Journal of Undergraduate Research
Volume 3, Issue 7 - April 2002

Micrococcus Lysodeikticus sensitivity to lysozyme treatment from the hemolymph of the American oyster, Crassostrea virginica

Natalie Vincent

ABSTRACT

Hemolymph from the oyster, Crassostrea virginica, contains a lysozyme-like enzyme that may function as a defense mechanism against many of the same bacteria that cause human infections. The open circulatory system of the oyster enables an easy extraction of the lysozyme via a sterile syringe. The lysozyme can then be maintained in a phosphate buffer of pH 5.0-5.5. The lysozyme possesses lytic abilities towards the Gram-positive bacteria Micrococcus lysodeikticus. Numerous assays were performed in order to determine the precise amount and concentration of oyster lysozyme needed to completely inactivate the Micrococcus cell culture. In order to establish the optimal lysozyme/bacteria ratio the absorbance of various mixtures will be measured by means of a spectrophotometer. The results of this experiment could probably be applied to other assays containing bacteria associated with ready-to-eat produce.

INTRODUCTION

Patients Oysters are filter feeders and free-living bacteria can be a major source of nutrition. These bivalves require a digestive system capable of hydrolyzing prokaryotic cells while maintaining a defense mechanism against pathogenic bacteria (1). It is known that the hemolymph of the oyster, Crassostrea virginica, contains a "lysozyme like" enzyme serving as part of an internal defense mechanism (2). This enzyme is produced in the hemocytes and secreted into the mantle hemolymph. Studies conducted on this enzyme have led to its further classification as a lysozyme (3). In order for an enzyme to be classified as a lysozyme it must reduce the turbidity of intact bacterial cell walls, release reducing sugars, and liberate amine sugars and/or muramic acid (4). Along with the oyster lysozyme, egg white lysozyme fulfills all three requirements and therefore is used as a control in enzyme studies (5).

Oysters have the capacity to filter 3-4 gallons of water per hour (6). This process removes particles and pollutants from surrounding waters exposing the oyster to a constant challenge by pathogenic microbes. These microbes are degraded intracellularly by the oyster lysozyme, EC 3.2.1.17, N-acetylmuramide glycanohydrolase. Inactivation occurs at the cell wall's thick peptidoglycan layer of many Gram-positive bacteria, including Micrococcus lysodeikticus (3). The purpose of these experiments were to determine the optimal concentration of oyster lysozyme necessary to completely inactivate the bacterial cell culture. This study will initially determine this concentration active against M. lysodeikticus. A bovine serum albumin will be used as a standard, as it has presented lysis activity towards M. lysodeikticus cultures in past experiments (7).

METHODS AND MATERIALS

Oyster Collection

During the course of the study the oysters, C. virginica, were collected from Cedar Key Bay off the coast of Cedar Key, FL. Within fifteen hours of collection Northwest Seafood Company delivered the oysters to University of Florida. The oysters were stored for up to four days in a walk in cooler at a temperature of 5º C. The oysters were not used after four days out of the water because of the possibility of decreased hemolymph activity.

Hemolymph collection and substrate preparation

Hemolymph was withdrawn from the abductor muscle located one half an inch anterior to the hinge plate. A coarse file was used to provide a small opening in the shell, allotting for the insertion of a sterile 18-guage half inch hypodermic needle. The oyster's open circulatory system provided a homogenous hemolymph composition throughout the oyster, thus samples taken from all sites were assumed to be identical. The hemocytes were separated from the hemolymph by centrifuging at 4000 x g for 15 minutes at 4ºC. The resulting supernatant (serum), containing more active enzymes was decanted and placed on ice. The lysozyme was diluted with a phosphate buffer of pH 5.5. The sample was the frozen at –6ºC and thawed prior to experimental procedures. M lysodeikticus, obtained from Sigma, was sustained in a phosphate buffer, pH 6.2, and 4mg/mL of substrate in the form of dried M. lysodeikticus cells. The purified egg white lysozyme, also provided by Sigma was used as a standard.

