Characterization of Serum Phospholipase A2 Activity in Three Diverse Species of West African Crocodiles

Secretory phospholipase A2, an enzyme that exhibits substantial immunological activity, was measured in the serum of three species of diverse West African crocodiles. Incubation of different volumes of crocodile serum with bacteria labeled with a fluorescent fatty acid in the sn-2 position of membrane lipids resulted in a volume-dependent liberation of fluorescent probe. Serum from the Nile crocodile (Crocodylus niloticus) exhibited slightly higher activity than that of the slender-snouted crocodile (Mecistops cataphractus) and the African dwarf crocodile (Osteolaemus tetraspis). Product formation was inhibited by BPB, a specific PLA2 inhibitor, confirming that the activity was a direct result of the presence of serum PLA2. Kinetic analysis showed that C. niloticus serum produced product more rapidly than M. cataphractus or O. tetraspis. Serum from all three species exhibited temperature-dependent PLA2 activities but with slightly different thermal profiles. All three crocodilian species showed high levels of activity against eight different species of bacteria.


Introduction
The phospholipase A 2 (PLA 2 ) family is a diverse set of enzymes represented by 15 different groups that include five specific types: cytosolic (cPLA 2 ), secreted (sPLA 2 ), Ca 2+independent (iPLA 2 ), platelet-activating factor acetylhydrolases (PAF-AH), and the lysosomal PLA 2 s [1,2] These enzymes are characterized by their ability to cleave fatty acids from the sn-2 position of membrane lipids. Some of the cPLA 2 s have long been recognized for their roles in the biosynthesis of eicosanoids, paracrine hormones that mediate a broad spectrum of physiological processes [3,4] In general, the PLA 2 family of enzymes can be divided into two main groups: intracellular and extracellular (secreted) PLA 2 forms.
Higher eukaryotes must continuously defend against infection by potentially infectious microbes. Innate immunity is the first line of defense against infection [5]. It is typically nonspecific in its action, thus allowing for broadspectrum activity. Secretory PLA 2 (sPLA 2 ) has been shown to exhibit substantial innate immune activity [6,7]. PLA 2 is an enzyme that catalyzes the hydrolysis of fatty acids from the sn-2 position of membrane lipids [6]. The sPLA 2 isoform was first described by Vadas et al. [3,4], and was later described as having antibacterial properties [8][9][10]. The antibacterial activity of sPLA 2 has been largely attributed to the cationic nature of the enzyme, thus allowing for interaction with, and disruption of, microbial membranes [11]. Other studies have shown that the antimicrobial effects of sPLA 2 can be modulated by antimicrobial peptides [12]. The presence of this enzyme in inflammatory fluids [6], human tears [13], intestinal Paneth cells [14], and macrophages [15] is consistent with its role as an important component of innate immunity.
Three diverse crocodilian species, each belonging to a different genus, can be found sympatrically in western Central Africa. These three species vary greatly with respect to morphology and general ecology. The slender-snouted crocodile (Mecistops cataphractus) and the African dwarf crocodile (Osteolaemus tetraspis) are considered the most poorly understood crocodilians in the world, with respect to nesting, feeding, and general ecology [16]. M. cataphractus is a medium-bodied species that inhabits forested rivers and other wetlands in Central and West Africa. As its name suggests, the slender-snouted crocodile is a longirostrine species adapted for the speed necessary of a highly aquatic, piscivorous lifestyle [17][18][19]. O. tetraspis is a small, stocky crocodilian that can be found in a broad spectrum of habitats including inundated forests, mangrove swamps, and papyrus marshes. The Nile crocodile (Crocodylus niloticus) is among the most studied of all crocodilian species. The current distribution of C. niloticus in the study area is limited to coastal lagoons surrounded by savannah-forest mosaic, including frequent use of the marine environment for dispersal and seasonal movements [20, M. J. Eaton and M. H. Shirley pers. obs.]. Previous studies have shown that these three divergent species exhibit different immune activities toward different bacterial species [21].
Considering their protected status, little is known about crocodilian physiology and biochemistry. However, during the past five years, several studies have focused on the innate immunity of crocodilians [21][22][23][24][25][26]. Nevalainen et al. [27] first showed that crocodilians (Crocodylus porosus and Crocodylus siamensis) express serum PLA 2 activity. Later, Merchant et al. showed that the American alligator (Alligator mississippiensis) [28] exhibited serum PLA 2 activity. This study was undertaken to determine the serum PLA 2 activity of three diverse species of Centeral African crocodiles.

