The ring-infected erythrocyte surface antigen protein of Plasmodium falciparum is phosphorylated upon association with the host cell membrane

The ring-infected erythrocyte surface antigen (RESA) is a 155-kDa malarial polypeptide which is released from merozoites and becomes associated with the erythrocyte membrane at the time of invasion. Inside-out vesicles (IOVs) prepared from Plasmodium falciparum-infected erythrocytes contain RESA, presumably bound to the membrane skeleton, as it is largely insoluble in Triton X-100. When these IOVs were incubated with [gamma-32P]ATP, a 155-kDa polypeptide was labeled in IOVs from infected, but not from uninfected erythrocytes. Immunoprecipitation using specific rabbit antisera confirmed that RESA is indeed a phosphoprotein. Phosphoamino acid analysis revealed phosphoserine and a small amount of phosphothreonine, but no phosphotyrosine. Labeling of intact parasitized erythrocytes with inorganic [32P]phosphate for several hours in culture resulted in RESA in Triton-insoluble extracts being phosphorylated. Labeling of synchronized parasites showed that RESA was phosphorylated only when it became associated with the erythrocyte membrane, and although RESA was abundant in mature parasites, it was not phosphorylated. RESA, released into the culture supernatants during the growth of P. falciparum, bound to IOVs prepared from normal uninfected erythrocytes, and subsequent labeling with [gamma-32P]ATP resulted in the phosphorylation of RESA. The evidence suggests that RESA is phosphorylated by an erythrocyte membrane kinase and probably not by a parasite-encoded enzyme.     

Movement of a falciparum malaria protein through the erythrocyte cytoplasm to the erythrocyte membrane is associated with lysis of the erythrocyte and release of gametes.

Erythrocytes containing mature gametocytes of Plasmodium falciparum circulate in the blood until they are ingested by a mosquito, an event that triggers gametogenesis and lysis of the infected erythrocyte. It was previously shown that a parasite protein (Pf155/RESA) accumulates in the erythrocyte cytoplasm next to the parasitophorous vacuolar membrane (S. Uni, A. Masuda, M. J. Stewart, R. Nussenzweig, and M. Aikawa, Am. J. Trop. Med. Hyg., 36:481-488, 1987). Using a monoclonal antibody to Pf155/RESA and rabbit sera to two different repeat peptides of Pf155/RESA, we have studied the location of Pf155/RESA after induction of gametogenesis. Five minutes after triggering gametogenesis, the parasitophorous membrane no longer surrounded the parasite, bringing the parasite membrane in contact with the erythrocyte cytoplasm. Clear spaces appeared throughout the hemoglobin-rich host cytoplasm; Pf155/RESA was now localized in the cytoplasm directly surrounding the spaces. No membrane existed between the spaces and the erythrocyte cytoplasm. The spaces with surrounding Pf155/RESA protein extended to the erythrocyte membrane. After lysis of the erythrocyte membrane (15 min after triggering gametogenesis), the protein was distributed along the erythrocyte membrane and throughout the space between the gamete and the erythrocyte membrane. The mechanism by which Pf155/RESA remained aggregated around the spaces and its role in erythrocyte lysis are unknown. It is of interest that the parasite appeared to use the same molecule during invasion of erythrocytes and during release of gametes from infected erythrocytes.     

Antibodies to the ring-infected erythrocyte surface antigen of Plasmodium falciparum elicited by infection with Plasmodium malariae.

The ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum (RESA-P), found in the membrane of erythrocytes infected with young asexual stages of P. falciparum, is a promising vaccine candidate. Antibodies to RESA-P were inducible by infection with another human malaria species, P. malariae. Of 298 serum samples from inhabitants of three isolated localities in Peru where P. vivax and P. malariae were endemic and P. falciparum had never been reported, 26% had anti-RESA-P antibodies as evidenced by a modified immunofluorescent-antibody assay and confirmed by Western blot (immunoblot) analysis. These seroepidemiologic observations were corroborated by the fact that of six chimpanzees infected with P. malariae, three developed anti-RESA-P antibodies after infection. The modified immunofluorescent-antibody-reactive antibodies, purified by adsorption and elution on monolayers of glutaraldehyde-fixed and air-dried P. falciparum-infected erythrocytes, reacted in an immunofluorescent-antibody assay with both parasite structures and erythrocyte membrane in P. falciparum antigen preparations, but only with parasite structures in P. malariae antigen preparations. This serologic cross-reactivity between P. falciparum and P. malariae is of interest in view of the importance of RESA-P as a vaccine candidate and because the two species are coendemic in many areas.     

