Characterization of haemolytic activity from adult Haemonchus contortus

Internolionalfournolfor Prrmsilolog~,Vol.27.N0. Cl 1997 Austraban Society for Parasitology. Published

Pergamon

PII:

RESEARCH

9,pp. 1037-1040,

002&7519/97

SOO20-7519(97)00078-7

1997

by Elsevier Science Ltd Pnnted in Great Britain

Sl7.00+0.00

NOTE

Characterization

of Haemolytic Activity Haemonchus contortus R. H. FETTERER*

from Adult

and M. L. RHOADS

Parasite Biology and Epidemiology Laboratory, Livestock and Poultry Sciences Institute, Beltsville Agricultural Research Center, Beltsville, MD 20705, U.S.A. (Received 18 March 1997; accepted 27 May 1997)

Abstract-Fetterer R. H. & Rhoads M. L. 1997. Characterization of haemolytic activity from adult Huemonthus contortus. International Journalfor Parasitology 27: 1037-1040. Adult Haemonchus contortus contain a detergent-soluble factor that haemolyses sheep red blood ceils in a time- and concentration-dependent manner. This factor had comparable haemolytic activity at pH 5.0 and 8.0; activity was lower at pH 6.0 and 7.0. The activity was heat-stable, unaffected by proteolytic inhibitors, and inhibited by 20 mM polyethyleneglycol. Haemolytic activity was associated with the particulate fraction of the isolated intestine, suggesting an essential role for this activity in the acquisition of nutrients by disrupting host red blood cells. The data are consistent with the hypothesis that the mechanism of action of the haemolytic factor is as a pore-forming agent. 0 1997 Australian Society for Parasitology. Published by Elsevier Science Ltd.

Key words: Nematode; stomach worm; red blood cells; parasite; parasitic; blood feeding.

Haemolytic factors have been identified and partially characterized from several parasites, including the protozoans Leishmania amazonensis (Noronha et al., 1994) Entamoeba histolytica (Rosales-Encina et al., 1992), Naegleriafowleri (Young & Lowrey, 1989) and the blood fluke Schistosoma mansoni (Kasschau & Dresden, 1986); these factors disrupt host erythrocytes or other cells by forming pores in the plasma membrane. Haemolytic factors are common in nematophagous insects and include molecules with proteolytic or pore-forming activities (Spates, 1983; De Azambuja et al., 1983; Kirch et al., 1991). Haemolytic activity has not. however, been documented in parasitic nematodes. The present study identifies and partially characterizes a haemolytic factor from H. contortus adults and localizes this activity to the intestine. Adult H. contortus were recovered from the abomasum of sheep and washed extensively with phosphate buffered saline (PBS), rinsed with water and lyophilized. Lyophilized parasites were homogenized *To whom correspondence should be addressed. Fax: + 1 301 504 5306; E-mail: [email protected].

in 0.1 M sodium citrate buffer, pH 5.0 (40mg dry weightmll’) with a tissue grinder (Polytron, Brinkman Instruments, Westbury, New York, U.S.A.). The homogenate was centrifuged at 10 000 g for 15 min at 4°C. The supernatant fluid was removed and the pellet suspended in sodium citrate buffer to the original volume. The supernatant fluid and pellet suspension were frozen at -20°C in l.O-ml aliquots. In some experiments the pellet was further homogenized with a Polytron in PBS containing 0.5% (v/v) n-dodecylN,N-dimethyl-3-ammino-I-propane-sulfonate (SB12, Sigma, St. Louis, MO, U.S.A.) and centrifuged (12 000 g, 15 min, 4°C). The detergent-soluble fraction was dialysed for 72 h against 2 1 PBS; the detergentinsoluble pellet was washed 5 times with 5 ml PBS. Approximately 100 intestines were removed from adult H. contortus by microdissection. The remaining tissue, comprising muscle, cuticle and reproductive organs, was collected as a single pool. The intestine and the pooled tissues were homogenized separately in 0.1 M sodium citrate buffer, pH 5.0 with a conical, ground-glass homogenizer and centrifuged at 12 000 g for 15 min at 4°C. The supernatant fluids were removed and the pellets resuspended in 0.2 ml sodium citrate buffer. Protein concentrations were determined

