Inlernorional Journal/or Parasifology Vol. 21,No. 5,pP.609-61 I, 1991
OO20-7519/9l $3.00+ 0.00
Printed in Great Britain
0
Pergamon Press
p/c
1991 Arrsrralim Societyfor Pmmsifology
RESEARCHNOTE BABESIA BO VLS HOST CELL RECOGNITION
PROTEINS
G. R. BUSHELL,* B. GARRONE,* B. V. GOODGER,? I. G. WRIGHT? and B. P. DALRYMPLE~ *Division of Science and Technology, Griffith University, Nathan, Queensland 4111, Australia tCSIR0 Division of Tropical Animal Production, Private Bag No. 3 PO, Indooroopilly, Queensland 4068, Australia (Received 17 December 1990; accepted 29 April 1991) G. R., GARRONE B., GOODGERB. V., WRIGHT I. G. and DALRYMPLE B. P. 1991. Bubesia host cell recognition proteins. International Journal for Parasitology 21: 609-611. Babesiu bovis enters host erythrocytes by invagination but nothing is known of the proteins involved. By means of metabolic labelling, differential centrifugation in oil and salt elution, a number of babesial proteins have been shown to bind to bovine erythrocytes. Strong binding is evidenced only by a 38/19 kDa pair. Preliminary experiments indicate that these two proteins also bind to human erythrocytes, although apparently to a lesser extent. Abstract-BusmLL
bovis
INDEX KEY WORDS:
Babesia;
erythrocyte binding.
LITLE is known about the process of attachment to, and invasion of, erythrocytes by Babesia, and yet this is one of the most critical factors in the life cycle of this parasite. The merozoite, upon leaving a host erythrocyte, must then attach to, and invade, an uninfected erythrocyte. This is a very specific interaction; Babesia does not invade other host cells. The
specificity implies the presence of a receptor (or receptors) on the erythrocyte which is recognized by a complementary molecule (or molecules) on the parasite. In the related haemoparasite Plasmodium, a similar mechanism of ligand-receptor recognition has been proposed (Hadley, Klotz & Miller, 1986), although it is now clear that invasion of Plasmodium
is a multi-step process requiring a series of interactions involving several different host and parasite molecules, that is further complicated by alternative pathways for invasion (e.g. Mitchell, Hadley, Klotz, McGinniss & Miller, 1986; Hadley, Klotz, Pasvol, Haynes, McGinniss, Okubo & Miller, 1987). A 175 kDa erythrocyte binding antigen has been identified in P. falciparum (see Camus & Hadley, 1985; Orlandi, Kim Lee Sim, Chulay & Haynes, 1990). The binding of this antigen to sialic acid-containing determinants on the erythrocyte appears to correlate with invasion efficiency. The studies with Plasmodium have, in part, been aided by the availability of welldefined human blood groups, some of which are refractory to invasion. So far as is known, all bovine erythrocytes are susceptible to invasion by Babesia bovis. This report describes initial attempts to identify the molecules involved in the host cell recognition process by B. bovis. Blood from a B. bovis (Kv strain)-infected cow was collected
into heparin,
and the plasma and buffy coat 609
were removed after centrifugation at 1000 g for 10 min at 22°C. The cells were washed three times in RPM1 1640 medium, then incubated in RPM1 medium containing either 7.4 x lo6 Bq “Smethionine per ml infected cells or a mixture of ‘?amino acids (3.7 x lo5 Bq ml-‘) and ‘H-amino acids (8.14 x lo6 Bq ml-‘) for 2 h or overnight at 37°C in 5% CO,. Infected erythrocytes which were enriched to about 80% parasitized cells by differential lysis of non-infected erythrocytes (Mahoney, 1967) were also metabolically labelled by this method. A control culture of uninfected bovine erythrocytes was similarly prepared. At the end of the culture period, the medium was removed by centrifugation at 1000 g for 20 min and stored at - 70°C. The labelled parasitized erythrocytes were washed three times in phosphate buffered saline (PBS), resuspended in 5 vol. of distilled water and centrifuged at 10,000 g for 1 h at 4°C. The pellet was suspended in 5 vol. of cold PBS, sonicated and centrifuged at 100,000 g for 1 h at 4°C. Labelled sonicated supematant was collected and stored at - 7o’C. Normal erythrocytes for use in the erythrocyte binding assay were collected in EDTA, washed three times in PBS and stored at 4°C. Metabolically labelled sonicate or culture medium (0.5 ml) was added to 0.1 ml of packed erythrocytes. After incubation for 20 min at 22°C the suspension was layered over silicone oil (Dow 550 or Sure-Sep II, General Diagnostics) (Miller, Hudson & Haynes, 1988), and centrifuged in an Eppendorf microfuge for 2 min. The cell pellet was washed once in PBS, then resuspended in 1.5 M-NaCl (0.02 ml), After a 10 min incubation the suspension was centrifuged, the NaCl eluate was removed and electrophoresed on a 10% SDS-polyacrylamide gel under reducing con-
