Characterization of serum complement activity in serum of the Komodo dragon (Varanus komodoensis)

Abstract

Incubation of different volumes of serum from the Komodo dragon (Varanus komodoensis) with sheep red blood cells (SRBCs) resulted in volume-dependent hemolysis, as measured spectrophotometrically at 540 nm. The hemolysis occurred rapidly, with almost 90% of the hemolytic activity occurring within 20 min of incubation. A thermal profile showed that Komodo dragon serum exhibited low activity from 5- 20℃, but exerted maximum activity at 35℃, which was substantially reduced at 40℃. The maximum activity was observed near optimal temperatures to which Komodo dragons thermoregulate. Mild heat treatment of Komodo dragon serum (56℃, 30 min) depleted the ability to hemolyze SRBCs. In addition, preincubation of Komodo dragon serum with only 5 mM EDTA or phosphate, both chelators of divalent metal ions, reduced the hemolytic activity sharply. These results indicate that the hemolytic activity was due to the presence of a potent serum complement system. Incubation of Komodo dragon serum with 5 mM EDTA and 15 mM Ca2+ or Mg2+, but not Ba2+, Zn2+, or Fe2+, completely restored activity. These results indicate that Komodo dragon serum complement activity requires the presence of Mg2+ or Ca2+. This is the first assessment of innate immune activity of a Varanid.

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Merchant, M. , Henry, D. , Falconi, R. , Muscher, B. and Bryja, J. (2012) Characterization of serum complement activity in serum of the Komodo dragon (Varanus komodoensis). Advances in Biological Chemistry, 2, 353-359. doi: 10.4236/abc.2012.24043.

1. INTRODUCTION

Innate immunity comprises that part of microbial defense which responds in a non-specific manner to infiltration by microbes. Serum complement is one of the first lines of defense against infection, and is made up of approximately 11 different proteins that can be activated by three different mechanisms, the classical (antibody-dependent) pathway [1], the alternative pathway [2], and the lectin-dependent pathway [3]. Once activated, all three mechanisms work in a proteolytic cascade fashion, resulting in the eventual formation of a multi-protein “membrane attack complex” [4] in the outer membrane of the microbe, causing leakage of cellular contents and lysis. Some of the proteolytic complement protein fragments also act to opsonize microbes for phagocytosis [5], serve as attractants for the chemotaxis of macrophages and neutrophils to the site of infection [6], and function as anaphylactic factors [7], causing mast cell degranulation and vascular permeability [8]. Deficiencies in complement proteins have been associated with susceptibility to bacterial infection [9], Leiner’s disease [10], fulminant hepatic failure [11], and many other clinical conditions [12]. Serum complement is among the most important components of innate immunity, and has been identified and characterized across a broad spectrum of diverse taxa, including mammals [13] and ancient invertebrates [14,15].

The Komodo dragon (Varanus komodoensis) is the world’s largest lizard. Its range is restricted to several small islands in the Indonesian archipelago. These animals are carnivorous reptilians, and are known to feed on prey items much larger than themselves. Komodo dragons typically lie at ambush points and bite their prey, delivering a heavy load of infectious bacteria from their saliva [16,17], and then often follow their prey for days, or even weeks, before they succumb to systemic infection. In addition, Komodo dragons have been reported to deliver potent toxins in their bites that prevent coagulation and cause prey items to succumb to systemic shock [18]. Although these animals exhibit aggressive behaviors toward members of their own species during territorial disputes, and often bite each other during feeding frenzies [19], Komodo dragons do not seem to experience the same fate as their prey. This seems to indicate that these animals have developed an immune system that allows them to combat potentially high loads of pathogenic bacteria. However, because of their remote location and endangered status, little is known concerning the physiology, biochemistry, and immunology of these apex predators. Recent studies in our laboratory have shown that the serum from Komodo dragons exhibits potent and broad-acting antibacterial properties [20]. This study was undertaken to examine the serum complement system of the Komodo dragon, and is the first characterization of an innate immune component in a Varanid.

2. MATERIALS AND METHODS

Chemicals and biochemicals: Sheep red blood cells (SRBCs) were purchased from Rockland Immunochemicals (Gilbertsville, PA, USA). Ethylene diamine tetraacetate (EDTA), CaCl2, MgCl2, BaSO4, FeCl2, and ZnCl2 were purchased from Sigma Chemical Company (St. Louis, MO, USA).