1037

1038

R. H. Fetterer and M. L. Rhoads

with a modified Lowry method (BCA, Pierce, Rockford, IL, U.S.A.). Sheep red blood cells (RBCs) were isolated from sodium citrate-treated whole blood by centrifugation and stored as a 50% suspension in PBS at 4°C. Immediately prior to assay, RBCs were washed 3 times with PBS and suspended at a concentration of 1% (v/v) in 0.1 M sodium citrate buffer, pH 5.0. The assay mixtures consist of RBC (1%) suspension and 1&lo0 ml of parasite extract or supernatant fluid in a total volume of 1 ml. Assays using isolated parasite tissue were run in 0.5 ml total volume. Control tubes contained only RBC suspension. To determine thermal stability, the parasite extract was placed in a water bath at 100°C for 5 min prior to assay. In some experiments, a cocktail of proteolytic inhibitors (Complete, Boehringer Mannheim, Mannheim, Germany) was dissolved in 0.1 M citrate buffer, pH 5.0 and added to assays at the maximum recommended concentration. In other experiments, polyethyleneglycol (PEG 800; Sigma. St. Louis, MO. U.S.A.) was added to assays at a final concentration of 20mM. Assay tubes were incubated at 37°C for 0.54 h. After incubation, tubes were centrifuged at 12 OOOg for 5 min to remove intact cells. A 200~~1 aliquot of each supernatant fluid was added to 0.8ml Drabkins reagent (Sigma, St. Louis, MO, U.S.A.) to stabilize the haemoglobin and, after 15-min incubation at room temperature, the absorbance at 540nm was determined. Results were expressed as mg released haemoglobin per assay. Assays were conducted in duplicate and results expressed as the mean. The pellet fraction from an H. CO~~~Y~US homogenate caused a time- and concentration-dependent haemolysis of sheep RBCs (Fig. 1). No activity (< 0.2 mg haemoglobin released) was associated with the supernatant fluid fraction (data not shown). The pellet (0.62 mg protein) released 4.0 mg haemoglobin after a 1-h incubation. Lower concentrations of pellet protein (0.48 and 0.32 mg) resulted in a lag in the time of onset of haemolysis. Less than 0.25mg haemoglobin was released by 0.16 mg of pellet suspension or 0.5mg of homogenate supernatant. In controls, less than 0.2 mg haemoglobin was released after a 4-h incubation (data not shown). Complete haemolysis of a 1% RBC suspension in 1 ml distilled water released 5.3mg(S.E.M. =0.15, N= 14) ofhaemoglobin. AtpH 8.0, the time course of haemolysis was nearly identical to that observed at pH 5.0. However, at pH 6.0 and 7.0 the onset of haemolysis was slowed as compared with pH 5.0. At pH values lower than about 4.5, RBCs were not stable. Heating the pellet (100°C for 5 min) prior to assay slowed the onset of haemolysis; however, after a 3-h incubation the released haemoglobin (3.8mg) was nearly equal to that of the unheated pellet (Fig. 2). A proteolytic inhibitor cock-

-3 0.65 m g 0.46ny 0.32 m g 0.16 m g 0.0 m g

0

2

1 Incubation

3 Time

4

(hr)

Fig. 1. Haemolytic activity of resuspended pellet from homogenized adult H. conform. Protein concentrations of resuspended pellet ranged from 0.0 to 0.65mg per assay (1 .Oml volume). For all figures, values are means for duplicate measurements of released haemoglobin and vertical bars represent the range but are not shown when less than 0.2. tail did not alter haemolysis (Fig. 2). PEG (20mM) prevented haemolysis (Fig. 2) with less than 0.45 mg haemoglobin released after a 4-h incubation. SB-12 (0.5%) solubilized the haemolytic activity (Fig. 3); about 6.5% of the pellet protein was solubilized. The

0

1

2 Incubation

3

4

time (hr)

Fig. 2. The effect of various treatments on haemolytic activity of H. cot~tortu~ pellet. All assays contained 0.52mg protein (l-ml assay volume). Control: homogenate alone: 100°C: homogenate heated at 1OO’C for 5 min prior to incubation: inhibitors: co-incubated with cocktail of proteolytic inhibitors; PEG: co-incubated with 20mM polyethylene glycol.

