IDENTIFICATION OF PROTEINS WITH NEMATICIDAL ACTIVITY USING AN AXENIC CHEMICALLY-DEFINED NUTRIENT MEDIA ASSAY.

Hocker, James Randolph and Karel Schubert.

Department of Botany-Microbiology, University of Oklahoma,

770 Van Vleet Oval, Norman, OK 73069-9984.

 

This Poster was presented at the Society of Nematologist's meeting in St.Louis, MO on June 22, 1998.

Abstract

An assay was developed to identify proteins with biological activity capable of reducing nematode reproduction. An axenic chemically-defined culture media was used to test several isolated protein fractions from seeds of a tropical legume. The nematode Caenorhabditis elegans was cultured at 20 ° C in 2.5 ml replicated stationary cultures. Populations were observed and recorded every 3 to 4 days over a period of 2-3 weeks. Controls and non-active fractions contained comparable populations. An active fraction was identified after separation by gel-filtration chromatography. Maximum nematode populations containing the active fractions were reduced 70% below the control populations. The reduction in population potential by the biologically active fraction was dependent on concentration. Haemagglutination activity of rabbit, pig and human red blood cells and bioactivity were both observed in the protein fraction with an estimated molecular weight of 50,000 or greater.

Introduction

After initial biological control studies on several agriculturally important pests, a protein extract prepared from the seed of a tropical legume was tested for biological activity against the nematode C. elegans. A portion of the tested protein extract was known to be biologically active toward insect larvae. The following experiments were conducted to determine if these extracts were capable of inhibition nematode growth and reproduction.

A small culture defined media assay was developed to test for biologically active proteins. Large culture volumes are easily contaminated and may be subject to other factors affecting growth, such as aeration. Small culture volumes (2.5 ml) and bottles through which the entire contents could be observed were used to increase ease of population determination and culture manipulation. A chemically defined nutrient media was used in the culture system. The clarity of the media, which was maintained throughout the culture period, allowed a complete count of each population within a culture bottle. The use of a chemically defined media insured the changes observed in population growth would only be a result of the protein extract additive.

Materials and Methods

Culture: Axenic C. elegans cultures were initially provided by Dr. Nancy Lu and maintained in non-shaken liquid cultures at room temperature. Stock cultures of the nematodes were maintained on a soy-yeast-heated liver extract media (HS-YE-HLE). An osmotic buffer solution, M-9 (Brenner, 1974) was used to wash nematodes free of nutrients in the HS-YE-HLE solution before they were introduced into the chemically defined media (CBMM) for testing.

Stock Culture Media: HySoy-Yeast Extract (HS-YE): 40.0 g of HY-Soy Powder (Quest International) and 10.0 g of Yeast Extract (Difco) per liter.

Heated Beef Liver Extract (HLE): Frozen beef liver was purchased from a local market, diced into 1 inch squares and allowed to autolyse at 4 ° C for 24 hours. Liver was homogenized with an equal volume of water and the homogenate was filtered through two layers of Miracloth cloth. Filtrate was heated to 52 - 53° C and maintained for exactly 6 min. The homogenate was cooled and centrifuged at 39,000 x g for 30 min. The supernatant fluid was collected and filtered through two layers of Miracloth and through a 90 mm 1.0 micron Gelman Science 61664 type A/E glass fiber filter in a Buchner vacuum filter apparatus then filter sterilized using Nalgene sterilization filter units.

HySoy-Yeast-Heated Liver Extract: HS-YE-HLE consisted of a ratio of 1 part HLE to 9 parts HS-YE.

Osmotic Buffer (M-9): 6.0 g of Na2HPO4, 5.0 g of KH2PO4, and 0.25 g of MgSO4*7H2O per liter.

CbMM Defined Media: 2X CbMM Defined Media was prepared according to laboratory procedures established in the LU laboratory at San Jose State University. The concentrated solution was stored refrigerated until use. The 2 X CbMM was prepared and the pH was adjusted to 5.9 + 0.1 with 10 % KOH and filter sterilized using 0.22 micron pore size sterile filter units. CbMM was stored in a refrigerator at 2 ° C in dark until used.

TEA [triethanolamine (2,2',2''-nitrilotriethanol)] Soluble Components Stock Solution (TSC): A 250-ml solution of TSC was composed of: 0.1500 g of biotin, 0.1500 g of dl-thioctic acid (lipoic acid), 0.3000 g of niacin (nicotinic acid), 0.3000 g of p-aminobenzoic acid, 0.3000 g of pteroylglutamic acid (folic acid), 13.00 ml of 10% TEA solution. Exactly 6.25 ml per liter is contained in the final CBMM volume.