Collection of samples: Blood samples were collected from Komodo dragons at the Houston and San Antonio zoos. Blood was drawn from the tail vein, transferred to Vaccutainer™ tubes, and allowed to clot for at least five h before the serum was collected. The amount of blood collected from each individual depended on the size of the animal, and was at the discretion of the attending veterinarian at each institution. Blood was collected from the tail caudal veins three adults (20 - 81.5 kg) and five juveniles (1.5 - 6.2 kg), transferred to Vaccutainer™ tubes, and allowed to clot for at least five hr before serum was collected by centrifugation. The serum was pooled so that average antibacterial values for this species could be generated. The collection of blood from these animals was conducted in accordance with the Animal Care and Use institutional policies of the Houston and San Antonio Zoos.

Serum volume-dependent SRBC hemolysis: The functionality of the Komodo dragon serum complement system of proteins was examined using a SRBC lysis assay modified from the method of Mayer [21], and previously described for crocodilians [22]. To measure the volume dependence of SRBC activity on Komodo dragon serum, different volumes of serum (0 - 15 µL) were diluted to 150 µL total volume with a 0.9% saline solution and then 150 µL of 2% SRBCs was added to the solution. The samples were allowed to incubate for 60 min, followed by centrifugation at 16,000 × g for 5 min at ambient temperature. The optical densities of the resulting supernatants were measured using the Bio-Rad Benchmark Plus™ microplate spectrophotometer at 540 nm.

Kinetic analysis of SRBC hemolysis: For the determination of the hemolytic kinetic profile, a solution composed of 250 µL of Komodo dragon serum, 5.15 mL of a 0.9% saline solution, and 5.40 mL of a 1% SRBC was prepared. In quadruplicate, 290 µL of solution was dispensed into separate vials. At different time intervals (0 - 120 min), the samples were centrifuged and transferred to microtiter plates to measure optical density (540 nm) as described above.

Temperature-dependent SRBC hemolysis: Komodo dragon-mediated serum complement SRBC hemolysis activity was also assayed at different temperatures. Aliquots of Komodo dragon serum (5 μL) and 145 μL of saline were incubated at different temperatures (5˚C - 40˚C in increments of 5˚C) for 10 min. The reaction was initiated by the addition of 150 μL of 2% SRBCs, and allowed to continue for 30 min. The samples were then centrifuged (16,000 × g) and transferred to microtiter plates to measure optical density (540 nm) as described above.

Effects of divalent metal ions and heat on Komodo dragon complement: To examine the effects chelators of divalent metal ions on the Komodo dragon complement protein system, 100 μL of serum solution with SRBC and saline solution was spiked with either 1 μL of 500 mM EDTA or 500 mM Na3PO4. The resulting serum samples (5 μL), which contained 5 mM EDTA or Na3PO4, were incubated (30 min) with 145 μL of saline and 150 μL of 1% SRBCs. Another aliquot of serum (100 μL) was heated to 56˚C for 30 min. The serum was allowed to cool to ambient temperature, and then 5 μL was incubated (30 min) with 145 μL of saline and 150 μL of 1% SRBCs. These samples were centrifuged at 16,000 × g for 5 min, and the optical density of each sample was measured using the Bio-Rad Benchmark Plus™ microplate spectrophotometer at 540 nm.

To determine the specific requirement of Komodo dragon serum complement activity for divalent metal ions, serum was treated with 5 mM EDTA in the absence, and in the presence, of 15 µM CaCl2, MgCl2, ZnCl2, BaSO4, and FeCl2 (Table 1). The samples (150 μL) were then incubated with 150 μL of 2% SRBCs for 30 min at ambient temperature. The samples were centrifuged, and the optical densities of the supernatants were measured at 540 nm as described above.

Statistics and controls: All results presented in this study represent the means ± standard deviations for four independent determinations. The results are expressed as the % maximum of a positive control for SRBC hemolysis, which was generated by treating SRBCs with 0.1% Triton-X detergent (v/v), and passing the cells repeatedly through a TB syringe until all of the cells had been disrupted (10 passes), as determined by examination under 400× magnification. The statistical comparisons between groups were conducted using analyses of variance with Duncan’s post-hoc comparisons, and p < 0.05 was chosen as the standard for statistical significance.

Table 1. Effects of heat treatment and EDTA on hemolytic activity of serum from the Komodo dragon. Komodo dragon serum was treated with EDTA, or subjected to mild heat treatment, and then exposed to 1% SRBCs for 30 min at ambient temperature. The results are expressed as the percentage of maximum lysis, and represent the means ± standard deviation for four determinations.