Research note

0

+

0.067 m g soluble

fraction

-A-

0.040 m g soluble

fraction

+

0.067 m g pellet fraction

I

I

I

I

1

2

3

4

Incubation

Time

(hr)

Fig. 3. Detergent extraction (SB-12, 5%) of haemolytic activity from H. confortus. The detergent-soluble fraction was assayed at 0.067 mg and 0.040 mg protein. The detergentinsoluble fraction was assayed at 0.067 mg. detergent-soluble fraction (0.067 mg protein) released 3.6 mg haemoglobin after a 2-h incubation. The detergent-insoluble pellet at the same protein concentration did not release haemoglobin. The pellet fraction from a homogenate of isolated H. contortus intestines haemolysed RBCs (Fig. 4); 0.15 mg protein released 1.3 mg haemoglobin after a 3-h incubation and 0.06 mg protein released 0.37 mg haemoglobin. The 1.5 r

0.0

0

-S-

0.16 m g pellet

t

0.06 m g pellet fraction

fraction

I

I

I

J

1

2

3

4

Incubation

Time

(hr)

Fig. 4. The haemolytic activity of pellet and supernatant fluid fractions of H. contortus intestine. The assay volume was 0.5ml. The pellet was assayed at 0.16 and 0.06mg protein. The supernatant was assayed at 0.07mg protein.

1039

supernatant (0.07 mg protein) released less than 0.12 mg haemoglobin after a 4-h incubation. The supernatant fluid and pellet fractions from the combined cuticle, muscle and reproductive tissues released less than 0.1 mg haemoglobin (data not shown). A haemolytic mechanism is a prerequisite for an effective blood-feeding parasite. Previous studies have demonstrated the ability of H. contortus to digest the blood proteins haemoglobin and albumin (Rhoads & Fetterer, 1995; Fetterer & Rhoads, in press), but a mechanism for lysis of RBCs has not been demonstrated. Lytic factors have been characterized from microbes (Buckley et al., 1981). invertebrates (Boyer et al., 1979; Mollay et al., 1976; Spates, 1983) and vertebrates (Louw & Visser, 1977); the majority of these are soluble factors active at neutral pH. In contrast, the haemolytic factor from H. contortus is membrane-associated and is active at acidic pH. The H. contortus haemolytic factor resembles those from S. mansoni (Kasschau & Dresden, 1986), L. amazonensis (Noronha et al., 1994) and N. fowleri (Young & Lowrey, 1989); the S. mansoni and L. amazonensis factors are active at acidic pH and the N. jhvleri haemolytic factor is a membrane-bound, hydrophobic protein. However, unlike the other parasites, the haemolytic activity from H. contortus was as active at pH 8.0 as at pH 5.0. The only previous report of a poreforming agent from a parasitic nematode is a 47 kDa protein from the whipworm Trichuris murk, which was shown to produce ion-conducting channels in planar lipid bilayers (Drake et a/., 1994). The T. rnztris pore-forming protein was present in excretions/ secretions and was postulated to aid in penetration of host intestinal epithelium. The H. contortus haemolytic factor was localized to the intestine. The pH of the intestine is unknown, but cysteine proteases present in the intestines of H. contortus are active at a pH 5.5, suggesting that the intestine is acidic. The activity of the haemolytic factor at pH 5.0 may be an adaptation to the gut environment where it functions as a component of a mechanism for cell lysis and proteolysis. In addition, the intestinal localization of the haemolytic factor supports its function in parasite nutrition. The haemolytic factor of H. corztortus is unlikely to be an enzyme since its activity was heat-stable and not altered by the presence of protease inhibitors. Although the activity has not been purified. the data are consistent with the hypothesis that this haemolytic factor exerts its activity through a pore-forming mechanism. The lag time before onset of haemolysis and the inhibition of haemolysis by PEG are consistent with the colloidal-osmotic effects caused by pore-forming action of a haemolytic factor, resulting in ionchannel formation or an increase in general membrane

