Date Published: June 6, 2019
Publisher: Public Library of Science
Author(s): Sarah Baker, Ethan Kessler, Lancia Darville-Bowleg, Mark Merchant, Sebastian D. Fugmann.
Reptiles are declining worldwide yet our understanding of their immune function lags far behind other taxa. The innate immune system is the primary mode of defense in reptiles, and the serum complement cascade is its major component. We assessed serum complement activity of plasma in two closely related aquatic turtle species, the common snapping turtle (CST; Chelydra serpentina) and alligator snapping turtle (AST; Macrochelys temminckii). We used a sheep red blood cell (SRBC) hemolysis assay to assess serum complement activity. Although the antibacterial activities of the plasma of these turtle species are similar, the hemolytic activity was much stronger in CST than AST. Treatment with inhibitors of the serum complement cascade indicated differences in the mechanisms of complement activation between the turtle species. We subjected plasma from both turtle species to mannan affinity chromatography and analyzed the eluate with SDS-PAGE, which revealed that plasma from the CSTs contained only small amounts of one C-type lectin protein while the AST plasma contained high concentrations of two C-type lectins (31.0 and 35.9 kDa). Edman degradation analyses confirmed that the two AST proteins contained identical N-terminal sequences. Thus, the CST appears to rely more heavily on the alternative mechanism of serum complement activation, while the AST appears to rely more on the lectin-mediated pathway, which is a pattern recognition response to prokaryotes not activated by the SRBCs. These results are unique in that the use of serum complement pathways are generally assumed to be conserved within clades.
The animal immune system is divided into two branches, innate and adaptive immunity, which work both individually and in tandem to provide defense from pathogens. Innate immunity is nonspecific, requires no previous exposure, and responds rapidly. In contrast, adaptive immunity requires previous exposure, is very specific, and may take 48 hours or more to launch a coordinated response. The primary mechanism of the innate immune response is the serum complement system. Activation of the serum complement cascade leads to eventual construction of a multiprotein membrane attack complex in the membrane of microbes that causes leakage of cellular contents, and eventual lysis . In addition, serum complement exhibits a wide array of other functions, such as opsonization, phagocytosis, chemotactic recruitment of leukocytes, inflammation, and expression of proinflammatory cytokines, which facilitate a multifaceted immune response . Serum complement components are also crucial for coordinating some important events in adaptive immune response .
Chemicals and Biochemicals–Ethylene diamine tetraacetate (EDTA), magnesium chloride, calcium chloride, methylamine, salicylaldoxime, Coomassie blue protein stain, mannose-agarose, and a protease isolated from Streptomyces griseus (P8811) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sheep red blood cells (10%, v/v) were purchased from Rockland Immunochemicals (Rockland, MD, USA).
The plasma from ASTs and CSTs both exhibited volume-dependent hemolysis of 1% SRBCs (Fig 1). Plasma from CSTs showed much higher activity than that from ASTs. The plasma from CSTs showed a biphasic, asymptotic-like response with linear hemolysis between 0 and 20 μL (slope = +3.75% hemolysis/μL plasma) and gradually declining increases thereafter. In comparison, the plasma from ASTs showed a near linear response from 10–60 mL, but with a much lower slope of +1.2% hemolysis/μL plasma. The hemolytic activity of the 100% AST plasma (90.7 ± 1.5%) was similar to that of the 50% CST plasma (90.6 ± 0.9%).
Incubation of plasma from CSTs or ASTs caused a volume-dependent hemolysis of SRBCs (Fig 1). The hemolytic activity was much more potent with the plasma of the CST, which was surprising because the results from a previous study found antibacterial activities of plasma from these turtle species was similar, with CSTs showing only slightly higher activity . In addition, the kinetic analysis of the SRBC hemolysis by CST plasma was much faster and more potent than that from the AST (Fig 2). The activity of AST plasma remains low throughout the experiment, likely due to low concentrations of complement proteins preventing increased activity . Although the hemolytic activities were very different, they are both mediated by the serum complement system of proteins because they were heat labile, sensitive to proteases, and inhibited by chelators of divalent metal ions (Table 1). These characteristics eliminate the possibility that the hemolysis was due to heat-stable antimicrobial cationic peptides , or lytic enzymes such as lysozyme that are not dependent on Mg2+ or Ca2+ for activity .