Correct promoter control is needed for trafficking of the ring-infected erythrocyte surface antigen to the host cytosol in transfected malaria parasites

Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependent on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.     

Antibodies raised against receptor-binding domain of Plasmodium knowlesi Duffy binding protein inhibit erythrocyte invasion

Erythrocyte invasion by malaria parasites requires specific receptor-ligand interactions. Plasmodium vivax and Plasmodium knowlesi are completely dependent on binding the Duffy blood group antigen to invade human erythrocytes. P. knowlesi invades rhesus erythrocytes by multiple pathways using the Duffy antigen as well as alternative receptors. Plasmodium falciparum binds sialic acid residues on glycophorin A as well as other sialic acid-independent receptors to invade human erythrocytes. Parasite proteins that mediate these interactions belong to a family of erythrocyte binding proteins, which includes the P. vivax Duffy binding protein, 175 kDa P. falciparum erythrocyte binding antigen (EBA-175), P. knowlesi alpha protein, which binds human and rhesus Duffy antigens, and P. knowlesi beta and gamma proteins, which bind Duffy-independent receptors on rhesus erythrocytes. The receptor-binding domains of these proteins lie in conserved, N-terminal, cysteine-rich regions that are referred to as region II. Here, we have examined the feasibility of inhibiting erythrocyte invasion with antibodies directed against receptor-binding domains of erythrocyte binding proteins. Region II of P. knowelsi alpha protein (Pk(alpha)RII), which binds the Duffy antigen, was expressed as a secreted protein in insect cells and purified from culture supernatants. Rabbit antibodies raised against recombinant Pk(alpha)RII were tested for inhibition of erythrocyte binding and invasion. Antibodies raised against Pk(alpha)RII inhibit P. knowlesi invasion of both human and rhesus erythrocytes. These data provide support for the development of recombinant vaccines based on the homologous binding domains of P. vivax Duffy binding protein and P. falciparum EBA-175.     

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum associates with the erythrocyte membrane skeletal protein, band 4.1

Several proteins synthesized by mature asexual stages of Plasmodium falciparum interact with the erythrocyte membrane skeleton. One of these is the mature-parasite-infected erythrocyte surface antigen (MESA; also called PfEMP2), a phosphoprotein of 250-300 kDa, which is found on the internal face of the erythrocyte membrane. When MESA is precipitated with anti-MESA antibodies, another phosphoprotein of 80 kDa is co-precipitated. This 80-kDa phosphoprotein was identified by peptide mapping as the erythrocyte membrane component band 4.1. Thus, MESA is apparently anchored at the erythrocyte membrane through an association with band 4.1. Band 4.1 is more intensely phosphorylated in infected erythrocytes and is increased in relative molecular mass in erythrocytes infected by isolates of P. falciparum that cytoadhere.     

Affinity-purified antibodies to ring-infected erythrocyte surface antigen do not correlate with merozoite invasion inhibition in Plasmodium falciparum.

We affinity purified, from malaria-immune serum, antibody to the ring-infected erythrocyte surface antigen (RESA), using petri dishes containing a monolayer of Plasmodium falciparum ring-infected erythrocytes. Except for one out of eight samples, the purified antibody positive by RESA-immunofluorescent assay was not inhibitory to the in vitro invasion of merozoites into erythrocytes in three geographically distinct strains of P. falciparum. However, the initial high level of merozoite-inhibiting antibodies of the intact serum samples remained in the immunoglobulin G fraction from which the RESA antibodies had been removed by affinity chromatography. These results suggest that, although in some cases RESA-immunofluorescent assay-positive antibodies may be inhibitory to merozoite invasion, there are more important antibodies capable of merozoite invasion inhibition.     