1040

R. H. Fetterer

permeability (Heustis, 1977). Further purification of the H. contortus haemolytic factor will be needed to more precisely elucidate its properties and more clearly define its physiological role within the parasite.

Acknowledgements-We are indebted Ndika for expert technical assistance.

to R. Barfield

and A.

REFERENCES Boyer J. M., D’Antonio L. E. & Schiavone W. A. 1979. Lipid composition and activity of a lytic factor isolated from Plasmodium berphei. Infection and Immunity 25: 805-809. Buckley J. T., Hal&a L. N. & Lund K. D. 1981, Purification and some properties of the hemolytic toxin aerolysin. Canadian Journal of Biochemistry 59: 43G435. De Azambuja P., Guimaraes J. A. & Garcia E. S. 1983. Hemolytic factor from the crops of Rhodnius prohixus: evidence and partial characterization. Journal of Insect Physiology> 29: 833-837. Drake L., Korchev Y., Bashford L., Djamgoz M.. Wakelin D., Ashall F. & Bundy D. 1994. The major secreted product of the whipworm, Trichuris, is a pore-forming protein. Proceedings of Royal Society London B 257: 255-261. Fetterer R. H. & Rhoads M. L., in press. The in vitro uptake and incorporation of hemoglobin by adult Haemonchus contortus. Veterinary ParasitologJj 69: 77-W. Heustis W. H. 1977. A sodium-specific membrane permeability defect induced by phospholipid vesicle treatment of erythrocytes. Journal of Biological Chemistry 252: 6764 6768.

and M. L. Rhoads Kasschau M. R. & Dresden M. H. 1986. Schistosoma mansoni: characterization of hemolvtic activity from adult worms. Experimental Parasitology 61: 201-209. Kirch H. J.. Snates G.. Droleskev R.. Kloft W. J. & DeLoach J. R. 1991,Mechanism of hemolysis of erythrocytes from Stonmxys calcitrans (L) (Diptera: Muscidae). Journal of Insect Ph.vsiology 31: 85 l-861. Louw A. I. & Visser L. 1977. Kinetics of erythrocyte lysis by snake venom cardiotoxin. Biochimica et Biophysics Acta 498: 143-153. Mollay C., Kreil G. & Berger H. 1976. Action of phospholipases on the cytoplasmic membrane of Escherichia coli: stimulation by melittin. Biochimica et Bioph.vsica Acta 426: 3 177324. Noronha F. S. M., Ranalhjo-Pinto F. J. & Horta M. F. 1994. Identification of a putative pore-forming hemolysin active at acid pH in Leishmania amazonetlsis. Brazilian Journal of Medical and Biological Research 21: 477482. Rhoads M. L. & Fetterer R. H. 1995. Developmentally regulated secretion of cathepsin L-like cysteine protease by Haemonchus contortus. Journal of Parasito1og.v 81: 501505. Rosales-Encina J. L., Schile-Guzman M. A., Jimenez-Delgadillo B.. Talmas-Rohana P. & Rojkind Matluk M. 1992. Purification and partial characterization of an hemolytic activity from Entamoeba histolytica. Archives of Medical Research 23: 243-248. Spates G. E. 1983. Midgut proteolytic and hemolytic activities and feeding habits of young adult stable flies Stomoxys calcitrans (L.). The Southwestern Entomologist 8: 178-185. Young J. D. & Lowrey D. M. 1989. Biochemical and functional characterization of a membrane associated poreforming protein from pathogenic ameboflagelate Nagleria fowleri. Journal of Biological Chemistr.v 264: 107771083.