Type II Secretion Is Essential for Virulence of the Emerging Fish Pathogen, Hypervirulent Aeromonas hydrophila

Abstract

Hypervirulent Aeromonas hydrophila (vAh) is an emerging pathogen in freshwater aquaculture systems. In the U.S.A., outbreaks of motile aeromonad septicemia associated with vAh result in the loss of over 3 million pounds of channel catfish from Southeastern production systems each year. A. hydrophila is a well-known opportunistic pathogen that secretes degradative and potentially toxigenic proteins, and the rapid mortality that occurs when catfish are challenged with vAh by intraperitoneal injection suggests that vAh-induced motile aeromonad septicemia may be, in part, a toxin-mediated disease. While vAh isolates from carp isolated in China possess complete Type I, Type II, and Type VI secretion systems, many of the US catfish isolates only possess complete Type I and Type II secretions systems. In order to determine the role of secreted proteins in vAh-induced disease, and to determine the extent of protein secretion by the Type II secretion pathway, an exeD secretin mutant was constructed using a recombineering method in the well-characterized US vAh strain, ML09-119. Wild-type and mutant secretomes were analyzed for protein content by SDS-PAGE and by assays for specific enzymes and toxins. Type II secretion-deficient mutants had a near complete loss of secreted proteins and enzyme/toxin activity, including hemolytic and proteolytic activity. The intact Type II secretion system was cloned and used to complement the deletion mutant, ML09-119 exeD, which restored protein secretion and the degradative and toxigenic potential. In vivo challenges in channel catfish resulted in complete attenuation of virulence in ML09-119 exeD, while the complemented mutant was observed to have restored virulence. These results indicate that secreted proteins are critical to vAh virulence, and that the Type II secretion system is the primary secretory pathway utilized for multiple effectors of vAh pathogenesis.

Differential production and secretion of potentially toxigenic extracellular proteins from hypervirulent Aeromonas hydrophila under biofilm and planktonic culture

“…Biofilm media was prepared by adding 0.2% agar powder (AlfaAesar) to TSB media prior to sterilization, and 70 ml of molten agar was poured into deep well petri dishes (Fisher), as previously described [ 64 ].. An aliquot of vAh ML09–119 was removed from cryogenic storage and cultured in TSB overnight at 30 °C on an orbital shaker. This culture was then used to prepare planktonic and biofilm cultures, as previously described [ 64 ]. Briefly, 70 ml of fresh TSB media was inoculated with 1 ml of the overnight culture and grown at 30 °C with shaking to mid-log phase.…”

“…Secreted proteins of planktonically-cultured vAh ML09–119 were purified from cell-free supernatants, prepared as previously described [ 64 ]. Briefly, vAh was cultured in TSB media as described above, cells were pelleted by centrifugation, and supernatant was decanted and retained.…”

“…Secreted proteins of biofilm-cultured vAh ML09–119 were purified from cell-free supernatants, as previously described [ 64 ]. In brief, cells were removed from the biofilm media surface and washed twice with cold, sterile PBS as described above.…”

“…Extracellular proteins (ECPs) were precipitated from cell-free supernatants by ammonium sulfate precipitation, as previously described [ 30 , 64 ]. Briefly, ammonium sulfate crystals (Fisher Scientific) were added to cell-free supernatants to achieve 65% saturation and incubated at 4 °C with gentle mixing for 24 h. Precipitated proteins were collected by centrifugation, resuspended in Tris buffer, and dialyzed twice against the same buffer in 10 Kda dialysis cassettes (Slide-A-Lyzer (Thermo Fisher)).…”

“…Non-specific proteolytic activity was measured using HiLyteFluor 488-labeled casein as the substrate, following manufacturer’s protocol with minor modifications (Sensolyte Green Fluorimetric Protease Assay Kit, AnaSpec, Inc.), as previously described [ 64 ]. Briefly, a suitable concentration of protein was added to triplicate wells of black, flat-bottom 96-well plates with non-binding surface (Greiner Bio-One).…”