MESA is a Plasmodium falciparum phosphoprotein associated with the erythrocyte membrane skeleton

The mature-parasite-infected erythrocyte surface antigen (MESA) of Plasmodium falciparum is an antigenically variable, high molecular weight protein of trophozoites and schizonts that is located at the erythrocyte surface membrane. It is first synthesized at the late ring stage and continues to be synthesized until late schizogony. MESA cannot be detected on the external surface of erythrocytes infected by trophozoites and early schizonts but is located at the internal surface in association with the erythrocyte membrane skeleton. The degree of association with the membrane skeleton varies among parasite lines, being greater in knobby parasite lines. MESA is phosphorylated and is present in a similar location to another phosphoprotein, the ring-infected erythrocyte surface antigen (RESA). However, it differs from RESA in being detected at a later stage of asexual development of the parasite.     

Phosphorylation of erythrocyte membrane and cytoskeleton proteins in cells infected with Plasmodium falciparum

Phosphorylation changes in the erythrocyte membrane and cytoskeletal proteins as a consequence of infection by the malarial parasite Plasmodium falciparum were examined. Spectrin, band 3, band 4.1, ankyrin and glycophorin are phosphorylated in normal erythrocytes. As a consequence of invasion by the merozoite, the extracellular stage of the parasite, into 32P-prelabeled normal erythrocytes, all the major 32P-labeled erythrocyte proteins are dephosphorylated. As the parasite develops intracellularly from the immature ring stage to the mature schizont stage, selective phosphorylation of certain host proteins, spectrin, ankyrin and band 3 is observed. Band 4.1 does not appear to incorporate [32P]phosphate at any stage of parasite development. These observed phosphorylation changes may be important in the regulation of the cytoskeletal organization in P. falciparum-infected cells.     

Possible relationship between Plasmodium falciparum ring-infected erythrocyte surface antigen (RESA) and host cell resistance to destruction by chemicals

Repeated incubation of Plasmodium falciparum culture in 0.015 % saponin solution for a total of 35 min destroys most of the uninfected cells, leaving only the ring-infected erythrocytes (RIEs). Parasites concentrated by this method can subsequently complete the asexual cycle and infect other erythrocytes. It is possible that resistance to saponin is mediated by one or more of the numerous parasite proteins present in the host erythrocyte membrane. We have found that schizonts are as susceptible as uninfected erythrocytes to saponin, indicating that the protective protein is parasite stage specific. Studies with cultured parasites have shown that ring-infected erythrocyte surface antigen (RESA) strengthens host erythrocyte membrane and protects against destruction. Therefore, we hypothesize that RESA could be involved in resistance to saponin. Here, we have carried out PCR test on RESA gene, using three different primers. One of them showed that P. falciparum isolates collected directly from infected humans and cultured only for a few days, or not at all, have amplicon sizes ranging from 372 to 510 bp. However, the amplicon size changed to 873 bp when in vitro growth was continued for one or more weeks. This genetic transformation precedes acquisition of the ability to confer saponin resistance to RIEs.     

Selective association of a fragment of the knob protein with spectrin, actin and the red cell membrane

The knob protein of Plasmodium falciparum is essential for the formation of knob-like protrusions on the host erythrocyte membrane. A functional domain of the knob protein was identified. This peptide formed stable complexes with the two major red cell skeletal proteins, spectrin and actin. When introduced into resealed normal erythrocytes, the peptide associated selectively with the cytoplasmic surface of the membrane and formed knob-like electron dense deposits. Knobs are thought to play an important role in the immunopathology of P. falciparum infections. Our findings provide a first step towards understanding the molecular basis for selective membrane changes at knobs.     

