Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3′,5′‐monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss)

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 aetiologic agent of furunculosis in marine and freshwater fish. The Crp protein 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 ∆crp showed a higher ability of cell invasion compared to the wild type. Fish challenges showed that A. salmonicida ∆crp is ~6 times attenuated in Oncorhynchus mykiss and conferred protective immunity against the intraperitoneal challenge with A. salmonicida wild type. We concluded that deletion of A. salmonicida crp influences 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.

Aeromonas salmonicida infects Atlantic salmon (Salmo salar) erythrocytes

“…The number of intraerythrocytic A. salmonicida at 2 hr (0.047%; 5.90 × 10 3 CFU) and 3 hr (0.032%; 2.10 × 10 3 CFU) post‐infection was significantly lower than the intracellular A. salmonicida founded in different type of cells at the same infection times. For instance, the number of intracellular A. salmonicida founded at 2 hr and 4 hr post‐infection in CHSE‐214 cells was 0.47% and 0.29%, respectively (Valderrama et al, ). The number of intracellular A. salmonicida founded at 2 hr and 3 hr post‐infection in primary Atlantic salmon cells was 0.42% and 0.26%, respectively, and in Atlantic cod primary macrophages, the number of intracellular A. salmonicida founded at 2 hr and 3 hr post‐infection was 0.42% and 0.37%, respectively (Soto‐Dávila et al, ).…”

“…A. salmonicida was routinely incubated at 15ºC aerobically (180 rpm). Bacterial growth was monitored spectrophotometrically and/or by plating (Valderrama et al, ). A single colony of A. salmonicida J223 was grown routinely in 3 ml of Trypticase Soy Broth (TSB, Difco) at 15ºC in a 16‐mm diameter glass tube and placed in a roller for 24 hr.…”

“…Virulent A. salmonicida J223 (NZ_LSGV00000000) (Valderrama et al, 2017) was utilized in this study. Bacteriological media and components were from Difco.…”

“…Although its natural route of infection is through skin lesions and mucosal epithelia (e.g. gut and gills), intraperitoneal experimental doses of 30 bacterial cells can result in a lethal infection (Valderrama, Saravia, & Santander, ).…”

“…In addition, A. salmonicida can invade and replicate in non-phagocytic epithelial cells (Garduño et al, 2000;Valderrama et al, 2017).…”