Treatment of Animals.
Crocodiles were captured from the N'Dougou Lagoon, Bongo River, and Nyanga River areas of the Gamba Complex region in Gabon, Africa. Animals were captured using standard crocodilian capture techniques, including by hand, tongs, locking snare, or darting [29]. Blood was collected from crocodiles via the spinal vein [30,31]. Five mL of whole blood were collected from animals that were less than one meter in total length, and larger volumes were collected from larger crocodilians, commensurate with length and body condition. All animals were released at the site of capture. All of the activities were approved by McNeese State University and University of Florida Animal Care and Use Committees. The serum samples for each species were pooled (14-30 per species) such that average enzymatic activities could be obtained for each species.

Labeling of Bacteria.
One-mL cultures of E. coli bacteria were grown overnight at 37 • C in nutrient broth. These cultures were used to inoculate one liter cultures. These cultures were incubated for 24 hours in the presence of 1 mg of BODIPY FL C 16 (dissolved in one mL of DMSO). 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, the bacterial pellet was resuspended in 30 mL of sterile isotonic saline and frozen at −20 • C until ready for use. 2 Assay. The method used for the determination of crocodile serum PLA 2 enzyme activity has been recently described [28]. Crocodile serum 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 serum volume on PLA 2 activity, different amounts of crocodile serum 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 mL of stop buffer (100 mM Tris-HCl, pH 7.4 with 15 mM EDTA) and were then centrifuged at 16,000 ×g to pellet the labeled bacteria, and 650 μL of each reaction were removed to a one 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 crocodile PLA 2 activities.

Statistics and Controls.
The fluorescent intensity of each sample was compared to a standard curve of pure product to determine the nmols of product formed. The fluorescent intensities of each sample were corrected for background fluorescence by subtraction of a reagent blank in the absence of serum. Each data point represents the means ± standard deviation for four independent determinations. The results obtained from each experiment were subjected to analysis of variance using Scheffe's post hoc comparisons [32].

Results
The results of the serum volume-dependent PLA 2 activity for O. tetraspis, M. cataphractus, and C. niloticus are displayed in Figure 1. Substantial PLA 2 activity (P < 0.01) was measured in all three crocodilian species with the use of only one μL of serum. The PLA 2 activities measured for Osteolaemus and Mecistops were similar to each other throughout the entire range of serum volumes. However, the PLA 2 activity for C. niloticus was substantially higher than that of the other two species at serum volumes of 20 μL and higher (P < 0.01). At low serum volumes, the activity increased rapidly and then increased more slowly at volumes greater than 20 μL.
The effect of BPB, a specific PLA 2 enzyme inhibitor, on the PLA 2 activity of crocodile serum is illustrated in Figure 2.   2342 ± 263, and 3919 ± 204 nmol, respectively. However, incubation of serum from O. tetraspis, M. cataphractus and C. niloticus serum with 2 mM BPB resulted in a 65%, 55%, and 75% inhibition of PLA 2 activity, respectively. An increase in the BPB concentration to 5 and 10 mM only increased the enzyme inhibition slightly. Figure 3 shows the time-dependent PLA 2 -dependent hydrolysis of BODIPY by three species of Central African crocodiles. Accumulation of product occurred rapidly during the first 20 min. of incubation with substrate, and then decreased after 20 min. Serum from C. niloticus exhibited a higher rate of product formation than O. tetraspis and M. cataphractus serum. Enzyme activity was detected as early as one minute after incubation of substrate with serum from all three crocodilian species. Within 10 minutes of incubation with substrate, the C. niloticus serum exhibited statistically higher activity (P < 0.01) than that from O. tetraspis or M. cataphractus. Figure 4 illustrates the temperature-dependent PLA 2 serum exhibited by the three African crocodile species. The thermal profile for C. niloticus serum PLA 2 showed an increase in activity throughout the temperature range. However, the accumulation of product at the highest temperature (3299 nmol, 40 • C) was only 41% higher than that recorded at the lowest temperature (2342 nmol, 5 • C). In contrast, M. cataphractus serum PLA 2 activity increased from 5-30 • C, where it peaked and then decreased at 35 and 40 • C. The thermal profile for O. tetraspis serum PLA 2 increased steadily from 5-20 • C, where it remained relatively constant from 20-40 • C.
The effects of crocodile serum PLA 2 on cleavage of fatty acids from eight different bacterial species are shown in Figure 5. In general, the serum PLA 2 from all three species of crocodilians showed high activity against Enterobacter cloacae, Klebsiella oxytoca, and Streptococcus faecalis, while the lowest activities were recorded against Staphylococcus aureus. Serum from C. niloticus was more active (P < 0.01) against Enterobacter cloacae and Salmonella typhi than that from the other two crocodilian species. However, serum from O. tetraspis exhibited higher activity against Providencia stuartii and Streptococcus pyrogens, relative to serum from the other two African crocodilians.