Water Soluble Components: 0.2400 g of thyamine, 0.2400 g of riboflavin-5'-PO4(Na) *2H2O (flavin mono-nucleotide), 0.2400 g of pyridoxine hydrochloride, 0.1200 g of pyridoxamine di-hydrochloride, 0.1200 g of pyridoxal 5-phosphate, 0.2400 g of pantothenate (Ca) pantothenic acid hemicalcium salt, 0.1200 g of pantethine, 0.2400 g of niacinamide, 0.4800 g of N-acetylglucosamine and 0.1200 g of canocobalamine (vitamin b-12) in a 200-ml solution. Exactly 6.25 ml is contained in 1-liter of CBMM.

Beta-Sitosterol: Exactly 6.25 ml Tween 80 was added to 0.2500 g beta-sitosterol and brought to volume in a 50-ml volumetric flask. 10 ml of beta-sitosterol is contained in 1-liter final solution CBMM.

Salts: 2.2050 g of CaCl2 di-hydrate with 0.0650 g of CuCl2 di-hydrate, 0.2220 g of MnCl2 tetra-hydrate, 0.1020 g of ZnCl2 , 0.5880 g of Fe(NH4)2(SO4)2 hexa-hydrate, 4.8600 g of K3citrate mono-hydrate, 12.2550 g of KH2PO4, 6.3030 g of citric acid mono-hydrate and 1.7400 g of Mg(OH)2 c

Essential Amino Acids: 1.0200 g of L-valine, 0.1840 g of L-tryptophan, 0.7170 g of L-threonine, 0.6230 g of L-phenylalanine, 0.3890 g of L-methionine, 1.2830 g of L-lysine hydrochloride, 1.4390 g of L-leucine, 0.8610 g of L-isoleucine, 0.2830 g of L-histidine and 0.9750 g of L-arginine is contained in 1-liter of CbMM.

Nucleic Acid Substitutes: (0.3652 g of adenosine 2’&3’-monophosphate (mixed isomers, free acid), 0.3232 g of cytidine 2’ & 3’-monophosphate mixed isomers (free acid, cytdylic acid), 0.3632 g of guanosine 2’ & 3’-monophosphate (mixed isomers, sodium salt, guanylic acid), 0.3681 g of uridine 2’ & 3’-monophosphate (mixed isomers, free acid), 0.1261 g of thyamine and 0.617 ml of 10% KOH is contained in 1-liter of CbMM.

Growth Factors and Energy Source: Glutathione (reduced), 0.204 g and cytochrome-c, 0.05 g, choline dihydrogen citrate, 0.0885 g and myo-inositol, 0.0645 g. D-glucose, 32.5 g, was used as an energy source in 1-liter of CbMM.

Non-Essential Amino Acids: 0.2720 g of L-tyrosine. 0.6530 g of L-proline, 0.1800 g of L-phenyalanine, 1.4630 g of L-glutamine, 0.5500 g of L-glutamate monohydrate (sodium salt), 0.7880 g of L-serine, 0.0280 g of L-cysteine hydrochloride monohydrate, 1.6200 g of L-aspartic acid, 1.3950 g of L-alanine and 0.7220 g of glycine is contained in 1-liter of CbMM.

Setup: Using sterile technique 5 ml of nematodes approximately 30 days old were removed from an existing culture. The nematodes were washed 4 times in sterile M-9 and 2 times in sterile CbMM before they were used as an inoculum. Nematode density in inoculation solutions was determined to deliver at least 10 to 20 nematodes into each culture. C. elegans was cultured in a modified Caenorhabditis brigssae Maintenance Medium (CbMM). Generally, five to ten replicates of 2.5 ml total volume were setup in 1/2 oz square screw-top bottles. Standard experimental volume of tested extracts was 0.50 ml, 0.40 ml or 0.20 ml. Nematode populations were determined within 24 hours after culture initiation and every three to four days thereafter until the nematode control populations became too high to precisely count. This counting method ensured the recording of populations prior, during and after their logarithmic-growth phase. Only living nematodes were counted. Any movement of the nematode not consistent with brownian motion or associated with movements of the culture fluid was scored as a live nematode. Non-moving nematodes were presumed dead and not counted. Nematodes retained inside cadavers were not counted. Eggs were not counted as nematodes. Any contamination of the culture by bacterial or fungal growth was recorded and populations not included in statistical analysis. Nematode populations were observed using a Nikon binocular inverted microscope model TMS equipped with a Nikon Microflex HFZ-DX photomicrographic attachment and a Nikon FX35DX dark box. Light micrographs taken from a Leitz Dialux 20 microscope equipped with Differential Contrast filters.