3. RESULTS

Exposure of different dilutions of serum from Varanus komodoensis to SRBCs resulted in volume-dependent hemolysis (Figure 1). Treatment of a 500 μL suspension of 1% SRBCs (v/v) with only 3 μL of serum resulted in measureable (p < 0.5) hemolysis, as 5.5% ± 0.9% of the SRBCs were hemolyzed. Significant hemolysis of SRBCs occurs with infusion of a small quantity of Komodo dragon serum. Increased volumes of 5 and 7 μL of serum produced 60.0% ± 6.5% and 93.6% ± 2.6% hemolysis, respectively. However, a further increase to 10 μL of serum produced 99.3% ± 0.6% hemolysis, which indicated that a near-maximal response could be obtained by the use of only 7 μL of serum.

Figure 2 shows the kinetic response of the hemolytic activity of serum from Varanus komodoensis, at two different volumes, toward SRBCs. At the lower volume (3 mL), an increase in SRBC hemolysis was not observed until 30 min of incubation with Varanus komodoensis serum. The activity increased to only 15.9% ± 0.3% of maximum activity at 60 min. However, the activity increased sharply to 91.2% ± 0.8% at 120 min. In contrast, the higher serum volume (7 mL) produced a sharp increase (26.0% ± 2.7%) at only 5 min of incubation with 1% SRBCs. The activity increased rapidly, in a linear fashion, to 88.5% ± 2.8% at 20 min. The linear relation of the increase (y = 0.0464x – 0.011) exhibited a correlation coefficient of 0.985.

Incubation of 1% SRBCs with serum from Varanus komodoensis at different temperartures (5˚C - 40˚C) resulted in temperature-dependent hemolysis (Figure 3). Hemolysis at 5˚C - 20˚C was low (7.4% - 9.5% of maximum), and was not statistically different between these

Figure 1. Titer-dependent hemolysis of SRBCs by Komodo dragon serum. Incubation of 1% (v/v) SRBCs with different volumes of Komodo dragon serum for 30 min resulted in a volume-dependent hemolysis, as measured spectrophotometrically as described in the Materials and Methods. The results are expressed as % of maximum hemolysis, relative to a positive control, and represent the means ± standard deviations, and represent the results of four independent determinations.

Figure 2. Kinetic analysis of SRBC hemolysis by Komodo dragon serum. Incubation of 1% SRBCs (v/v) with Komodo dragon serum, diluted in normal saline, for different amounts of time resulted in a time-dependent hemolysis. The results are expressed as the % of maximum hemolysis, relative to a positive control, and represent the means ± standard deviations, and represent the results of four independent determinations.

temperatures (p > 0.05). The hemolytic activity increased (p < 0.05) at 25˚C (28.7% ± 2.8%), 30˚C (53.3% ± 2.3%), and 35˚C (68.7% ± 5.1%). However, the activity at 40˚C resulted in decreased (p < 0.05) activity, to 52.0% ± 2.6% of maximum activity.

Table 1 illustrates the effects of mild heat treatment and chelators of divalent metal cations on the hemolysis of SRBCS by Komodo dragon serum. Incubation of Komodo dragon serum at 56˚C for 30 min decreased its

