Profiling of acylated homoserine lactones of Vibrio anguillarum in vitro and in vivo: Influence of growth conditions and serotype.
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.
Buchholtz
Christiane
C
Department of Seafood Research, Danish Institute for Fisheries Research, Søltofts Plads, DTU bldg. 221, DK-2800 Kgs. Lyngby, Denmark.
Nielsen
Kristian Fog
KF
Milton
Debra L
DL
Larsen
Jens Laurits
JL
Gram
Lone
L
D015107:4-Butyrolactone / Q000031:analogs & derivatives* / Q000032:analysis / Q000302:isolation & purification; D000222:Adaptation, Physiological; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005393:Fish Diseases / Q000382:microbiology; D005798:Genes, Bacterial; D007501:Iron / Q000378:metabolism; D013058:Mass Spectrometry; D017686:Oncorhynchus mykiss; D012703:Serotyping; D012965:Sodium Chloride / Q000378:metabolism; D013696:Temperature; D014733:Vibrio / Q000737:chemistry* / Q000145:classification / Q000254:growth & development / Q000502:physiology; D014735:Vibrio Infections / Q000382:microbiology / Q000662:veterinary
C088386:homoserine lactone; D012965:Sodium Chloride; D007501:Iron; D015107:4-Butyrolactone