Specific activity of lysozyme

Lysozyme activity against M. lysodeikticus was determined and compared to the avian control. Numerous assays were performed to determine the minimal amount of lysozyme needed to completely deactivate the bacteria culture. Absorbency was read by a SPECTRAmax PLUS384 spectrophotometer at 450 nm. Assays were run using 96-well microtiter plates and results were quantitated with SPECTRAmas PLUS software installed on a connected PCU. Absorbency readings were documented every five minutes for sixty minutes. The following ratios of M. lysodeikticus:avian lysozymeand M. lysodeikticus:oyster lysozyme were tested; 175:25, 150:50, 125:75, 100:100, 75:125, 50:150, 25:175. Protein concentrations of hemolymph and avian lysozyme were determined by finding a 280/260 _ ratio (8).

RESULTS

Ratios that displayed a continual decrease in absorbency were as follows; 190:10, 175:25, 150:50, 125:75, 100:100. After multiple runs results indicated that the 175:25 M. lysodeikticus:oyster lysozyme ratio provided the minimal amount of lysozyme necessary, resulting in a steady decrease in absorbency. Figure 1 shows the gradual decrease in absorbance, represented by a negative, decreasing curve.

Figure 1. Oyster lysozyme versus time (175:25)

DISCUSSION

Lysozyme is a ubiquitous enzyme that is widely distributed in the animal kingdom (9). The biological function of this enzyme is believed to be for defense against bacterial infection. This occurs due to the lysozyme's ability to induce bacterial cell lysis by hydrolyzing β1,4 linked glycosidic bonds of the cell wall's peptidoglycan layer (10). Studying the activity of such an efficient defense mechanism can provide a number of applications to the food industry. Human pathogenic bacteria have caused numerous foodborne infections throughout the past decade. In the United States alone, there are on average twenty million pathogenic foodborne cases annually (11). Although a large number of chemicals have been presented as potential food preservatives, only a relatively small number are FDA approved (12). Therefore, finding a means of naturally cleansing raw produce, specifically fruits and vegetables would be of great benefit to the food industry.

A wide variety of human pathogenic bacteria can be found on the surfaces of fruits and vegetables (13). The incidence of microorganisms on these land-grown foods may be expected to reflect the sanitary quality of the processing steps and the biological conditions of the raw product at the time of processing (12). Outbreaks of human disease associated with the consumption of raw fruits and vegetables are frequent in underdeveloped countries and have increased the frequency in developed countries over the past decade (14). Factors contributing to the occurrence of these diseases can be associated with the high volume of international trade, the increasing prevalence of environmental bacteria found in regions of underdeveloped farming, the lack of personal hygiene of those handling the product and the immunotolerance to foreign diseases (14). Also, growing populations have lead to an increased demand for more efficient processing, essentially producing more product in a shorter amount of time. This results in products being held longer and shipped farther before reaching the level of consumption (12). For optimal consumer safety, the sanitation of ready-to-eat foods must be continuously employed and regulated. New approaches are needed to ensure safe products.

