospores of the green alga Ulva to bacterial biofilms.
- 2005
-
Ingår i: Environ Microbiol. - 1462-2912. ; 7:2, s. 229-40
-
Tidskriftsartikel (refereegranskat)abstract
- Zoospores of the eukaryotic green seaweed Ulva respond to bacterial N-acylhomoserine lactone (AHL) quorum sensing signal molecules for the selection of surface sites for permanent attachment. In this study we have investigated the production and destruction of AHLs in biofilms of the AHL-producing marine bacterium, Vibrio anguillarum and their stability in seawater. While wild type V. anguillarum NB10 was a strong attractor of zoospores, inactivation of AHL production in this strain by either expressing the recombinant Bacillus lactonase coding gene aiiA, or by mutating the AHL biosynthetic genes, resulted in the abolition of zoospore attraction. In seawater, with a pH of 8.2, the degradation of AHL molecules was temperature-dependent, indicating that the AHLs produced by marine bacterial biofilms have short half-lives. The Ulva zoospores sensed a range of different AHL molecules and in particular more zoospores settled on surfaces releasing AHLs with longer (>six carbons) N-linked acyl chains. However, this finding is likely to be influenced by the differential diffusion rates of AHLs from the experimental surface matrix. Molecules with longer N-acyl chains, such as N-(3-oxodecanoyl)- L-homoserine lactone, diffused more slowly than those with shorter N-acyl chains such as N-(3-hydroxy-hexanoyl)- L-homoserine lactone. Image analysis using GFP-tagged V. anguillarum biofilms revealed that spores settle directly on bacterial cells and in particular on microcolonies which we show are sites of concentrated AHL production.
|
|
| 31. |
- Wang, Su-Yan, et al.
(författare)
-
A ToxR homolog from Vibrio anguillarum serotype O1 regulates its own production, bile resistance, and biofilm formation
- 2002
-
Ingår i: Journal of Bacteriology. - : American Society for Microbiology. - 0021-9193 .- 1098-5530. ; 184:6, s. 1630-1639
-
Tidskriftsartikel (refereegranskat)abstract
- ToxR, a transmembrane regulatory protein, has been shown to respond to environmental stimuli. To better understand how the aquatic bacterium Vibrio anguillarum, a fish pathogen, responds to environmental signals that may be necessary for survival in the aquatic and fish environment, toxR and toxS from V. anguillarum serotype O1 were cloned. The deduced protein sequences were 59 and 67% identical to the Vibrio cholerae ToxR and ToxS proteins, respectively. Deletion mutations were made in each gene and functional analyses were done. Virulence analyses using a rainbow trout model showed that only the toxR mutant was slightly decreased in virulence, indicating that ToxR is not a major regulator of virulence factors. The toxR mutant but not the toxS mutant was 20% less motile than the wild type. Like many regulatory proteins, ToxR was shown to negatively regulate its own expression. Outer membrane protein (OMP) preparations from both mutants indicated that ToxR and ToxS positively regulate a 38-kDa OMP. The 38-kDa OMP was shown to be a major OMP, which cross-reacted with an antiserum to OmpU, an outer membrane porin from V. cholerae, and which has an amino terminus 75% identical to that of OmpU. ToxR and to a lesser extent ToxS enhanced resistance to bile. Bile in the growth medium increased expression of the 38-kDa OMP but did not affect expression of ToxR. Interestingly, a toxR mutant forms a better biofilm on a glass surface than the wild type, suggesting a new role for ToxR in the response to environmental stimuli.
|
|
| 32. |
- Wang, Su-Yan, et al.
(författare)
-
Role for the major outer-membrane protein from Vibrio anguillarum in bile resistance and biofilm formation.
- 2003
-
Ingår i: Microbiology. - : Microbiology Society. - 1350-0872 .- 1465-2080. ; 149:Pt 4, s. 1061-1071
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum, a fish pathogen, produces a 38 kDa major outer-membrane porin, which may be involved in environmental adaptation. The gene encoding the 38 kDa porin was cloned and deleted. The deduced protein sequence was 75 % identical to that of the major outer-membrane protein (OMP), OmpU, from Vibrio cholerae. LacZ expression from an ompU : : lacZ transcriptional gene fusion was increased 1.5-fold in the presence of bile salts and was decreased 50- to 100-fold in a toxR mutant compared to that in the wild-type, showing that ompU expression is positively regulated by ToxR and induced by bile salts. Similar to a toxR mutant, an ompU mutant showed a slight decrease in motility, an increased sensitivity to bile salts and a thicker biofilm with better surface area coverage compared to that of the wild-type. When ompU was expressed under a ToxR-independent promoter in the toxR mutant, the phenotypes for bile resistance and biofilm formation, but not motility were complemented to that of the wild-type. In rainbow trout, the ompU mutant showed wild-type virulence via immersion into infected seawater and intraperitoneal injection. The ompU mutant produced two colony morphologies: opaque, which did not grow at 0.2 % bile, and translucent, which grew at 2 % bile. The translucent ompU mutant strain produced a second major OMP that was induced by bile. All ompU mutants showed variations in the amount and length of smooth LPS. In V. anguillarum, OmpU is not required for virulence, possibly due to a second OMP also critical for resistance to bile; however, outside of the fish host, OmpU limits the progression of biofilm formation.
|
|
| 33. |
- Weber, Barbara, 1980-, et al.