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] Cooper, N.R. (1985) The classical complement pathway: Activation and regulation of the first complement component. Advances in Immunology, 37, 151-216. doi:10.1016/S0065-2776(08)60340-5
[2] Pillemer, L., Blum, L., Lepow, I., et al. (1954) The properdin system and immunity: I. Demonstration and isolation of a new serum protein, properdin, and its role in immune phenomena. Science, 120, 279-285. doi:10.1126/science.120.3112.279
[3] Fujita, T., Matsushita, M., and Endo, Y. (2004) The lectin- complement pathway: Its role in innate immunity and evolution. Immunological Reviews, 198, 185-202. doi:10.1111/j.0105-2896.2004.0123.x
[4] Muller-Eberhard, H.J. (1986) The membrane attack complex of complement. Annual Reviews in Immunology, 4, 503-528. doi:10.1146/annurev.iy.04.040186.002443
[5] Tofte, R.W., Petersen, P.K., Kim, Y., et al. (1980) Opsonization of four Bacteroides species: Role of the classical complement pathway and immunoglobulin. Infection and Immunity, 2, 693-696.
[6] Ward, P.A., Chocrane, C.G. and Mueller-Eberhard, H.J. (1965) The role of serum complement in chemotaxis of leukocytes in vitro. Journal of Experimental Medicine, 122, 327-346. doi:10.1084/jem.122.2.327
[7] Rhyne, M.B. and Germuth F.G. (1961) The relationships between serum complement activity and the development allergic lesions in rabbits. Journal of Experimental Medicine, 114, 633-646. doi:10.1084/jem.114.5.633
[8] Muller-Eberhard, H.J. (1988) Molecular organization and function of the complement system. Annual Reviews in Biochemistry, 57, 321-347. doi:10.1146/annurev.bi.57.070188.001541
[9] Miller, M.E. and Nillson, U.R. (1970) A familial deficiency of the phagocytosis-enhancing activity of serum related to a dysfunction of the fifth component of complement (C5). New England Journal of Medicine, 282, 354-358. doi:10.1056/NEJM197002122820702
[10] Jacobs, J. and Miller, M. (1972) Fatal familial Leiner’s disease: A deficiency of the opsonic activity of serum complement. Pediatrics, 49, 225-232.
[11] Wyke, R.J., Rajkovic, I.A., Eddleston, A.L., et al. (1980) Defective opsonisation and complement deficiency in serum from patients with fulminant hepatic failure. Gut, 21, 643-649. doi:10.1136/gut.21.8.643
[12] Morgan, B.P. and Walport, M.J. (1991) Complement deficiency and disease. Immunolology Today, 12, 301- 306. doi:10.1016/0167-5699(91)90003-C
[13] Ruddy, S., Gigli, I. and Austen, K.F. (1971) The complement system of man. New England Journal of Medicine, 287, 489-495. doi:10.1056/NEJM197209072871005
[14] Day, N., Gewurz, H., Johannsen, R., et al. (1970) Complement and complement-like activity in lower vertebrates and invertebrates. Journal of Experimental Medicine, 132, 941-950. doi:10.1084/jem.132.5.941
[15] Gigli, I. and Austen, K.F. (1971) Phylogeny and function of the complement system. Microbiology, 25, 309-332. doi:10.1146/annurev.mi.25.100171.001521
[16] Pianka, E.R. and King, D.R. (2004) Introduction. In: Pianka, E.R., King, D.R. and King, R.A., Eds., Varanoid Lizards of the World, Indiana University Press, Bloom- ington, 3-9.
[17] Montgomery, J.M., Gillespie, D., Sastrawan, P., Freeking et al. (2002) Aerobic salivary bacteria in wild and captive Komodo dragons. Journal of Wildlife Diseases, 38, 545- 551.
[18] Fry, B.G., Wroe, S., Teeuwisse, W., et al. (2009) A central role for venom in predation by Varanus komodoensis (Komodo Dragon) and the extinct giant Varanus (Mega- lania) priscus. Proceedings of the National Academy of Science, 106, 8969-8974. doi:10.1073/pnas.0810883106
[19] Earley, R.L., Attum, O. and Eason, P. (2002) Varanid combat: Perspectives from game theory. Amphibia-Reptilia, 23, 469-485. doi:10.1163/15685380260462374
[20] Merchant, M., Henry, D, Falconi, R., et al. (2012) Anti-bacterial activities of serum from the Komodo dragon (Varanus komodoensis). Microbiology Research, in Press.
[21] Mayer, M.M. (1967) Complement and complement fixation. In: Kabat, E.A. and Mayer, M.M., Eds., Experimental Immunochemistry, 2nd Edition, Thomas, Springfiled, 133-240.
[22] Merchant, M., Hammack, T., Dronette, J., et al. (2006) Assessment of innate immune activity of crocodilians using a spectroscopic assay based on the hemolysis of sheep red blood cells. Spectroscopy Letters, 39, 337-343. doi:10.1080/00387010600781290