Association of Plasmodium berghei proteins with the host erythrocyte membrane: binding to inside-out vesicles

Two acidic phosphoproteins of Plasmodium berghei origin, of 65 and 46 kDa, are associated with the plasma membrane of the host mouse erythrocyte. The 65-kDa protein partitions between a soluble and particulate phase upon host cell lysis, whereas the 46-kDa protein is localized exclusively in the particulate fraction. Both proteins bind to inside-out vesicles derived from erythrocyte ghosts and the conditions of the reassociation reaction indicate that the binding is specific and that the proteins interact only with the cytoplasmic face of the erythrocyte membrane. The 65-kDa protein appears to exist in two membrane-associated states; one loosely bound, which readily dissociates from the membrane, and a more tightly associated state, which does not dissociate under non-denaturing conditions. The 46-kDa protein is tightly bound to the host erythrocyte membrane and does not dissociate. Cross-linking studies suggest that both of these parasite proteins interact with the submembrane cytoskeleton of the erythrocyte, and that the 65-kDa protein also appears to interact simultaneously with the lipid bilayer and erythrocyte membrane proteins. However, direct interaction between the malarial proteins and distinct erythrocyte membrane proteins could not be demonstrated. In summary, these findings indicate that the acidic phosphoproteins of the malarial parasite interact with the cytoplasmic face of the erythrocyte membrane both in vivo and in vitro.     

The C-terminal domain of the Plasmodium falciparum acyl-CoA synthetases PfACS1 and PfACS3 functions as ligand for ankyrin

Infection of erythrocytes by the malaria parasite Plasmodium falciparum results in the export of several parasite proteins into the erythrocyte cytoplasm establishing novel interactions between host and parasite proteins, particularly at the membrane skeleton that modifies both the structural and functional properties of the red cell. We present evidences that two members of the P. falciparum acyl-CoA synthetase (PfACS) family, responsible for the activation of long-chain fatty acids by thio-esterification with CoA, are transported in vesicle-like structures toward the host erythrocyte cytoplasm where they interact with the cytoskeletal protein ankyrin. Carboxyl-terminal domain (CTD) overlay studies indicated that PfACS1 and PfACS3 bind to the 78-kDa fragment of ankyrin corresponding with its spectrin-binding domain. Co-immunoprecipitation of ankyrin and PfACS1/3 indicates that at least a fraction of these proteins are physically associated in the infected erythrocytes and provide evidence for a novel specific interaction which suggest that such a binding may bring these enzymes closer to the host erythrocyte membrane where exogenous fatty acids are available.     

Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and their possible role in invasion

Rhoptry proteins of Plasmodium falciparum merozoites, of 140, 130, and 110 kDa, identified by co-precipitation with Mab.1B9, bind selectively to mouse erythrocytes and reticulocytes. The properties of binding are shown to correlate with invasion of P. falciparum into mouse erythrocytes. Invasion of two strains of P. falciparum 7G8 and FCR-3, into mouse erythrocytes was examined, and was found to differ significantly. The 7G8 strain invades mouse erythrocytes at a rate of 40-60% compared to invasion into human erythrocytes, whereas FCR-3 invades at a rate of 5-15%. Both strains of P. falciparum preferentially invade reticulocytes in the in vitro invasion assay. This correlated with an increase in the amount of rhoptry protein of the 7G8 strain bound to mouse erythrocytes, compared to the FCR-3 strain and an increased binding to reticulocytes compared to mature erythrocytes. Binding of the rhoptry proteins and merozoite invasion into the erythrocyte is blocked in erythrocytes treated with trypsin and chymotrypsin but not in neuraminidase-treated erythrocytes, suggesting that the putative receptor site is exposed and accessible on the erythrocyte surface. Rabbit antiserum against gp3, the major glycophorin of mouse erythrocytes, blocks binding of the rhoptry proteins to erythrocytes and reduces merozoite invasion into mouse erythrocytes by 50%. Binding of rhoptry proteins to mouse reticulocytes was not blocked by alpha gp3 indicating a receptor difference between reticulocytes and erythrocytes. Mab.1B9 reduces merozoite invasion but does not decrease binding of the rhoptry proteins to the mouse erythrocyte. The mouse erythrocyte serves as a useful model to study the receptor-ligand interaction of rhoptry proteins and host surface proteins and to define the role of the rhoptry proteins during the invasion process.     

