M. AIKAWA
Institute of Pathology
Case Western Reserve University
Cleveland, Ohio 44106, USA
Erythrocyte Changes Induced by P. falciparum
Movement of the P. falciparum Protein to the Erythrocyte Membrane Resulting in Lysis of the Erythrocyte and Release of Gametes
Erythrocyte Changes Induced by P. brasilianum Infection
Erythrocyte Changes Induced by P. vivax
Conclusions
References
Morphological and functional changes occur in erythrocytes infected with malarial parasites. These erythrocyte alterations appear to relate to the capability of malarial parasites to change the properties of the erythrocyte and its membrane in order to export malarial proteins from the parasite to the host erythrocyte membrane. Although the significance of these changes are not clear, they appear to be involved in the development of malaria-related complications in the host.
In my presentation, the host cell alterations induced by primate parasites, including Plasmodium falciparum, P. brasilianum, P. vivax and P. malariae infections, will be discussed and the trafficking of these malarial proteins from the parasite to the erythrocyte membrane will be described.
Plasmodium falciparum infection induces morphological changes in erythrocytes that include (1) knob-like protrusions of the erythrocyte membrane, (2) clefts in the erythrocyte cytoplasm and (3) electron dense material (EDM) in the erythrocyte cytoplasm. 3,8
Electron dense material and clefts appear to be associated with the formation of knobs. EDM is seen associated with the plasma membrane of intracellular parasites and is also associated with unit membrane-bounded Maurer's clefts. This EDM has the same density and appearance as the material located under knobs at the erythrocyte membrane.4 Immunoelectron microscopy has demonstrated that EDM and knobs contain P. falciparum antigens. Thus, the parasite-derived EDM appears to be transported from the parasite plasmalemma to the erythrocyte membrane via clefts in the erythrocyte cytoplasm.
At least four malarial proteins (HRP1, HRP2, EMP1 and EMP2) have been identified in the surface of P. falciparum-infected erythrocytes. Three of these proteins - HRP1, EMP1 and EMP2 - are localized in knobs as demonstrated by immunoelectron microscopy. The presence of histidine-rich proteins in HRP1 is an important factor in knob formation,9 and a histidine analogue, 2-fluoro-L-histidine, reduces the formation of knobs and increases the amount and density of ELM.5 This indicates that this histidine analogue blocks export of EDM from the parasite to knobs, thereby inhibiting knob formation.
The knobs have cytoadherence activity to the endothelial cells11 and are responsible for sequestration of infected erythrocytes in organs such as the spleen, pancreas, heart and brain. Host cell molecules such as OKM56 and thrombospondin 12 have been suggested as endothelial cell surface receptors for the knobs of P. falciparum-infected erythrocytes. By acting independently or together, these proteins could play a role in cytoadherence of knobs in vitro.1
During erythrocyte invasion by merozoites, a molecule (Pf155/RESA) is injected by the parasite into the erythrocyte membrane and the entire membrane becomes covered with Pf155/RESA.14 During differentiation of the parasite to the trophozoite stage, the antigens, as detected by immuno-gold labelling, are no longer detectable on the erythrocyte membrane, while gold particles become more numerous within the parasite and in the erythrocyte cytoplasm adjacent to the parasite. As the parasites develop into schizonts, more antigen appears within the parasite and some of it appears to diffuse into the erythrocyte cytoplasm. At the segmented schizont stage, many intraparasitic gold particles are associated with rhoptries and micronemes of developing merozoites.14
In addition, Pf155/RESA appeared to be associated with gametocytes. Therefore, using a MAb to Pf155/RESA and rabbit sera to two different repeat peptides of Pf155/RESA, we studied the location of Pf155/RESA after induction of gametogenesis. Five minutes after gametogenesis was triggered, the parasitophorous membrane no longer surrounded the parasites, bringing the parasite membrane in contact with erythrocyte cytoplasm. Clear spaces appeared throughout the haemoglobin-rich host cytoplasm. Pf155/RESA was now localized in the haemoglobin directly surrounding the spaces. No membrane existed between the spaces and the haemoglobin. The spaces with surrounding malarial protein extended to the erythrocyte membrane. After lysis of the erythrocyte membrane, the antigen was distributed along the erythrocyte membrane and throughout the space between the gamete and the erythrocyte membrane.
Our study, therefore, indicates that Pf155/RESA antigen is responsible for disrupting the red blood cell cytoplasm and lysing the erythrocyte plasma membrane, thereby allowing release of P. falciparum gametes from their host cells.
Plasmodium brasilianum is a quartan malarial parasite of New World monkeys. A close evolutionary relationship between P. brasilianum and the human malaria parasite, P. malariae, is suggested by analogies in their morphology and course of their development in primate hosts.
Erythrocyte changes include the formation of knobs on the infected erythrocyte membrane and clefts in the erthrocyte cytoplasm.7 The knobs are similar to those seen in P. falciparum-infected erthrocytes. The cytoplasmic clefts can be divided into three morphological types, namely, short, long and circular clefts. Recently a series of monoclonal antibodies (MAbs) against blood stages of P. brasilianum has been developed that reacts with knobs and clefts as detected by immunoelectron microscopy.