Discussion
Lipases, which are subsets of esterase enzymes, constitute a large group of enzymes that are capable of hydrolysis of lipid ester bonds [33]. Lipases are utilized for a broad variety of biological functions, including signal transduction, synthesis of hormones, cholesterol metabolism, serum lipoprotein balance, innate immunity, and so forth. Because of the possibility that the hydrolysis activities observed were due to enzymes other than PLA 2 , the effects of BPB, a specific inhibitor of PLA 2 activity [34], were observed on the cleavage of fluorescently labeled fatty acid from the surface of E. coli bacteria (Figure 2). The data clearly show that the BPB inhibits fluorescent product formation in the serum of all three species in a concentration-dependent manner. These data indicate that the lipolysis activities measured are likely due to the presence of PLA 2 in crocodilian serum. It is worthy to note that another enzyme with PLA 2 -like activity, PAFacetylhydrolase, is not inhibited by BPB. However, it has also been shown that this enzyme does not require Ca 2+ and thus would not be inhibited by PLA 2 [35]. Therefore, any PAF-acetylhydrolase activity in the serum would have been subtracted as background activity due to the fact that the reaction would not have been stopped by the addition of Stop Buffer, which contained EDTA. All three West African crocodilian species exhibited high levels of serum PLA 2 , relative to other crocodilian species [28]. However, C. niloticus showed overall higher PLA 2 activity than O. tetraspis and M. cataphractus with respect to Product formed (nmol)

