Broad spectrum antimicrobial activity of leukocyte extracts from the American alligator (Alligator mississippiensis)

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Abstract

Leukocytes were isolated from whole blood of wild alligators by differential sedimentation. The leukocytes were disrupted in 5% AcOH and the crude extracts processed by ultrafiltration. The extracts were subjected to solvent exchange (0.1% AcOH) and the fraction that contained macromolecules between 1 and 10 kDa were subjected to further analyses. The acid extracts of the alligator leukocytes exhibited substantial antimycotic activities against six of eight species of Candida yeast tested. In addition, the alligator leukocyte extracts were effective as antimicrobial agents against 10 of 12 bacterial species, and displayed moderate activity against two enveloped viruses (human immunodeficiency virus-1 and herpes simplex virus-1HF). Kinetic analyses revealed that the antimycotic effects of the leukocyte extract occurred rapidly, with 64% fungal growth inhibition within 3 min of exposure. The molecule(s) responsible for the antimicrobial activities were sensitive to proteases, heat-stable, acid soluble, and in the 1–10 kDa range. These data suggest that alligator leukocytes express cationic peptides that are responsible for their antimicrobial properties.

Introduction

Anecdotal evidence suggests that the American alligator (Alligator mississippiensis) is resistant to microbial infection. Alligators often sustain serious injuries but, despite the aseptic conditions of the marshes and swamps in which they live, heal rapidly and seldom with serious infection (Merchant, unpublished observations). Shaharabany et al. (1999) reported that tissue extracts of the Nile crocodile (Crocodilus niloticus) exhibited antibacterial activities. Furthermore, previous studies in our laboratory indicated that the American alligator (A. mississippiensis) exhibits potent innate immunity against various species of bacteria (Merchant et al., 2003), amoebae (Merchant et al., 2004), and three enveloped viruses (Merchant et al., 2005a). However, these antimicrobial activities were attributed to the serum complement protein system (Merchant et al., 2003, Merchant et al., 2005a, Merchant et al., 2005b).

Antimicrobial peptides have been isolated from the leukocytes from a wide variety of eukaryotic organisms (Yeaman and Yount, 2003). This study was conducted to determine whether the leukocytes of alligators produce compounds that exhibit antimicrobial properties. The data presented in this study demonstrate that alligator leukocyte extracts display antibacterial, antifungal, and antiviral properties, and that these activities are most likely due to the presence of cationic antimicrobial peptides.

Section snippets

Treatment of animals

Adult alligators were captured at night from a boat using a spotlight and a cable snare. Blood samples were drawn from the supravertebral branch of the internal jugular vein (Olson et al., 1977, Zippel et al., 2003) using 3.8 cm 18 gauge heparinized needles and 60 mL syringes, and transferred immediately to 250 mL bottles containing 15 mL of 0.5 M EDTA. The animals were released unharmed within minutes of capture. The whole blood from 15 adult alligators (>1.5 m) was pooled for a total volume of 3 L.

Results

The protein concentration of the alligator leukocyte extract was determined to be 114 μg protein/mL extract using the Bradford assay (Bradford, 1976). This extract was used, undiluted, to investigate its antifungal properties. Alligator leukocyte extract was effective as an antimycotic agent against six of eight pathogenic Candida yeast spp. species tested (Fig. 1). The zones of fungal growth inhibition are expressed in mm and represent the means ± S.D. for four independent determinations. The

Discussion

Antimicrobial peptides have been isolated from a broad variety of phylogenetically diverse organisms (Ganz and Lehrer, 2001). These peptides can be segregated into a variety of classes based on their structures (Boman, 1995). These classes exhibited different mechanisms of antimicrobial action (Yeaman and Yount, 2003). However, all antimicrobial peptides have several factors in common. They all tend to be cationic in nature so that they are able to interact with the negatively charged

Acknowledgements

The authors wish to thank Dr. Michael Berger, Wildlife Division Director of the Texas Parks and Wildlife Department, for access to public lands on which to capture wild alligators.

This research was supported by the McNeese State University Shearman Research Fund and Pfizer Global Research Summer Undergraduate Research Fund.

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