(författare)
-
Colonization of fish skin is vital for Vibrio anguillarum to cause disease
- 2010
-
Ingår i: Environmental Microbiology Reports. - : Society for Applied Microbiology and Blackwell Publishing Ltd.. - 1758-2229. ; 2:1, s. 133-139
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum causes a fatal haemorrhagic septicaemia in marine fish. During initial stages of infection, host surfaces are colonized; however, few virulence factors required for colonization of the host are identified. In this study, in vivo bioluminescent imaging was used to analyse directly the colonization of the whole rainbow trout animal by V. anguillarum. The wild type rapidly colonized both the skin and the intestines by 24 h; however, the bacterial numbers on the skin were significantly higher than in the intestines indicating that skin colonization may be important for disease to occur. Mutants defective for the anguibactin iron uptake system, exopolysaccharide transport, or Hfq, an RNA chaperone, were attenuated for virulence, did not colonize the skin, and penetrated skin mucus less efficiently than the wild type.These mutants, however, did colonize the intestines and were as resistant to 2% bile salts as is the wildtype. Moreover, exopolysaccharide mutants were significantly more sensitive to lysozyme and antimicrobial peptides, while the Hfq and anguibactin mutants were sensitive to lysozyme compared with the wildtype. Vibrio anguillarum encodes several mechanisms to protect against antimicrobial components of skin mucus enabling an amazingly abundant growth on the skin enhancing its disease opportunities.
|
|
| 34. |
- Weber, Barbara, et al.
(författare)
-
RpoS induces expression of the Vibrio anguillarum quorum-sensing regulator VanT
- 2008
-
Ingår i: Microbiology. - Reading : Society for General Microbiology. - 1350-0872 .- 1465-2080. ; 154:3, s. 767-780
-
Tidskriftsartikel (refereegranskat)abstract
- In vibrios, regulation of the Vibrio harveyi-like LuxR transcriptional activators occurs post-transcriptionally via small regulatory RNAs (sRNAs) that destabilize the luxR mRNA at a low cell population, eliminating expression of LuxR. Expression of the sRNAs is modulated by the vibrio quorum-sensing phosphorelay systems. However, vanT mRNA, which encodes a LuxR homologue in Vibrio anguillarum, is abundant at low and high cell density, indicating that VanT expression may be regulated via additional mechanisms. In this study, Western analyses showed that VanT was expressed throughout growth with a peak of expression during late exponential growth. VanO induced partial destabilization of vanT mRNA via activation of at least one Qrr sRNA. Interestingly, the sigma factor RpoS significantly stabilized vanT mRNA and induced VanT expression during late exponential growth. This induction was in part due to RpoS repressing expression of Hfq, an RNA chaperone. RpoS is not part of the quorum-sensing regulatory cascade since RpoS did not regulate expression or activity of VanO, and RpoS was not regulated by VanO or VanT. VanT and RpoS were needed for survival following UV irradiation and for pigment and metalloprotease production, suggesting that RpoS works with the quorum-sensing systems to modulate expression of VanT, which regulates survival and stress responses.