Bacterial species
Crocodylus niloticus Mecistops cataphractus Osteolaemus tetraspis the volume of serum (Figure 1), more rapid accumulation of product in kinetic experiments (Figure 3), and higher activity across a broad range of temperatures ( Figure 4). This could potentially be a result of the more aggressive lifestyle of C. niloticus, relative to the other two species. C. niloticus is a highly social species with well-established breeding and dominance hierarchies, through aggressive maintenance of territories, which often results in extensive injury [36][37][38]. This might lead to an increased rate of injury during feeding and/or territory defense, compared to Osteolaemus and Mecistops, and thus the need for more potent innate immunity. The physiology, biochemistry, and metabolic rates of ectothermic vertebrates are largely dependent on the temperatures of their environments [39]. Serum enzyme activities for crocodilians have been shown to be extremely temperature dependent [40]. For instance, the serum complement system in Alligator mississippiensis [22], Crocodylus porosus and Crocodylus johnstonii [41], Caiman latirostris [42], and Crocodylus acutus [43] are all temperature dependent. It is interesting to note that serum PLA 2 activity for both O. tetraspis and M. cataphractus increased from 5-20 • C ( Figure 4) and then remained relatively constant or decreased slightly at higher temperatures (35 and 40 • C). However, the PLA 2 activity for C. niloticus increased throughout the entire range of temperatures observed. This might be a result of the temperature differences in the habitat selection for the three species in this study.
Biochemistry Research International 5 While all three crocodilians in this study can be found syntopically, C. niloticus, M. cataphractus, and O. tetraspis exhibit niche partitioning through differential foraging, nesting ecology, habitat preference, and morphology. O. tetraspis is usually found in flooded forests and other small wetlands/waterways under canopy cover in heavily forested areas [18,44,45]. In addition, this species makes extensive use of underground burrows [16,18,19]. Similarly, M. cataphractus typically inhabits rivers, flooded forests, and swampy wetlands in heavily forested areas [17,18,44]. In contrast, ideal C. niloticus habitat is more open and includes exposed water and ample open shoreline for basking and nesting [46,47]. The habitat chosen by C. niloticus is more prone to higher temperatures due to the lack of shaded areas, while the forested habitats most often selected by O. tetraspis and M. cataphractus would tend to be more shaded and thus lower in temperature. Therefore, observed differences in temperature profiles and optima for the PLA 2 activities in these species may reflect microclimatic variation in preferred habitats.
This observed pattern may be supported by the enzyme thermodynamic profiles seen in crocodilians that inhabit more temperate versus tropical latitudes. Temperate species exhibit lower temperature optima than more tropical species [22]. Although serum PLA 2 activities for both O. tetraspis and M. cataphractus were lower than C. niloticus, they were both approximately 30% higher than that measured in Alligator mississippiensis [28]. A. mississippiensis is one of the most temperate crocodilians, even commonly found in regions with frequent winter freeze events [47]. Though, while the absolute amount of product formation was higher in the African crocodile species, the rate of product formation was lower than that for A. mississippiensis (Figure 3) [28]. It may be that species occurring in colder climates are adapted to quicker immune response to counteract the immune suppression caused by lower core body temperatures. Lower absolute product formation with high initial formation rates are mirrored in other temperately distributed species (e.g., Caiman latirostris) [42].
The species relationships in enzymatic response observed in this study could also represent underlying evolutionary relationships in innate immunity [48,49]. Recent studies in molecular phylogenetics have confirmed a sister taxa relationship between Osteolaemus and Mecistops, which form a clade sister to the true crocodiles of the genus Crocodylus [50][51][52][53][54]. PLA 2 temperature profiles and optima for Crocodylus acutus mirrored that of C. niloticus, while Osteolaemus and Mecistops are intermediate from that seen in alligatorids (Alligator mississippiensis [28], Caiman latirostris and Caiman yacare (Merchant, unpublished observations)). Molecular and fossil dating estimates confirm that the family Crocodylidae, and in particular the genus Crocodylus, is the newest and most derived member of the Crocodylia, while the Alligatoridae is the oldest and most ancestral clade [50,[52][53][54][55][56]. These results suggest an evolutionary basis for enzyme activity which supports results from previous studies using immunological data to inform phylogenetics of the Crocodylia [57,58].
Serum from all three West African crocodilians exhibited substantial PLA 2 activities toward all eight bacterial species tested. This is not surprising, considering that PLA 2 is a nonspecific enzyme with activity that is not dependent on the presence of particular antigens. The activities of PLA 2 enzymes depend on the content (lipid content, fluidity, charge density, etc.) of the membrane [59][60][61]. It is interesting to note that all three crocodiles exhibited PLA 2 activities against all eight bacteria, which included both Gram-positive (n = 3) and Gram-negative (n = 5) species. These results indicate that crocodilian PLA 2 is potentially effective against a broad spectrum of diverse bacteria.
This study has provided evidence of differential PLA 2 expression in three species of diverse African crocodilian species. The PLA 2 activity was serum volume-, time-, and temperature-dependent. The PLA 2 activities were broad spectrum in character, affecting eight different bacterial species and also inhibited by BPB. Although this investigation did not include in vivo studies, it is reasonable to expect that serum PLA 2 constitutes an important component of crocodilian innate immunity.