The appropriate buffer was used as the standard control unless otherwise stated. All water was tissue culture grade water from a Nanopure water system providing ultra filtered type I water. Experimental solutions included a phosphate buffer control, water and several plant protein extracts. Statistical significance determined using Chi-square and ANOVA tests with p>0.05. Percent population change determined as the difference between a population for two consecutive recording periods. Tabled data is presented as a negative population percentage compared to the control population where a reduction of growth will appear as a positive number.

The presence of a test substance and its effect on population growth were compared to control population growth levels. Protein concentration of samples was determined using the Bradford analysis (Bradford, 1976). Protein samples were subjected to SDS-polyacrylamide gel electrophoresis on a 12.5% gel (Laemmli, 1970). The protein banding patterns were revealed after staining with Coomassie Blue R250 and silver stain (Bloom, Beier and Gross, 1987). Selected fractions were size fractionated and concentrated using Centricon concentrating filters following the manufacturer’s protocols. The concentrated fractions were used to construct a dose response curve. Haemagglutination of rabbit, pig and human red blood cells was observed (Rudiger, 1993).

Results and Discussion

Preliminary investigation of a biologically active crude and more purified extract helped identify biologically active fractions for further study (Table 1). A more detailed testing of the extract identified a biological active protein fraction which inhibited reproduction of the nematode C. elegans (Tables 1, 2, 3 & Figure 1) and caused an apparent anatomical change (Figure 2).

The heat treatment did not destroy all of the extract bioactivity but decreased the impact of these fractions on population growth. This is consistent with the hypothesis that the active component is a protein. After further testing, it became apparent the activity in KRS-1297 increased from fraction 3 to fraction 6 (Table 2). This suggests the bioactive component may be spread over a range of fractions as is often the case with fractions isolated by chromatographic methods. The greatest activity was present in fractions 5 and 6.

Haemagglutination is a general characteristic of lectins. Some lectins can influence the behavior of nematodes. The ingestion of lectins may cause damage to the epithelial cells and interfere with nematode metabolism. Lectins are toxic to nerve tissue and may inhibit or inactivate the binding affinity of olfactory tissues (Pusztai, 1991). Possible lectin binding and growth inhibitory activity was greater than 50,000 in molecular weight based on separation by Centricon ultrafiltration of KRS-1297-5&6. Stimulatory activity without lectin binding was present in the fraction below 10,000 molecular weight. All Centricon retentates possessed inhibitory activity and haemagglutination, while stimulatory activity with out haemagglutination was observed with the Centricons filtrates. This stimulatory activity apparently has a molecular weight below 10,000. SDS-PAGE analysis was performed on the protein extracts and heavy banding above the 50,000 M.W. size range was observed corresponding to inhibitory activity in the Centricon fractions.

An abnormal growth on the nematodes in cultures with KRS-1297-5&6 was ventral and post-anal on the tail of hermaphrodite C.elegans. Although a few males were observed in other tested extracts, there were no males present in cultures containing KRS-1297-5&6. Preliminary investigations have located an enlarged cell near the enlarged region in nematodes from cultures containing KRS-1297-5&6. Haemagglutination was also observed with these extracts.

 

Table 1. Change in Nematode Population in Cultures with KRS-Fractions.

0.2 ml Additions of

% Change in Population

Sample ID

Day 10

Day 13

Day 17

KRS-1297-34s (8.2)

16

18

28

KRS-1297-56s (11.0)*

80

79

78

KRS-1297-crude (n.d.)

63

3

-25

0.4 ml Additions of

% Change in Population

Sample ID

Day 10

Day 13

Day 17

Water (0)

-8

-8

-23

KRS-1297-34s (20.5)

40

39

38

KRS-1297-56s (28.5)*

69

54

-20

KRS-1297-crude (n.d.)

-8

-67

-104

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the fraction. Protein concentrations in micrograms (n.d. is not determined). * Post anal bump present (Figure 2).

Table 2. Change in Nematode Population in Cultures with Individual Fractions.

Sample ID (protein)

% Change in Population

 

Day 8

Day 11

Day 15

KRS-1297-6 (10.06)*

71.10

53.20

24.62

KRS-1297-5 (10.14)*

70.67

59.38

28.43

KRS-1297-4 (9.92)

41.74

18.02

8.33

KRS-1297-3 (6.30)

28.85

24.32

11.67

KRS-1297-6 heated

36.65

33.79

17.75

KRS-1297-5 heated

41.93

36.07

14.00

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the fraction. Protein concentrations in micrograms. Heated extracts were taken to 100 C for 5 minutes. * Post anal bump present (Figure 2).