G. R. BUSHELL~~~
610
9467-
1
2
3
FIG.1. Autoradiograph of ‘5S-methionine-labelled B. bovis proteins and material eluted from uninfected erythrocytes. Lane I: ‘%-methylated protein standards (Amersham); lane 2: 3sS-methionine-la~Il~ B. bovis sonicate; lane 3: 1.5 MNaCl eluate from erythrocytes treated with medium from %-methionine B. bovis culture and centrifuged once through silicone oil. Major bands at 67,48 and 38 kDa are marked on the print.
67-
1
2
FIG. 2. 35S-Methionine-labelled B. bovis sonicate bound to uninfected bovine erythrocytes and layered through silicone oil twice. Lane 1: “C-methylated protein standards (Amersham); lane 2: 1.5 M-NaCl &ate.
ditions. The gel was processed for autoradiography using Enhance (Amersham). For some experiments, the erythrocyte suspension was layered twice through the silicone oil. Three major 3SS-labelled proteins with mol. wts of 47, 48 and 38 kDa were eluted from the uninf~ted along with several minor bands, erythrocytes, including one at 19 kDa (Fig. 1). Many of these proteins were apparently only weakly attached to the erythrocytes, since when the suspension was layered through the silicone oil twice, only two protein bands with mol. wts of 38 and 19 kDa were eluted with 1.5 M-NaCl (Fig. 2). The results were the same whether labelled sonicate or medium from the labeiling culture was used, and also when erythrocytes were pre-treated with 1.5 M-NaCl prior to binding the labelled babesial proteins. When the ‘H/‘%-amino acid-labelled parasite sonicate was bound to bovine erythrocytes, only one protein of mol. wt 38 kDa was eluted with 1.5 M-NaCl. Uninfected bovine erythrocytes similarly cultured with ?S-methionine or the 3H/‘4C-amino acid mixture did not produce any detectable protein labelling and no labelled bands were detected from binding experiments using this material. In one experiment, human erythrocytes were used to bind the ?Gmethionine and ‘H/“Camino acid-la~lled parasite proteins. Two very faint bands with mol. wts 38 and 19 kDa were eluted when medium from %methionine labelled cultures was used as absorbing ligand, but no bands were detected when whole sonicated 35S-methionine-labelled parasites were bound to the human erythrocytes. There were also no bands detected when the ‘H/%-amino acid-la~lled parasites were bound to the human erythrocytes. Further characterization of these babesial erythrocyte binding proteins is underway in our laboratory. It is interesting that of the three major bands (67,48 and 38 kDa) initially seen to be binding to the bovine erythrocytes, only one, the 38 kDa protein, survives a second treatment through silicone oil. Apparently, the binding of the other two proteins is weaker, and therefore probably less specific. (We have not determined whether the 19 kDa band which also remains after two separations through the oil represents a monomeric unit of the 38 kDa protein.) Western blots of these eluted proteins probed with defined monoclonal antibodies indicate that the 67 kDa protein may be a previously reported babesial protease (Commins, Goodger & Wright, 1985). The eluted babesial erythrocyte binding proteins are much smaller than the equivalent molecule identified in P. falciparum using a similar technique, but in some of our experiments, a high molecular weight labelled protein remained associated with the bovine erythrocytes after the N&I treatment. This protein may represent an equivalent Babesia molecule which binds more tightly to the erythrocyte than the corresponding Plasmodium molecule. Further experiments are underway to investigate the nature of this
Research protein. Since both the normal and infected erythrocytes used in the assays were washed prior to use, we are also investigating whether the presence of plasma components affects the number and/or type of Babesia proteins which can bind to erythrocytes. Acknowledgements-We are grateful to Dr W. Jorgensen, Tick Fever Research Centre, Wacol, for kindly supplying the B. bovis-infected blood. This work was supported by the Australian Research Council.
