Characterization of plasma secretory phospholipase A2 activity in the prairie rattlesnake (Crotalus viridis)

Plasma phospholipase A2 (PLA2) enzyme activity is a key component of innate immunity in most vertebrates. We evaluated circulating secreted PLA2 activity of prairie rattlesnakes (Crotalus viridis) by incubation of plasma with bacteria labeled with fluorescent membrane lipids. Incubation of bacteria with increasing volumes of plasma resulted in volume-dependent lysis of fatty acids from bacterial membranes. The activity was rapid, with substantial activity recorded after only 5 min of incubation with labeled bacteria, and a linear response for 20 min. In addition, the lysis activity was temperature-dependent, increasing activities from 5 to 20 °C, peak activities at 25–30 °C, and then decreasing activities from 35 to 40 °C. Furthermore, the activity was inhibited in a concentration-dependent manner by p-bromophenacyl bromide, a specific inhibitor of PLA2 activity, which indicated that the observed activities were due to the presence of PLA2 in the plasma of C. viridis. This study represents the first description of secretory PLA2 activity in the plasma of a snake. Our study shows that in addition to being an important component of snake venom, PLA2 enzymes play an important role in the snake’s immune response.


Background
Phospholipase A 2 (PLA 2 ) is a member of a superfamily of enzymes, the phospholipases, that function to remodel membranes (Rocha et al., 2014) and liberate fatty acids from the sn-2 position of membrane lipids to generate a broad spectrum of paracrine hormones (Murakami & Kudo, 2002;Dennis, 2000). In general, members of the PLA 2 family of enzymes can be divided into two main groups: intracellular and extracellular (secreted) PLA 2 forms. Intracellular PLA 2 serves the important function of membrane remodeling (Brown et al., 2003) and the generation of arachidonic acid (Balsinde et al., 2002, Fonteh et al., 1994 for the biosynthesis of a wide array of eicosanoids that serve a broad spectrum of biological functions (Dennis, 2000). Extracellular PLA 2 enzymes also play diverse biological roles, and some bind to extracellular receptors that mediate biological responses (Murakami et al., 2014).
The antibacterial activity of PLA 2 has been attributed to the cationic properties of this enzyme, which allows it to selectively utilize prokaryote membranes as substrates (Buckland & Wilton, 2000). The presence of this enzyme in human tears (Qu & Lehrer, 1998), and inflammatory fluids (Dennis, 1994), and its expression in macrophages (Murakami et al., 1997) and intestinal Paneth cells (Harwig et al., 1995) are consistent with its role as a modulator of immune activity.
Secretory PLA 2 has been identified as an immune component in a variety of ectothermic vertebrates. Members of the families Alligatoridae (Merchant et al., 2009;Siroski et al., 2013) and Crocodylidae (Merchant et al., 2011;Merchant et al., 2017) express high levels of circulating secretory PLA 2 enzymes. In addition, Komodo dragons (Varanus komodoensis, Merchant et al., 2018) and several species of turtles (Merchant, unpublished results) also exhibit relatively high circulating levels of this enzyme. This study was conducted to identify and characterize PLA 2 activity in the plasma of the prairie rattlesnake (Crotalus viridis).

Treatment of animals
Plasma samples were obtained from 21 (9F:12M) adult long-term captive C. viridis. Rattlesnakes were housed in plastic enclosures (Neodesha Plastics, Inc.) on newspaper substrate. Temperature was maintained between 18 and 24°C and photoperiod on a 12L:12D cycle. Water was provided ad libitum, and snakes were fed pre-killed mice biweekly. Blood samples were collected from the caudal vein and did not exceed 0.08% of total snake body mass per sampling event. Whole blood was centrifuged to separate plasma from red blood cells, and the collected plasma was pooled and stored at − 20°C until use.

Labeling of bacteria
One-milliliter cultures of E. coli bacteria were grown overnight at 37°C in nutrient broth. These cultures were used to inoculate 1-L cultures. These cultures were incubated for 24 h in the presence of 1 mg of BODIPY FL C16 (dissolved in 1 mL of dimethyl sulfoxide). The bacteria were centrifuged at 8000×g for 15 min, the cultures were decanted, and the bacteria were resuspended in 30 mL of sterile isotonic saline. The bacteria were again centrifuged 8000×g for 15 min to remove unincorporated BODIPY, and the bacterial pellet was resuspended in 30 mL of sterile isotonic saline, divided into 2-mL aliquots, and frozen at − 20°C until ready for use.

PLA 2 assay
The method used for the determination of PLA 2 in the plasma enzyme activity of C. viridis was described by Merchant et al. (2009). Rattlesnake plasma was incubated with 250 μL of assay buffer (1 mM Ca 2+ in 100 mM tris-HCl, pH 7.4) and 100 μL of BODIPY-labeled bacteria. The balance of the 750-μL reaction consisted of isotonic saline. For the determination of the effects of plasma volume on PLA 2 activity, different volumes of C. viridis plasma (1-100 μL) were incubated with 50 μL of BODIPY-labeled E. coli bacteria in assay buffer for 30 min ambient temperature. The reactions were terminated by the addition of 750 μL of stop buffer (100 μM tris-HCl, pH 7.4 with 15 mM EDTA) and were then centrifuged at 16,000×g to pellet the labeled bacteria, and approximately 1500 μL of each reaction was removed to a 1-mL plastic cuvette. The fluorescent intensity of each reaction was measured at an excitation λ of 500 nm and an emission λ of 510 nm (excitation and emission slit widths = 1 nm) in a Horiba Jobin Yvon Fluoromax-4 fluorimeter. The same procedure was followed to determine the effects of time, temperature, and inhibitors on PLA 2 activities in plasma from C. viridis.

