Serine proteinase of Renibacterium salmoninarum digests a major autologous extracellular and cell-surface protein

Abstract

Renibacterium salmoninarum is a pathogen of salmonid fish that produces large amounts of extracellular protein (ECP) during growth. A proteolytic activity present in ECP at elevated temperatures digested the majority of the proteins in ECP. This digestion was also associated with the loss of ECP immunosuppressive function. In vitro activity of the proteinase in ECP was temperature dependent: it was not detected in an 18-h digest at 4 and 17 degrees C but became readily apparent at 37 degrees C. Proteinase activity was detected at bacterial physiological temperatures (17 degrees C) in reactions incubated for several days. Under these conditions, digestion of partially purified p57, a major constituent of ECP and a major cell-surface protein, yielded a spectrum of breakdown products similar in molecular weight and antigenicity to those in ECP. This pattern of digestion suggests that most of the immunologically related constituents of ECP are p57 and its breakdown products. The proteolytic activity was sensitive to phenylmethylsulfonyl fluoride, methanol, and ethanol and to 10-min incubation at temperatures above 65 degrees C. Electrophoretic analysis of the proteinase on polyacrylamide gels containing proteinase substrates indicated the native form to be 100 kDa or greater. The enzyme was active against selected unrelated substrates only when coincubated with a denaturant (0.1% lauryl sulfate) and (or) a reducing agent (20 mM dithiothreitol).

Antigenic and functional characterization of p57 produced by Renibacterium salmoninarum

“…Cell-free ascites fluid from phobic amino acid composition (Jones & Isaacson 1983, group I MAbs (4Cl1, 4H8, and 4D3), group I1 MAbs Daly & Stevenson 1990, Dubreuil et al 1990a, Wiens & (2G5 and 3H1) and group 111 MAbs (1Al and 4D10) Kaattari 1991). In vitro, the secreted form of p57 has were collected and purified by protein A chromatograimmunomodulatory effects as it is able to bind and phy as described previously (Wiens & Kaattari 1991). agglutinate salmonid leucocytes and suppress antiProtein content was determined by the method of body production to the unrelated antigens, trinitro- Lowry et al (1951).…”

“…phenylated lipopolysaccharide and human gammaExtracellular protein (ECP) concentration. Renibacglobulin (Turaga et al 1987a, Rockey et al 1991b, and terium salmoninarum ATCC 33209 extracellular pro Wiens & Kaattari 1991, Fredriksen et al 1997. In additein was harvested from medium and concentrated as tion, injection of coho salmon eggs with p57 has been described previously (Wiens & Kaattari 1991).…”

“…Renibacglobulin (Turaga et al 1987a, Rockey et al 1991b, and terium salmoninarum ATCC 33209 extracellular pro Wiens & Kaattari 1991, Fredriksen et al 1997. In additein was harvested from medium and concentrated as tion, injection of coho salmon eggs with p57 has been described previously (Wiens & Kaattari 1991). The prereported to result in long term irnmunosuppression dominant component of ECP prepared in this manner (Brown et al 1996).…”

“…The prereported to result in long term irnmunosuppression dominant component of ECP prepared in this manner (Brown et al 1996). is p57 with associated proteolytic degradation fragWe have previously identified functional domains of ments (Rockey et al 1991b, Wiens & Kaattari 1991. p57 using a panel of 8 anti-p57 monoclonal antibodies Total protein was assessed by the method of Lowry et (MAbs) (Wiens & Kaattari 1991).…”

“…is p57 with associated proteolytic degradation fragWe have previously identified functional domains of ments (Rockey et al 1991b, Wiens & Kaattari 1991. p57 using a panel of 8 anti-p57 monoclonal antibodies Total protein was assessed by the method of Lowry et (MAbs) (Wiens & Kaattari 1991). Three of the MAbs al.…”