Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3’,5’monophosphate receptor protein (crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss) 1PhD
Katherinne Valderrama1,3, Melissa Saravia2 and Javier Santander2,3 Program in Aquaculture, Universidad Católica del Norte, Coquimbo, Chile; 2Universidad Mayor, Chile; 3Marine Microbial Pathogenesis and Vaccinology Laboratory, Memorial University of Newfoundland, Canada.
[email protected];
[email protected] ABSTRACT
Precise deletion of genes related to virulence can be used as a strategy to produce attenuated bacterial vaccines. Here, we study the deletion of the cyclic-3’,5’-adenosine monophosphate (cAMP) receptor protein (Crp) in Aeromonas salmonicida, the causative agent of furunculosis in marine and freshwater fish. Crp is a conserved global regulator, controlling physiology processes, like sugar utilization. Deletion of the crp gene has been utilized in live attenuated vaccines for mammals, birds, and warm water fish. Here, we characterized the crp gene and reported the effect of a crp deletion in A. salmonicida virulent and non-virulent isolates. We found that A. salmonicida Δcrp was not able to utilize maltose and other sugars, and its generation time was similar to the wild-type. A. salmonicida Δcrp showed a higher ability of cell invasion compared to the wild-type. Fish assays showed that A. salmonicida ∆crp is moderately attenuated in Oncorhynchus mykiss and conferred protective immunity against the intra coelomic (i.c.) challenge with A. salmonicida wild type. We concluded that deletion of A. salmonicida crp influence sugar utilization, cell invasion and virulence. Deletion of crp in A. salmonicida could be considered as part of an effective strategy to develop immersion live attenuated vaccines against furunculosis. MATERIALS AND METHODS. Bacterial strains, plasmids, media, and reagents. The bacterial strains and plasmids are listed in Table 1. Construction and characterization of A. salmonicida with defined deletions. The recombinant pEZ suicide vectors (Table 1) carrying the linked flanking regions to generate in-frame deletion of crp gene were constructed as described previously by Santander et al. (Santander et al. 2011). Complementation of A. salmonicida mutants. The crp gene were cloned independently into pEZ151 (Table 1) under Plac control at the AdhI restriction site. Table 1. Strains and plasmid utilized in this study Phenotypic tests: Crp expression was evalauted by Western Strain Relevant Characterisitics Reference blot, generation time was evalauted in TSB at 15ºC, sugar utilization was evalauted in OF media, brown pigment A. salmonicida J208 Wild type, A . non-virulent, smooth LPS, ATCC 33658 synthesis was evalauted in LB agar plates. s -1 s -1 Amp (100 µg ml ), Cm (25 µg ml ) NZ_LSGW00000000 Cell culture and Gentamicin exclusion assay. Chinook J210 J208 derivative, Δcrp-12, A-, non-virulent, salmon (Oncorhynchus tshawytscha) embryo cells (CHSE-214) smooth LPS, Amps (100 µg ml-1), Cms (25 µg This study were utilized for attachment and invasion assays. ml-1) J223
A
Wild type 3.173; Pittsford NFH, VT, USA; Atlantic salmon (Salmo salar) isolated, July, 1999; A+, smooth LPS, Amps (100 µg ml-1), Cms (25 µg ml-1) J223 derivate, Δcrp-12, A+, smooth LPS, Amps (100 µg ml-1), Cms (25 µg ml-1)
B J250
Dr. Rocco Cipriano NZ_LSGV00000000 This study
S. enterica
C
χ3761
S. Typhimurium UK-1
χ8132
S. Typhimurium UK-1, Δcya-27 Δcrp-27
χ3751
S. Choleraesuis crp-773::Tn10 Tet
r
(Hassan & Curtiss III 1996) (Curtiss III & Kelly 1987) (Kelly, Bosecker & Curtiss III 1992)
Figure 3. Complementation of Δcrp mutants with A. salmonicida crp gene. A. Growth of A. salmonicida Δcrp-12 in TSB broth at 15°C complemented with A. salmonicida crp gene; B. Growth of E. ictaluri in BHI broth at 28°C complemented with A. salmonicida crp gene; C. Growth of S. enterica serovar Typhimurium in LB broth at 37°C complemented with A. salmonicida crp gene. D. A. salmonicida Crp-cAMP dependence test in S. enterica Δcrp Δcya. The assays were repeated at least 3 times independently.
