Aeromonas salmonicida subsp. salmonicida is the causative agent of furunculosis, a severe systemic disease affecting salmonid fish. This bacterium contains a type III protein secretion system that is responsible for the secretion and translocation of the ADP-ribosylating toxin, AexT, into the cytosol of fish cells. This study showed that inactivation of the type III secretion system by marker-replacement mutagenesis of the gene ascV, which encodes an inner-membrane component of the type III secretion system, attenuated virulence in a rainbow trout model. The isogenic ascV deletion mutant was phagocytosed by peripheral blood leukocytes but the wild-type (wt) A. salmonicida subsp. salmonicida isolate was not. Histological examination of fish experimentally infected with the wt bacterium revealed extensive tissue necrosis and bacterial aggregates in all organs examined, including the heart, kidney and liver, indicating that the isolate established a systemic infection. Cumulative mortality of fish experimentally infected with the wt bacterium reached 88 %. In contrast, no mortality was observed among fish infected with the same dose of the ascV mutant, and histological examination of fish infected with this strain revealed healthy organs. The results indicate that the type III secretion system of A. salmonicida subsp. salmonicida is required to establish systemic infection.
AustinD. A.,
RobertsonP. A. W.,
WallaceD. K.,
DaskalovH.,
AustinB.
1998; Isolation of Aeromonas salmonicida in association with purple-pigmented bacteria in sediment from a Scottish loch. Lett Appl Microbiol 27:349–351[CrossRef]
AusubelF. M.,
BrentR.,
KingstonR. E.,
MooreD. D.,
SeidmanJ. G.,
SmithJ. A.,
StruhlK.
(editors) 1999Current Protocols in Molecular Biology New York: Wiley;
BellandR. J.,
TrustT. J.
1987; Cloning of the gene for the surface array protein of Aeromonas salmonicida and evidence linking loss of expression with genetic deletion. J Bacteriol 169:4086–4091
BullockW. O.,
FernandezJ. M.,
ShortJ. M.
1987; XL1-Blue: a high efficiency plasmid transforming recA Escherichia coli strain with beta-galactosidase selection. Biotechniques 5:376–378
BurrS. E.,
StuberK.,
WahliT.,
FreyJ.
2002; Evidence for a type III secretion system in Aeromonas salmonicida subsp.salmonicida
. J Bacteriol 184:5966–5970[CrossRef]
BurrS. E.,
StuberK.,
FreyJ.
2003; The ADP-ribosylating toxin, AexT, from Aeromonas salmonicida subsp.salmonicida is translocated via a type III secretion pathway. J Bacteriol 185:6583–6591[CrossRef]
FürsteJ. P.,
PansegrauW.,
FrankR.,
BlockerH.,
ScholzP.,
BagdasarianM.,
LankaE.
1986; Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48:119–131[CrossRef]
MatthysseA. G.,
StrettonS.,
DandieC.,
McClureN. C.,
GoodmanA. E.
1996; Construction of GFP vectors for use in Gram-negative bacteria other than Escherichia coli
. FEMS Microbiol Lett 145:87–94[CrossRef]
SharplesG. J.,
InglestonS. M.,
LloydR. G.
1999; Holliday junction processing in bacteria: insights from the evolutionary conservation of RuvABC, RecG, and RusA. J Bacteriol 181:5543–5550
SimonR.,
PrieferU.,
PuhlerA.
1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria. Biotechnology 1:784–791[CrossRef]
StuberK.,
FreyJ.,
BurnensA. P.,
KuhnertP.
2003; Detection of type III secretion genes as a general indicator of bacterial virulence. Mol Cell Probes 17:25–32[CrossRef]
VipondR.,
BricknellI. R.,
DurantE.,
BowdenT. J.,
EllisA. E.,
SmithM.,
MacIntyreS.
1998; Defined deletion mutants demonstrate that the major secreted toxins are not essential for the virulence of Aeromonas salmonicida
. Infect Immun 66:1990–1998
YuH. B.,
Sriniasa RaoP. S.,
LeeH. C.,
VilchesS.,
MerinoS.,
ThomasJ. M.,
LeungK. Y.
2004; A type III secretion system is required for Aeromonas hydrophila AH-1 pathogenesis. Infect Immun 72:1248–1256[CrossRef]
ZaharikM. L.,
GruenheidS.,
PerrinA. J.,
FinlayB. B.
2002; Delivery of dangerous goods: type III secretion in enteric pathogens. Int J Med Microbiol 291:593–603[CrossRef]