[23] St. Georgiev, V. (2009) Mammalian host defenses: Innate and adaptive immunity. National Institute of Allergy and Infectious Diseases, NIH Infectious Disease, Part III, 577-626.
[24] Cuchens, M.A. and Clem, L.W. (1979) Phylogeny of lymphocyte heterogeneity. IV. Evidence for T-like and B-like cells in reptiles. Developmental and Comparative Immunolology, 3, 465-475. doi:10.1016/S0145-305X(79)80042-7
[25] Merchant, M., Williams, S. and Hardy, R. (2008) Super- oxide production by leukocytes in the American alligator (Alligator mississippiensis). Comparative Biochemistry and Physiolology B, 152, 67-71. doi:10.1016/j.cbpb.2008.09.089
[26] Merchant, M., Heard, R. and Monroe, C. (2009) Characterization of phospholipase A2 activity in serum of the American alligator (Alligator mississippiensis). Journal of Experimental Zoology A, 311, 662-666. doi:10.1002/jez.553
[27] Merchant, M., McFatter, J., Mead, S., et al. (2009) Identification and characterization of serum complement activity in the American crocodile (Crocodylus acutus). Vetereinary Immunolology and Immunopathology, 133, 165-169. doi:10.1016/j.vetimm.2009.07.016
[28] Merchant, M., Monroe, C. and Falconi, R. (2009) Characterization of dipeptidyl peptidase IV enzyme activity in the blood of the American alligator (Alligator mississippiensis). Comparative Biochemistry and Physiolology B, 154, 341-345. doi:10.1016/j.cbpb.2009.07.010
[29] Siroski, P., Merchant, M., Parachú Marcó, V., et al. (2010) Characterization of serum complement activity of the broad snouted caiman (Caiman latirostris, Crocodilia: Alligatoridae). Zoological Studies, 49, 64-70.
[30] Boshra, H., Gelman, A.E. and Sunyer, J.O. (2004) Structural and functional characterization of complement C4 and C1s-like molecules in teleost fish: Insights into the evolution of classical and alternative pathways. Journal of Immunology, 173, 349-359.
[31] Sunyer, J.O. and Lambris, J.D. (1998) Evolution and diversity of the complement system of poikilothermic vertebrates. Immunological Reviews, 166, 39-57. doi:10.1111/j.1600-065X.1998.tb01251.x
[32] Sunyer, J.O., Zarkadis, I.K. and Lambris, J.D. (1998) Complement diversity: A mechanism for generating immune diversity. Immunolology Today, 19, 519-523. doi:10.1016/S0167-5699(98)01341-3
[33] Merchant, M., Roche, C., Sweeney, A., et al. (2005) Identification of serum complement activity in the American alligator (Alligator mississippiensis). Comparative Biochemistry and Physiology B, 141, 281-288. doi:10.1016/j.cbpc.2005.03.009
[34] Merchant, M. and Britton, A. (2006) Characterization of serum complement activity of the saltwater (Crocodylus porosus) and freshwater (Crocodylus johnstoni) crocodiles. Comparative Biochemistry and Physiology A, 143, 488-493. doi:10.1016/j.cbpa.2006.01.009
[35] Major, S., Fontenot Jr., C.L., Pojman Sr., J.A., et al. (2011) Serum complement activity in the three-toed amphiuma (Amphiuma tridactylum). Comparative Immunolology, Microbiology, and Infectious Disease, 34, 115- 121. doi:10.1016/j.cimid.2010.04.001
[36] Lutz, D. and Lutz, J.M. (1997) Komodo: The living dragon. Revised Edition, DIMI Press, Salem.
[37] Mayer, M.M. (1967) Complement and complement fixation. In: Kabat, E.A. and Mayer, M.M., Eds., Experimental Immunochemistry, 2nd Edition, R. Thomas, Springfield, 133-240.
[38] Merchant, T., Hammack, T., Sanders, P., et al. (2006) Rapid and inexpensive method for the spectroscopic determination of innate immunity of crocodilians. Spectroscopy Letters, 39, 337-343. doi:10.1080/00387010600781290
[39] Wright, S.D. and Levine, R.P. (1981) How complement kills E. coli: I. Location of the lethal lesion. Journal of Immunology, 127, 1146-1151.
[40] Soltis, R.D., Hasz, D., Morris, M.J., et al. (1979) The effect of heat inactivation of serum on aggregation of immunoglobulins. Immunology, 36, 37-45.
[41] Levine, L., Osler, A.G., et al. (1953) The role of calcium and magnesium ions in complement fixation and immune hemolysis: III. The respective roles of calcium and magnesium ions in immunehemolysis. Journal of Immunology, 71, 374-379.
[42] Merchant, M., Verret, B. and Elsey, R.M. (2005) Divalent metal requirements for serum complement activity in the American alligator (Alligator mississippiensis). Comparative Biochemistry and Physiology B, 141, 289-293.

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