E. coli
D
χ7213
Escherichia coli. thr-1 leuB6 fhuA21 lacY1 glnV44 recA1 ΔasdA4 Δ(zhf-2 :: Tn10) thi-1 RP4-2-Tc :: Mu [λpir]; Kmr Escherichia coli. endA1 hsdR17 (rK-, mk+) supE44 thi-1 recA1 gyrA relA1 Δ(lacZYAargF) U169 λpir deoR (f80dlacΔ(lacZ)M15)
χ7232
(Roland, Curtiss III & Sizemore 1999) (Santander, Wanda, Nickerson & Curtiss III 2007)
Plasmids r
pEZ151
ori pSC101 Gm
(Santander et al.
pEZ225
pMEG-375 Δcrp-12
This study
pEZ252
pEZ151 Pcrp-crp A. salmonicida, Gmr
This study
2011)
Figure 1. Fish growth and holding. A. Recirculatory tanks; B. Trout eggs; C. Trout larva; D. Trout fingerlings. 1
Evaluation of A. salmonicida virulence in rainbow trout (O. mykiss). Around 1000 certified disinfected rainbow trout eggs were hatched and reared under pathogen-free conditions. Trout challenges were performed by intracoelomic (i.c.) administration to anesthetized fish. The fish were acclimated during 2 weeks before experimentation. The water temperature was 18-20°C. Trout fingerlings (30-50 g) were sedated in 40 mg L-1 tricaine methanesulfonate (MS-222, Sigma), then i.c. injected with 3,6-36 CFU of A. salmonicida J223 wild type or 33-330 CFU of A. salmonicida J250 ∆ crp. The LD50 was calculated by the method of Reed & Muench (1938). Dead animals were necropsied to evaluate the internal signs of the infection. Immunization and challenges in rainbow trout (O. mykiss). Twenty five juvenile fish (100-150 g) were i.c. immunized (1,2x102 CFU), and twenty were orally inmmunized (1,2x106 CFU) with A. salmonicida J250 ∆crp. After 6 weeks post-immunization, the fish were i.c. challenged with 5,7x102 CFU A. salmonicida J223 wild type (~100 LD50). Statistical analysis software. The software utilized for all statistical analysis was Prism 7, version 7.02.
Figure 4. Gentamicin exclusion assay in CHSE-214 cells. A. Adhesion percentage of wild type and ∆crp mutant strains of Aeromonas salmonicida. B. Intracellular survival of both strains. C. Cell number and viability of CHSE-214 cells during the assay. Significant differences are explained in text. The assays were repeated at least 3 times independently. A
B
RESULTS Two parental strains were utilized, J208 a non-virulent isolated and J223 a virulent isolated (Table 1). The genotype was verified by PCR (Figs. 2A and 2B). A. salmonicida J210 ∆ crp mutant, a J208 derivative, showed a significant decrease of pigment synthesis and a longer generation time and in comparison to the wild type 223 (Figs.2E and 3A). In contrast, J250 ∆ crp, a J223 derivative, did not showed growth and pigment synthesis defects. Deletion of crp did not altered the LPS and outer membrane profiles.
A
C
B
D
E
WT Δcrp Δcrp
(pEZ252)
DISCUSSION AND CONCLUSIONS The development of live attenuated vaccines in aquaculture arises from the need for an accessible massive immunization that minimizes fish stress. The use of those vaccines would allow larvae and juveniles vaccination and boost immunizations. Here, we have applied a common methodology to genetically engineer A. salmonicida and delete the crp gene in absence of antibiotic-resistance genes in the final strain (Fig. 2). We determined that A. salmonicida ∆crp was attenuated and confers immune protection to the fish host, by i.c. administration. However, deletion of crp in A. salmonicida have a modest effect on fish virulence (Fig. 5A). This suggests that in contrast to other pathogens, A. salmonicida virulence genes are not totally regulated by Crp.
WT
Δcrp-12 (pEZ252)
Figure 5. Virulence and immune protection of A. salmonicida Δcrp in rainbow trout (O. mykiss). A. Virulence of A. salmonicida Δcrp in rainbow trout fingerlings; B. Immune protection of A. salmonicida Δcrp in rainbow trout juvenile.
Δcrp-12
Figure 2. Dele,on of crp gene, phenotype and complementa,on of A. salmonicida crp mutant. A. Gene dele(on map; B. PCR verifica(on of crp dele(on; C. Western blot of A. salmonicida Δcrp-12 mutant and A. salmonicida Δcrp-12 complemented with pEZ252 (Pcrp-crp); D. Maltose u(liza(on in OF- media. Yellow indicates u(liza(on of the maltose to acid products. Green-Blue indicates no u(liza(on of maltose; E. Synthesis of brown pigmenta(on on LB agar. The assays were repeated at least 3 (mes independently.
Deletion of crp gene in A. salmonicida is not enough to develop a live attenuated vaccine and further mutations are required. Nevertheless, deletion of crp could be considered as part of the genetic plan to construct a A. salmonicida live attenuated vaccine. ACKNOWLEDGEMENTS
FONDECYT Regular competition 1140330