|
|
| 35. |
- Weber, Barbara, 1980-
(författare)
-
Stress response and virulence in Vibrio anguillarum
- 2010
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Bacteria use quorum sensing, a cell to cell signaling mechanism mediated by small molecules that are produced by specific signal molecule synthases, to regulate gene expression in response to population density. In Vibrio anguillarum, the quorum-sensing phosphorelay channels information from three hybrid sensor kinases VanN, VanQ, CqsS that sense signal molecules produced by the synthases VanM, VanS and CqsA, onto the phosphotransferase VanU, to regulate activity of the response regulator VanO. VanO activates transcription of quorum-sensing regulatory RNAs (Qrr), which work together with the RNA chaperone Hfq to repress expression of the transcriptional regulator VanT. The work presented in this thesis characterizes quorum-sensing independent and quorum-sensing dependent mechanisms that regulate VanT expression. Moreover, an in vivo imaging system was established, as a means to study V. anguillarum infections in the rainbow trout infection model. Two quorum-sensing independent mechanisms regulating VanT expression were identified. First, the sigma factor RpoS indirectly activates VanT expression during transition into stationary growth phase by inhibiting hfq expression. Both, RpoS and VanT are crucial for stress response. Second, a type VI secretion system (T6SS) has a novel function as a signal sensing mechanism to regulate rpoS and vanT expression. Consequently, RpoS, quorum sensing and T6SS form a global network that senses stress and modulates stress response to ensure survival of the bacteria. Further analysis of the quorum-sensing dependent regulation of VanT expression by the phosphorelay system revealed that four qrr genes are expressed continuously during growth. The phosphotransferase VanU is suggested to activate two response regulators, VanO and a predicted second response regulator. Activated VanO induces expression of the Qrr sRNAs, whereas, the predicted response regulator represses expression of the Qrr sRNAs. Thus, VanU has a pivotal role in the regulation of VanT expression. The signal synthase VanM and VanT form a regulatory loop, in which VanM represses VanT by inducing expression of the Qrr sRNAs and VanT directly activates vanM expression to repress its own expression. Moreover, Hfq destabilizes vanM mRNA, repressing vanM expression. VanT forms another regulatory loop with the transcriptional regulator LuxT, in which LuxT activates vanT expression and VanT directly represses luxT expression. V. anguillarum is an opportunistic pathogen that causes vibriosis, a terminal hemorrhagic septicemia. The spatial and temporal progression of the infection was analyzed using the whole animal with an in vivo bioluminescent imaging method. Initial studies showed that colonization of the fish skin requires the siderophore, the RNA chaperone Hfq and the exopolysaccharide transport system, which protects against the innate immunity on the skin. Colonization of the fish skin is crucial for disease.
|
|
| 36. |
- Weber, Barbara, 1980-, et al.
(författare)
-
The phosphotransferase VanU represses expression of four qrr genes antagonizing VanO-mediated quorum-sensing regulation in Vibrio anguillarum
- 2011
-
Ingår i: Microbiology. - : Society for General Microbiology. - 1350-0872 .- 1465-2080. ; 157:12, s. 3324-3339
-
Tidskriftsartikel (refereegranskat)abstract
- Vibrio anguillarum utilizes quorum sensing to regulate stress responses required for survival in the aquatic environment. Like other Vibrio species, V. anguillarum contains the gene qui, which encodes the ancestral quorum regulatory RNA Owl, and phosphorelay quorum-sensing systems that modulate the expression of small regulatory RNAs (sRNAs) that destabilize mRNA encoding the transcriptional regulator VanT. In this study, three additional Orr sRNAs were identified. All four sRNAs were positively regulated by sigma(54) and the sigma(54)-dependent response regulator Van, and showed a redundant activity. The Orr sRNAs, together with the RNA chaperone Hfq, destabilized vanT mRNA and modulated expression of VanT-regulated genes. Unexpectedly, expression of all four qrr genes peaked at high cell density, and exogenously added N-acylhomoserine lactone molecules induced expression of the qrr genes at low cell density. The phosphotransferase VanU, which phosphorylates and activates VanO, repressed expression of the Orr sRNAs and stabilized van T mRNA. A model is presented proposing that VanU acts as a branch point, aiding cross-regulation between two independent phosphorelay systems that activate or repress expression of the Orr sRNAs, giving flexibility and precision in modulating VanT expression and inducing a quorum-sensing response to stresses found in a constantly changing aquatic environment.
|
|
| 37. |
- Weber, Barbara, et al.
(författare)
-
The transcriptional regulator VanT activates expression of the signal synthase VanM forming a regulatory loop in the Vibrio anguillarum quorum sensing system
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- In Vibrio anguillarum, one quorum-sensing regulatory sRNA, Qrr1, was previously shown to destabilize vanT mRNA repressing expression of the transcriptional regulator VanT. In this study, three additional Qrr sRNAs that destabilize vanT mRNA were identified. The expression of the Qrr sRNA depends on the active (phosphorylated) form of the σ54-dependent response regulator VanO and the sigma factor RpoN (σ54). The phosphotransferase VanU is predicted to activate VanO, but also a putative second response regulator that may repress qrr expression activating VanT expression. Signal production by the homoserine lactone synthase VanM inhibits the phosphorelay determining VanU activity. Therefore, qrr expression and VanT were analyzed in the ΔvanM mutant. Interestingly, VanM activated qrr expression and repressed VanT expression. Moreover, vanM expression is tightly regulated and peaks early in growth. Several putative Hfq binding sites are present in the vanM 5´-untranslated region. Thus, vanM expression and mRNA stability were measured in the Δhfq mutant. Hfq destabilizes vanM mRNA, but also regulates vanM transcription. The transcriptional effect occurs indirectly through VanT, which is derepressed in the Δhfq mutant. Additionally, VanT directly binds to a conserved binding motif in the vanM promoter to positively regulate vanM expression. In summary, VanM and VanT generate a regulatory loop, in which VanM induces qrr expression to repress VanT expression and VanT negatively regulates its own expression by activating vanM expression.
|
|
| 38. |
- Weber, Barbara, et al.