Purification and characterization of 37-kilodalton proteases from Plasmodium falciparum and Plasmodium berghei which cleave erythrocyte cytoskeletal components

Cytosoluble 100,000 X g extracts from Plasmodium berghei or Plasmodium falciparum infected red blood cells were shown to hydrolyze erythrocyte spectrin. By Fast Protein Liquid Chromatography (FPLC), these enzymes were purified and exhibited a pI of 4.5 and Mr of 37,000 using SDS-PAGE under reducing conditions. An immunochemical enzyme assay using anti-spectrin antibodies was developed. The optimal activity using spectrin as substrate was at pH 5.0, and the enzymes were strongly inhibited by HgCl2, ZnCl2, chymostatin, leupeptin and aprotinin, and moderately by pepstatin. These properties of the Pf37 and Pb37 proteases differ from the Plasmodium lophurae and P. falciparum 'cathepsin D-like' enzymes and from the serine or cysteine neutral proteases previously described in P. falciparum and P. berghei infected red blood cells. While the Pf37 and Pb37 enzymes cleaved spectrin preferentially, degradation of band 4.1 was also observed with high concentration of enzyme. The parasite origin of the Pf37 protease was clearly demonstrated, since purified radiolabeled enzyme was active on spectrin. A high-molecular-weight polymer (greater than 240 kDa) was often observed on incubating purified spectrin and Pf37 protease. The breakdown of erythrocyte cytoskeletal components could be of interest in the release of merozoites from segmented schizonts or during the process of invasion of erythrocytes by merozoites.     

Amino terminus of Plasmodium falciparum acidic basic repeat antigen interacts with the erythrocyte membrane through band 3 protein

The acidic basic repeat antigen (ABRA) of Plasmodium falciparum is localised in the parasitophorous vacuole, and associates with the merozoite surface at the time of schizont rupture. By virtue of its protease-like activity, it is implicated in the process of merozoite invasion and schizont rupture, and therefore, possibly interacts with erythrocyte membrane proteins to execute its function during these events. In this study, using Escherichia coli expressed recombinant fragments of ABRA, we have demonstrated that ABRA interacts with red blood cells through its N-terminus. Out of the four human erythrocyte proteins tested, namely, band 3, glycophorin A and B and spectrin, ABRA showed dose-dependent and saturable binding with the band 3 protein. This binding was lost on chymotrypsin treatment of erythrocytes or their membrane extract. Studies with the deletion constructs of the N-terminus revealed that the binding domain lies in the cysteine-rich N-proximal region of ABRA. In addition to the recombinant fragments, native ABRA derived from the P. falciparum-infected erythrocytes also showed binding to band 3 protein. Sequencing of the cysteine-rich 528 bp region, amplified from fifteen field isolates of P. falciparum, showed that not only the five cysteines of mature ABRA but also the whole sequence is fully conserved, even at the nucleotide level. This sequence conservation of the N-terminus and its role in RBC binding suggests that this region may be crucial for any putative function of ABRA, therefore emphasising its importance as a vaccine/drug target.     

Identification of Plasmodium knowlesi erythrocyte binding proteins

Plasmodium knowlesi, a malaria of Old World monkeys, invades all Duffy blood group positive human erythrocytes and various New World monkey erythrocytes except Cebus apella. We had previously identified a 135 kDa parasite protein in supernatants of P. knowlesi cultures that bound to Duffy positive but not to Duffy negative human erythrocytes [Haynes et al., J. Exp. Med. 167, 1873-1881 (1988)]. We now use New World monkey erythrocytes as a reagent to identify P. knowlesi proteins in culture supernatants that will bind to all New World monkey erythrocytes susceptible to invasion but not to C. apella erythrocytes, which are refractory to invasion. The 135 kDa protein binds to all New World monkey erythrocytes, including C. appella. Another protein of 155 kDa binds to all New World monkey erythrocytes except C. apella. The 155 kDa protein binds to Old World monkey erythrocytes, the natural host of P. knowlesi; it does not bind to human Duffy positive erythrocytes. This and the previous study are the beginning of the identification of parasite proteins of P. knowlesi that bind to erythrocytes in a receptor specific manner.     