REFERENCES CAMUSD. & HADLEYT. J. 1985. A Plasmodium falciparum antigen that binds to host erythrocytes and merozoites. Science 2301 553-556. COMMINSM. A., GRUDGERB. V. & WRIGHT I. G. 1985. Proteinases in the lysate of bovine erythrocytes infected with Babesia bovis: initial vaccination studies. International Journalfor Parasitology 15: 491495. HADLEYT. J., KLOTZF. W. & MILLER L. H. 1986. Invasion
Note
611
of erythrocytes by malarial parasites: a cellular and molecular overview. Annual Review of Microbiology 401 451477. HADLEY T. J., KLOTZ F. W., PASVOLG., HAYNESJ. D., MCGINNISSM. H., OKUBO Y. & MILLER L. H. 1987. Falciparum malaria parasites invade erythrocytes that lack glycophorin A and B (M’M’). Strain differences indicate receptor heterogeneity and two pathways for invasion. Journal of Clinical Investigation 80: 119&l 193. MAHONEYD. F. 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Experimental Parasitology 20: 232-241. MILLER L. H.. HUDXIN D. & HAYNESJ. D. 1988. Identification of ~lasmodium knowlesi erythrocyte binding proteins. Molecular and Biochemical Parasitology 31: 217-222. MITCHELLG. H., HADLEYT. J., KLOTZ F. W., MCGINNISS M. H. & MILLER L. H. 1986. Invasion of erythrocytes by Plasmodium falciparum malaria parasites: evidence for receptor heterogeneity and two receptors. Blood 67: 15191521. ORLANDIP. A., KIM LEE SIM B., CHULAYJ. D. & HAYNES J. D. 1990. Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum. Molecular and Biochemical Parasitology 40: 285-294.
OO20-7519/9l $3.00+ 0.00
Printed in Great Britain
0
Pergamon Press
p/c
1991 Arrsrralim Societyfor Pmmsifology
RESEARCHNOTE BABESIA BO VLS HOST CELL RECOGNITION
PROTEINS
G. R. BUSHELL,* B. GARRONE,* B. V. GOODGER,? I. G. WRIGHT? and B. P. DALRYMPLE~ *Division of Science and Technology, Griffith University, Nathan, Queensland 4111, Australia tCSIR0 Division of Tropical Animal Production, Private Bag No. 3 PO, Indooroopilly, Queensland 4068, Australia (Received 17 December 1990; accepted 29 April 1991) G. R., GARRONE B., GOODGERB. V., WRIGHT I. G. and DALRYMPLE B. P. 1991. Bubesia host cell recognition proteins. International Journal for Parasitology 21: 609-611. Babesiu bovis enters host erythrocytes by invagination but nothing is known of the proteins involved. By means of metabolic labelling, differential centrifugation in oil and salt elution, a number of babesial proteins have been shown to bind to bovine erythrocytes. Strong binding is evidenced only by a 38/19 kDa pair. Preliminary experiments indicate that these two proteins also bind to human erythrocytes, although apparently to a lesser extent. Abstract-BusmLL
bovis
INDEX KEY WORDS:
Babesia;
erythrocyte binding.