The food industry uses several means of reducing bacterial contamination in fruits and vegetables. Irradiation, chlorine, chlorine dioxide, trisodium phosphate and other chemical sanitizers are all means of inhibiting bacterial growth (15). However all of these man-made sanitizers possess some form of disadvantage, whether it is towards the environment, the processing machinery, the product or most important towards the consumer (15). A 1999 March issue of Consumer Reports stirred public concern when researches found unacceptably high levels of pesticides in a number of fruits and vegetables. The researches found that pesticide residues are almost always within legal limits, but many of the limits are higher than what the government considers safe for young children (16). Recent research has given the highest toxicity scores to apples, grapes, green beans, peaches, pears, spinach and winter squash (16). The posed risk of infection in eating these ready-to-eat foods could greatly be reduced by the production of a natural means of cleansing fruits and vegetables. As indicated in this study one possible agent that has gained sufficient attention for its activity towards Gram-positive bacteria is the lysozyme from the oyster, C. virginica. With an optimal bacteria:oyster lysozyme concentration found, assays may now be run on various bacterial substrates common to raw fruits and vegetables. Currently, studies are being conducted with the purpose of further purifying the lysozyme. Preliminary analysis of C.virginica hemolymph using SDS-PAGE revealed the presence of two lysozyme-like proteins with apparent molecular weight of 18.2 Kda and 38.8 Kda respectively (17). Applying this information to the experimental process may enable a finer purification of the lysozyme solution, assisting further testing against other Gram-positive bacteria. Should this lysozyme show inactivation of other Gram-positive bacteria, further testing may reveal its success as a pathogenic rinse reducing foodborne bacteria associated with raw produce.

REFERENCES

  1. Nilsen I., Overbo K., Sandaker E., Sandsdalen E. and K. Sletten. Protein Purification and gene isolation of chlamysin, a cold-active lysozyme0like enzyme with antibacterial activity. Federation of European Biochemical Studies, 1999.
  2. McDade, J. & M. Tripp. Lysozyme in the hemolymph of oyster, Crassostrea virginica. J. Invertebr. Pathol. 9:531-535, 1967.
  3. Rodrick, G. E. & Cheng, T.C. (1974) Kinetic properties of lysozyme from the hemolymph of Crassostrea virginica. J. of Invertebrate Pathology 24:41-48, 1974.
  4. Jolles P. Recent developments in the study of lysozymes. Angew. Chem. 3:28-36, 1964.
  5. Lowry, O., N. Rosebrough, A. Farr, & R. Randall. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951.
  6. Genthner, F., A. Volety, L. Oliver & W. Fisher. Factors influencing in vitro killing of bacteria by hemocytes of Eastern oyster (Crassostrea virginica). Appl. Environ. Microbiol. 65: 3015-3020, 1999.
  7. Shugar D. Measurement of lysozyme activity and the ultraviolet inactivation of lysozyme. Biochem. Biophys. Acta. 8: 302-308, 1952.
  8. Layne E. Spectrophotometric and turbidimetric methods for measuring proteins. Methods Enzymology 3: 47, 1957.
  9. Prager, E.M. & P. Jolles. Lysozyme: Model Enzymes in Biochem. & Biophys. Birkhauser-Verlag Basel, Switzerland 9-31, 1996.
  10. Imoto T., Johnson L. N., North A., Phillips D.C., & A. Rupley. Vertebrate lysozyme, the Enzymes. Academic Press, New York 35-64, 1972.
  11. Council for Agricultural Science and Technology (CAST). Food pathogens: Risks and Consequences, 1994.
  12. Jay J. Modern Food Microbiology, Fifth Edition. International Thomson Publishing. Florence, KY 408, 1996.
  13. Beuchat L. Pathogenic microorganisms associated with fresh produce. J. Food Prot. 59: 204-216, 1996.
  14. Alterkause S.F. & D.L. Swerdlow. The changing epidemiology of foodborne diseases. Am J. Med. Sci. 311: 23-29, 1996.
  15. Berbarde M., Snow W.B., Olivieri O. P. & B. Davidson. Kinetics and mechanisms of bacterial disinfection by chlorine dioxide. Appl. Microbiol. 15: 257-265, 1967.
  16. Child Health Alert. How Safe Are Our Fruits and Vegetables? (1999) <http://www.findartilcles.com/cf_dls/m0867/1999_April/54450677/print.jhtml>
  17. Chu F.E., Cruz-Rodriguez L. & A. Volety. Partial Purification and Characterization of Lysozyme-like Proteins from the Plasma of the Eastern Oyster, Crassostrea Virginia.

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