Interaction of Bartonella bacilliformis with human erythrocyte membrane proteins

Intracellular invasion is an important aspect of Carrión's disease caused by Bartonella bacilliformis. Both the hematic and tissue phases of the disease involve the initial attachment of the organism to erythrocytes and endothelial cells, respectively. Using two different approaches, preliminary evidence is provided that B. bacilliformis interacts with multiple surface-exposed proteins on human erythrocytes. Utilizing Western blot analysis, it was demonstrated that the organism binds several biotinylated erythrocyte proteins with approximate molecular masses of 230, 210, 100, 83 and 44 kDa. There was enhanced Bartonella binding to the 44 kDa protein and binding to a 25 kDa protein following exposure of intact red cells to trypsin. Moreover, there was a complete abrogation of binding to these proteins following exposure of erythrocytes to sodium metaperiodate oxidation, indicating the significance of carbohydrate moieties in the interactions of Bartonella with the erythrocyte. In a second approach, similar binding proteins or putative receptors were identified when Bartonella was co-incubated with isolated membrane proteins from red cell ghosts. A comparison of the molecular weights of these putative receptors with known erythrocyte proteins and their immunoreactivity to specific antisera suggested that the 230 and 210 kDa proteins are the alpha and beta subunits of spectrin; the 100 and 83 kDa proteins are band 3 protein and glycophorin A, respectively; and the 44 and 25 kDa proteins are the respective dimeric and monomeric forms of glycophorin B. Consistent with this notion was the binding of Bartonella to purified preparations of alpha and beta spectrin and glycophorin A/B.     

Plasmodium chabaudi antigen Pch105, Plasmodium falciparum antigen Pf155, and erythrocyte band 3 share cross-reactive epitopes.

By immunoblotting with a number of monoclonal antibodies raised in human and murine malaria systems, we have been able to establish the presence of cross-reactive epitopes on the Plasmodium falciparum vaccine candidate antigen Pf155/RESA and its proposed Plasmodium chabaudi analog Pch105. These findings were confirmed when the same antibodies were tested in an immunofluorescence assay. By using short synthetic peptides corresponding to repeated sequences in the C terminus of the Pf155 and enzyme-linked immunosorbent assays, the cross-reacting epitope was found to be localized to this repeat segment. Furthermore, a monoclonal antibody to murine erythrocyte band 3 which also cross-reacted with human band 3 bound to both Pch105 and Pf155 as well as to the synthetic peptides, suggesting that these proteins share a related epitope. The cross-reactions reflect the existence of sequence homologies of band 3 with these plasmodial proteins. This molecular similarity may be used by the parasite to disturb the rigidity of the erythrocyte membrane, thereby facilitating its entrance into the cell.     

Calcium is required for binding of Escherichia coli hemolysin (HlyA) to erythrocyte membranes.

The calcium requirement for hemolytic activity of Escherichia coli hemolysin was investigated by using hemolytic assays and immunoblotting of toxin-treated erythrocytes. The hemolytic activity of cell culture supernatants obtained during growth of E. coli in Luria-Bertani (LB) broth or calcium-free LB broth was calcium dependent. The hemolytic activity of culture supernatants obtained during growth in LB broth supplemented with calcium was calcium independent. Osmotic protection experiments using Dextran 4 to prevent cell lysis indicated that calcium was required for the binding of hemolysin to erythrocytes at both 4 and 37 degrees C. The binding efficiency at 4 degrees C was 50% of that occurring at 37 degrees C. The calcium-dependent binding was confirmed by immunoblotting saline-washed, toxin-treated erythrocytes with a monoclonal antibody after sodium dodecyl sulfate-polyacrylamide gel electrophoresis separation of membrane proteins. Bound hemolysin increased the calcium permeability of the cell membranes as evidenced by calcium-induced membrane protein alterations. The alterations in membrane proteins did not directly cause lysis of the cells. The results were consistent with a mechanism of lysis involving the formation of cation-selective pores in the membranes of target cells.