LITLE is known about the process of attachment to, and invasion of, erythrocytes by Babesia, and yet this is one of the most critical factors in the life cycle of this parasite. The merozoite, upon leaving a host erythrocyte, must then attach to, and invade, an uninfected erythrocyte. This is a very specific interaction; Babesia does not invade other host cells. The
specificity implies the presence of a receptor (or receptors) on the erythrocyte which is recognized by a complementary molecule (or molecules) on the parasite. In the related haemoparasite Plasmodium, a similar mechanism of ligand-receptor recognition has been proposed (Hadley, Klotz & Miller, 1986), although it is now clear that invasion of Plasmodium
is a multi-step process requiring a series of interactions involving several different host and parasite molecules, that is further complicated by alternative pathways for invasion (e.g. Mitchell, Hadley, Klotz, McGinniss & Miller, 1986; Hadley, Klotz, Pasvol, Haynes, McGinniss, Okubo & Miller, 1987). A 175 kDa erythrocyte binding antigen has been identified in P. falciparum (see Camus & Hadley, 1985; Orlandi, Kim Lee Sim, Chulay & Haynes, 1990). The binding of this antigen to sialic acid-containing determinants on the erythrocyte appears to correlate with invasion efficiency. The studies with Plasmodium have, in part, been aided by the availability of welldefined human blood groups, some of which are refractory to invasion. So far as is known, all bovine erythrocytes are susceptible to invasion by Babesia bovis. This report describes initial attempts to identify the molecules involved in the host cell recognition process by B. bovis. Blood from a B. bovis (Kv strain)-infected cow was collected
into heparin,
and the plasma and buffy coat 609
were removed after centrifugation at 1000 g for 10 min at 22°C. The cells were washed three times in RPM1 1640 medium, then incubated in RPM1 medium containing either 7.4 x lo6 Bq “Smethionine per ml infected cells or a mixture of ‘?amino acids (3.7 x lo5 Bq ml-‘) and ‘H-amino acids (8.14 x lo6 Bq ml-‘) for 2 h or overnight at 37°C in 5% CO,. Infected erythrocytes which were enriched to about 80% parasitized cells by differential lysis of non-infected erythrocytes (Mahoney, 1967) were also metabolically labelled by this method. A control culture of uninfected bovine erythrocytes was similarly prepared. At the end of the culture period, the medium was removed by centrifugation at 1000 g for 20 min and stored at - 70°C. The labelled parasitized erythrocytes were washed three times in phosphate buffered saline (PBS), resuspended in 5 vol. of distilled water and centrifuged at 10,000 g for 1 h at 4°C. The pellet was suspended in 5 vol. of cold PBS, sonicated and centrifuged at 100,000 g for 1 h at 4°C. Labelled sonicated supematant was collected and stored at - 7o’C. Normal erythrocytes for use in the erythrocyte binding assay were collected in EDTA, washed three times in PBS and stored at 4°C. Metabolically labelled sonicate or culture medium (0.5 ml) was added to 0.1 ml of packed erythrocytes. After incubation for 20 min at 22°C the suspension was layered over silicone oil (Dow 550 or Sure-Sep II, General Diagnostics) (Miller, Hudson & Haynes, 1988), and centrifuged in an Eppendorf microfuge for 2 min. The cell pellet was washed once in PBS, then resuspended in 1.5 M-NaCl (0.02 ml), After a 10 min incubation the suspension was centrifuged, the NaCl eluate was removed and electrophoresed on a 10% SDS-polyacrylamide gel under reducing con-
G. R. BUSHELL~~~
610
9467-
1
2
3
FIG.1. Autoradiograph of ‘5S-methionine-labelled B. bovis proteins and material eluted from uninfected erythrocytes. Lane I: ‘%-methylated protein standards (Amersham); lane 2: 3sS-methionine-la~Il~ B. bovis sonicate; lane 3: 1.5 MNaCl eluate from erythrocytes treated with medium from %-methionine B. bovis culture and centrifuged once through silicone oil. Major bands at 67,48 and 38 kDa are marked on the print.
67-
1
2
FIG. 2. 35S-Methionine-labelled B. bovis sonicate bound to uninfected bovine erythrocytes and layered through silicone oil twice. Lane 1: “C-methylated protein standards (Amersham); lane 2: 1.5 M-NaCl &ate.