Table 3. Change in Nematode Population in Cultures Containing Active Fractions

After Ultra-filtration.

Sample ID (n)

% Change in Population

 

Day 9

Day 12

Day 16

Agglutination

Protein

Retentate (1x)-Centricon -10K (4)*

73.96

77.04

62.27

r,p,h

5.6

Filtrate (1x)-Centricon -10K (3)

-67.36

-35.49

-24.72

none

~0

Retentate (1x)- Centricon -50K (4)*

47.83

49.31

29.91

r,p,h

5.4

Filtrate (1x) Centricon -50K (4)

-135.33

-32.56

-24.72

none

~0

Data shown in bold type indicate a significant rate of population growth compared to the previous recording interval. Underlined populations are significantly different from the control (p = 0.05). Negative numbers indicate a stimulatory or growth promoting effect of the fraction. Agglutination activity was tested on red blood cells of rabbit, human and pig (r=Rabbit, p=Pig and h=Human). Protein concentrations are in micrograms. Protein levels ~0 = below detectable limit. * Post anal bump present (Figure 2).

Figure 1. Change in Nematode Population Growth Rate in CbMM

As A function of Added Protein.

Reduction of reproductive success increases with additions of extract. The extract inhibitory activity is limited and appears to be concentration dependent. BSA additions at this concentration were not significantly different from controls (Data not shown * Post anal bump present (Figure 2).

Figure 2. Anatomical Abnormality in C. elegans Initiated by the Addition of a Protein.

The tail of C. elegans with an enlarged bump located post anal. Notice the enlarged dome shaped cell. This enlargement is conspicuously absent from cultures that did not contain the KRS-1297-56 protein extract. Magnification ~2,160 X.

Conclusions

Culture System: The use of smaller-culture tubes and lack of modified aeration may be reducing the nematode growth potential in the culture systems. Although these experiments were shorter in term and smaller in culture volumes, the extrapolated populations from control cultures appear to be similar to populations reported by Lu and Goetsch (1993) of 80,000 nematodes per milliliter. The major difference between this assay and other reported assays were the lack of indirect aeration, the use of complete chemically-defined media and the specific testing of proteins or peptides for their ability to inhibit reproduction.

Identified Inhibition: The presence of fractions KRS-1297-5 and KRS-1297-6 in cultures significantly reduced the average population below control levels and the inhibitory activity was consistent through five experimental sets. Further testing should be done to characterize and identify the active portions of these extracts. SDS-gels have revealed several protein bands which may be a related to the active portion of the fraction. There is no indication exactly which band is responsible for the observed activity. Size fractionation studies, however, suggest the active component has a molecular weight greater than 50,000.

Citations

Bradford, M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principles of dye binding. Analytical Biochemistry 72: 248-254.

Bloom, H., H. Beier, and H. S. Gross. 1987. Improved silver staining of plant protein, RNA and DNA in polyacrylamide gels. Electrophoresis 8: 93-99.

Brenner S. 1974. The genetics of Caenorhabditis elegans. Genetics 77: 71-94.

Lu NC; Goetsch KM. 1993. Carbohydrate requirement of Caenorhabditis elegans and the final development of a chemically defined medium. Nematologica 39: 303-311.

Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277: 680-685.

Lu, Nancy C. 1995. Laboratory manual axenic cultivation of Caenorhabditis elegans. Department of Nutrition and Food Science, San Jose State University. CA, USA. Personal Communication.

Puszati, A. 1991. Chemistry and pharmacology of natural products: plant lectins. Pp. 105-205 J. D. Phillipson, D. C. Ayres, H. Haxter, eds. New York: Cambridge University Press.

Rudiger, H. 1993. Isolation of plant lectins. Pp. 40-46 in H. J. Gabius and S. Gabius, eds. Lectins and glycobiology. New York: Springer-Verlag.

Acknowledgements:

Dr. Nancy C. Lu. 1995. Department of Nutrition and Food Science, San Jose State University. CA, USA.

Dr. Scott Russell. Department of Botany- Microbiology, The University of Oklahoma. Norman, Oklahoma.

Dr. Karel Schubert. Department of Botany- Microbiology, The University of Oklahoma. Norman, Oklahoma.

Dr. John Skvarla. Department of Botany- Microbiology, The University of Oklahoma. Norman, Oklahoma.

©Copyright by James Randolph Hocker 1998

All Rights Reserved.