(författare)
-
Type VI secretion modulates quorum sensing and stress response in Vibrio anguillarum.
- 2009
-
Ingår i: Environmental Microbiology. - : Wiley. - 1462-2912 .- 1462-2920. ; 11:12, s. 3018-3028
-
Tidskriftsartikel (refereegranskat)abstract
- Type VI protein secretion systems (T6SS) are essential for virulence of several Gram-negative bacteria. In this study, we identified a T6SS in Vibrio anguillarum, a marine bacterium that causes a hemorrhagic septicemia in fish. A partial operon vtsA-H (vibrio type six secretion) was sequenced and shown to encode eight proteins. VtsE-H are signature proteins found in other T6SSs, while VtsA-D are not associated with T6SS studied so far. In-frame deletions were made in each gene. Secretion of a haemolysin-co-regulated-like protein (Hcp), a protein secreted by all studied T6SSs, was decreased in VtsE-H. Unexpectedly, VtsA, VtsC and VtsD activated while VtsB and VtsE-H repressed hcp expression. The T6SS proteins also regulated expression of two extracellular proteases, EmpA and PrtV, but inversely to Hcp expression. This regulation was indirect as T6S positively regulated expression of the stress-response regulator RpoS and the quorum-sensing regulator VanT, which positively regulate protease expression. Moreover, VtsA-H proteins were not needed for virulence but did play a role in various stress responses. Thus, these data characterize a new role for T6S in the ecology of bacteria and we hypothesize this role to be a signal sensing mechanism that modulates the expression of regulators of the general stress response.
|
|
| 39. |
- Williams, P., et al.
(författare)
-
Quorum sensing and the population-dependent control of virulence
- 2000
-
Ingår i: Philosophical Transactions of the Royal Society of London. Biological Sciences. - : The Royal Society. - 0962-8436 .- 1471-2970. ; 355:1397, s. 667-680
-
Forskningsöversikt (refereegranskat)abstract
- One crucial feature of almost all bacterial infections is the need for the invading pathogen to reach a critical cell population density sufficient to overcome host defences and establish the infection. Controlling the expression of virulence determinants in concert with cell population density may therefore confer a significant survival advantage on the pathogen such that the host is overwhelmed before a defence response can be fully initiated. Many different bacterial pathogens are now known to regulate diverse physiological processes including virulence in a cell-density-dependent manner through cell-cell communication. This phenomenon, which relies on the interaction of a diffusible signal molecule (e.g. an N-acylhomoserine lactone) with a sensor or transcriptional activator to couple gene expression with cell population density, has become known as 'quorum sensing'. Although the size of the 'quorum' is likely to be highly variable and influenced by the diffusibility of the signal molecule within infected tissues, nevertheless quorum-sensing signal molecules can be detected in vivo in both experimental animal model and human infections. Furthermore, certain quorum-sensing molecules have been shown to possess pharmacological and immunomodulatory activity such that they may function as virulence determinants per se. As a consequence, quorum sensing constitutes a novel therapeutic target for the design of small molecular antagonists capable of attenuating virulence through the blockade of bacterial cell-cell communication.
|
|
| 40. |
- Wu, Z., et al.
(författare)
-
Vibrio cholerae hemagglutinin/protease (HA/protease) causes morphological changes in cultured epithelial cells and perturbs their paracellular barrier function
- 1996
-
Ingår i: Microbial Pathogenesis. - : Elsevier BV. - 0882-4010 .- 1096-1208. ; 21:2, s. 111-123
-
Tidskriftsartikel (refereegranskat)abstract
- In this report, we describe the cytotoxic activity of the cholera hemagglutinin/protease (HA/protease). A concentrated protein sample from the 37 degrees C overnight culture supernatant of CVD110, a delta ctxA, delta zot, delta Ace and hlyA::(ctxB mer) mutant of El Tor biotype Ogawa serotype strain E7946 caused morphological changes in cultured MDCK-I epithelial cells and altered their arrangement of filamentous actin (F-actin) and Zonula occludens-associated protein ZO-1. The drastic morphological changes can be inhibited by Zincov, a specific bacterial metalloprotease inhibitor. The cytotoxic fractions of the sample after FPLC gelfiltration fractionation showed two visible protein bands with molecular weights of approximately 34- and 32 kDa. Microsequencing of these two proteins revealed that they were the cholera HA/protease.
|
|