ditions. The gel was processed for autoradiography using Enhance (Amersham). For some experiments, the erythrocyte suspension was layered twice through the silicone oil. Three major 3SS-labelled proteins with mol. wts of 47, 48 and 38 kDa were eluted from the uninf~ted along with several minor bands, erythrocytes, including one at 19 kDa (Fig. 1). Many of these proteins were apparently only weakly attached to the erythrocytes, since when the suspension was layered through the silicone oil twice, only two protein bands with mol. wts of 38 and 19 kDa were eluted with 1.5 M-NaCl (Fig. 2). The results were the same whether labelled sonicate or medium from the labeiling culture was used, and also when erythrocytes were pre-treated with 1.5 M-NaCl prior to binding the labelled babesial proteins. When the ‘H/‘%-amino acid-labelled parasite sonicate was bound to bovine erythrocytes, only one protein of mol. wt 38 kDa was eluted with 1.5 M-NaCl. Uninfected bovine erythrocytes similarly cultured with ?S-methionine or the 3H/‘4C-amino acid mixture did not produce any detectable protein labelling and no labelled bands were detected from binding experiments using this material. In one experiment, human erythrocytes were used to bind the ?Gmethionine and ‘H/“Camino acid-la~lled parasite proteins. Two very faint bands with mol. wts 38 and 19 kDa were eluted when medium from %methionine labelled cultures was used as absorbing ligand, but no bands were detected when whole sonicated 35S-methionine-labelled parasites were bound to the human erythrocytes. There were also no bands detected when the ‘H/%-amino acid-la~lled parasites were bound to the human erythrocytes. Further characterization of these babesial erythrocyte binding proteins is underway in our laboratory. It is interesting that of the three major bands (67,48 and 38 kDa) initially seen to be binding to the bovine erythrocytes, only one, the 38 kDa protein, survives a second treatment through silicone oil. Apparently, the binding of the other two proteins is weaker, and therefore probably less specific. (We have not determined whether the 19 kDa band which also remains after two separations through the oil represents a monomeric unit of the 38 kDa protein.) Western blots of these eluted proteins probed with defined monoclonal antibodies indicate that the 67 kDa protein may be a previously reported babesial protease (Commins, Goodger & Wright, 1985). The eluted babesial erythrocyte binding proteins are much smaller than the equivalent molecule identified in P. falciparum using a similar technique, but in some of our experiments, a high molecular weight labelled protein remained associated with the bovine erythrocytes after the N&I treatment. This protein may represent an equivalent Babesia molecule which binds more tightly to the erythrocyte than the corresponding Plasmodium molecule. Further experiments are underway to investigate the nature of this
Research protein. Since both the normal and infected erythrocytes used in the assays were washed prior to use, we are also investigating whether the presence of plasma components affects the number and/or type of Babesia proteins which can bind to erythrocytes. Acknowledgements-We are grateful to Dr W. Jorgensen, Tick Fever Research Centre, Wacol, for kindly supplying the B. bovis-infected blood. This work was supported by the Australian Research Council.
REFERENCES CAMUSD. & HADLEYT. J. 1985. A Plasmodium falciparum antigen that binds to host erythrocytes and merozoites. Science 2301 553-556. COMMINSM. A., GRUDGERB. V. & WRIGHT I. G. 1985. Proteinases in the lysate of bovine erythrocytes infected with Babesia bovis: initial vaccination studies. International Journalfor Parasitology 15: 491495. HADLEYT. J., KLOTZF. W. & MILLER L. H. 1986. Invasion
Note
611
of erythrocytes by malarial parasites: a cellular and molecular overview. Annual Review of Microbiology 401 451477. HADLEY T. J., KLOTZ F. W., PASVOLG., HAYNESJ. D., MCGINNISSM. H., OKUBO Y. & MILLER L. H. 1987. Falciparum malaria parasites invade erythrocytes that lack glycophorin A and B (M’M’). Strain differences indicate receptor heterogeneity and two pathways for invasion. Journal of Clinical Investigation 80: 119&l 193. MAHONEYD. F. 1967. Bovine babesiosis: preparation and assessment of complement fixing antigens. Experimental Parasitology 20: 232-241. MILLER L. H.. HUDXIN D. & HAYNESJ. D. 1988. Identification of ~lasmodium knowlesi erythrocyte binding proteins. Molecular and Biochemical Parasitology 31: 217-222. MITCHELLG. H., HADLEYT. J., KLOTZ F. W., MCGINNISS M. H. & MILLER L. H. 1986. Invasion of erythrocytes by Plasmodium falciparum malaria parasites: evidence for receptor heterogeneity and two receptors. Blood 67: 15191521. ORLANDIP. A., KIM LEE SIM B., CHULAYJ. D. & HAYNES J. D. 1990. Characterization of the 175-kilodalton erythrocyte binding antigen of Plasmodium falciparum. Molecular and Biochemical Parasitology 40: 285-294.