Statistics and controls
The fluorescent intensity of each sample was compared to a standard curve of pure product to determine the nanomoles of product formed. The fluorescent intensities of each sample were corrected for background fluorescence by subtraction of a reagent blank in the absence of plasma. Each data point represents the means ± standard deviation for four independent determinations.

Results
The incubation of plasma derived from C. viridis with bacteria labeled with BODIPY resulted in a volumedependent increase in fluorescent product (Fig. 1). The relation between soluble fluorescence and plasma volume was biphasic, with a slow increase at low volumes (0-10 μL) and a near linear increase from 20 to 100 μL. At these higher volumes, an average increase of 1.35 nmol product formed/μL of plasma was observed (Fig. 1).
The accumulation of fluorescent product was relatively rapid, with substantial amounts of product (5.8 ± 5.6 nmol) detected after incubation of bacteria with C. viridis plasma for only 5 min (Fig. 2). The accumulation of product appeared to be asymptotic, while the initial formation of product was near linear (from 0 to 20 min), and product was generated at a rate of 2.5 nmol/min during this time frame. However, between 20 and 30 min, the rate of product formation fell to 0.9 nmol/min and dropped further to 0.5 nmol/min between 30 and 60 min. The thermal profile for PLA 2 activity was shown to be highly temperature-dependent (Fig. 3). From 5 to 10°C and from 10 to 15°C, the plasma exhibited a stepwise increase of 38 and 55 nmol of product formed, respectively. However, from 15 to 30°C, a relatively small increase of 36 nmol was observed for the 15°increase in temperature. At 35°C, the activity of 129 nmol was 22.8% lower than that measured at 30°C (167 nmol), which fell further to 113 nmol at 40°C.
The plasma-dependent generation of fluorescent product was inhibited in a concentration-dependent manner by BPB (Fig. 4). Incubation of C. viridis plasma with fluorescently labeled bacteria led to the generation of 71.5 ± 2.7 nmol of product. The inclusion of only 1 mm BPB inhibited the activity by 17.5 ± 4.6%. The inhibition was further increased to 35.7 ± 3.1 and 70.3 ± 7.8 when the concentration of BPB was increased to 2 and 5 mM, respectively. Furthermore, the addition of 50% unlabeled E. coli bacteria caused a 43.7 ± 7.3% reduction of activity relative to cultures of only labeled bacteria.

Discussion
Multicellular organisms must continuously defend against invasion and colonization by potentially infectious microbes. Innate immunity has been identified as a  primary mechanism of defense in ectotherms (Romo et al., 2016). However, our knowledge of the function of reptile immune systems lags behind other vertebrates (Warr et al., 2003). Recent studies have described the broad-acting antibacterial properties of plasma from C. viridis (Baker & Merchant, 2018a) and have assigned much of those properties to an active serum complement system of proteins (Baker & Merchant, 2018b). With the exception of crocodylians (Merchant et al., 2009), PLA 2 activity has not been well described in reptiles. The exception is the role of PLA 2 as a component of snake venoms. When injected, PLA 2 enzymes can contribute to envenomation morbidity via hemorrhage and edema, with the severity being highly species specific. However, PLA 2 enzymes also play an important role in immunity, with some venom PLA 2 showing bactericidal ability equivalent to that of commercially produced drugs (Samy et al., 2007). Fewer studies have examined the role of non-venom secretory PLA 2 in the snake's own immune system.
Activity of PLA 2 increases in a concentrationdependent manner but does not appear to reach an asymptote. Overall activity is less than that observed in American alligators, and higher concentrations of C. viridis plasma are needed to approach maximum activity (Merchant et al., 2009). We found PLA 2 activity is rapid, rising sharply from 5-20 min post exposure. Quick response to pathogens is an important component of the innate immune response. American alligators are frequently injured in combat with conspecifics and are found in swamps and marshes of the southeastern USA where temperatures remain warm year-round and potentially pathogenic organisms are common. In contrast, C. viridis inhabit dry regions of the western USA and must hibernate through the winter months throughout most of their range (Ernst & Ernst, 2003), reducing both the number of pathogenic organisms they are likely to encounter and the season when infection is likely. We found PLA 2 activity was maximal between 25 and 30°C, which approximates the preferred body temperature of C. viridis (28-32°C; Gannon & Secoy, 1985). Previous studies have found that the antibacterial ability of plasma and serum complement activity also peak at approximately 30°C (Baker & Merchant, 2018 a, b). At higher temperatures, PLA 2 activity is reduced. This is not the case in the American alligator, or in two species of caiman native to South America which show an increase in activity at temperatures above 30°C (Merchant et al., 2009;Siroski et al., 2013). This may be partially due to higher preferred body temperatures in crocodylians; however, neither American alligators nor spectacled caiman has been found to voluntarily tolerate temperatures above 38°C (Diefenbach, 1975;Johnson et al., 1978).

Conclusions
Our results indicate that secretory PLA 2 is an important component of the innate immune response in rattlesnakes. While the activity of this enzyme is well known as a component of venom, this is the first study to describe PLA 2 activity as it relates to the snake's own immune system.