MAbs that recognize an antigen of 38 kDa molecular weight reacted with short clefts. Immuno-gold particles appeared over short clefts and were not associated with long clefts, reflecting the specificity of binding of these MAbs to short cleft antigen.7 MAbs that recognize an antigen of 16 kDa molecular weight reacted with long clefts and also with the parasitophorous vacuole membrane (PVM). The long clefts appear to be continuous with both PVM and IRBC membranes. Immunoelectron microscopy identified the presence of 14, 16 and 19 kDa malarial proteins in knobs.
The difference in antigenic composition between short and long clefts has not been reported for any plasmodial species. That the long cleft antigen was associated with the PVM and the short cleft antigen was not suggests that different mechanisms incorporate the two antigens into their respective clefts.7 The clefts have been suggested to function in the transport of knob materials, as demonstrated in P. falciparum-infected erythrocytes. However, cleft and knob antigens of P. brasilianum are immunologically distinct. It is possible that the nature of antigens being transported via clefts varies with the stage of parasite development and that early in the parasite life cycle, knob proteins are principally transported. At a later stage of development, the antigenic composition of the clefts might change.
The relationship between knob proteins of P. brasilianum and P. falciparum remains unclear. Although the knobs of P. falciparum-infected erythrocytes have cytoadherent activity, those on P. brasilianum-infected erythrocytes do not appear to possess cytoadherent functions. Further studies may be required to determine the function of the knobs that appear on the membrane of erythrocytes infected with P. brasilianum.
Erythrocytes infected with vivax-type malaria parasites are characterized by Schuffner's dots, which appear as multiple small brick-red dots in Giemsa-stained thin films. Electron microscopy demonstrated that Schuffner's dots are composed of caveola-vesicle complexes (CVC).2 This structure consists of caveolae to which vesicles are attached in an alveolar fashion. Another host cell alteration observed within the infected erythrocyte cytoplasm is cytoplasmic clefts.
Recently investigators produced a series of MAbs against various antigens of erythrocytic stages of P. vivax. Among them, MAbs that identified a 95 kDa 35 S-methionine-labelled P. vivax protein produced a stippled pattern similar to Schuffner's dots in P. vivax-infected erythrocytes. Other MAbs that reacted with a 28 kDa 35S-methionine-labelled protein gave a linear pattern by immunofluorescent microscopy.10
To identify the precise location of P. vivax antigens that react with these MAbs, we perfommed post-embedding Immunoelectron microscopy. MAbs against a 95 kDa
35S-methionine-labelled protein gave a pattern similar to that of Schuffner's dots by the immunofluorescence test (IFA). Immunoelectron microscopically specific label was found by Immunoelectron microscopy to be associated with vesicles of the CVC, whereas only a few gold particles were associated with the caveolae. Vesicles scattered throughout the erythrocyte cytoplasm were also labelled with gold particles.
Other MAbs that react with a 28 kDa parasite protein gave a linear pattern in the cytoplasm of infected erythrocytes by IFA. Immunoelectron microscopy clearly revealed that the target antigen of these MAbs was located along the cytoplasmic clefts of infected erythrocytes. Immunoreactivity was also observed in association with vesicles scattered in the erythrocyte cytoplasm and vesicles of the CVC.
A double-labelling technique was applied to localize the 28 kDa and 95 kDa antigens in the same erythrocyte. The small gold particles identified the 95 kDa antigenic sites, while the large gold particles identified the 28 kDa antigenic sites. Immunoelectron microscopy demonstrated that small gold particles were associated only with vesicles and most of the large gold particles were seen in clefts. Some large particles, however, were found in association with vesicles together with small particles. Thus, double-labelling confirmed that the vesicles contained predominantly the 95 kDa antigen and some of the 28 kDa antigen, whereas clefts were associated only with the 28 kDa antigen.
The presence of P. vivax antigens in clefts and CVC indicates that these structures are related to trafficking of P. vivax antigen from the parasites to the erythrocyte surface membrane. Some P. vivax proteins, at least the 28 kDa protein, are transported from the parasite to the parasitophorous vacuole and to the clefts. The proteins are then transported along clefts and transferred to vesicles. The proteins moved toward the erythrocyte surface and into the caveola space. The proteins are then released from the caveola extracellulary. The vesicles might be formed by budding from the tips of lamellate clefts, similar to Golgi vesicles that are pinched off from the Golgi stack. On the other hand, a 95 kDa protein is present only within the vesicles and not in the clefts. This could indicate that the vesicles containing a 95 kDa protein may originate directly from the parasitophorous vacuole membrane. These observations indicate that host cell changes induced by P. vivax are involved in trafficking of P. vivax antigens to the erythrocyte membrane.
Host cell alterations induced by P. falciparum-, P. brasilianum- and P. vivax-infections were described by electron microscopy and post-embedding immunoelectron microscopy. Plasmodium falciparum infection induces knobs, electron dense material and clefts in the erythrocyte. Clefts are involved in exporting P. falciparum antigen from the parasite to the erythrocyte membrane. P. falciparum antigen is present in knobs that adhere to endothelial cells, causing the blockage of capillaries. Pf155/RESA, one of P. falciparum antigens, appears to lyse the erythrocyte cytoplasm and to assist in the release of gametes from the erythrocyte.
Plasmodium brasilianum infection induces knobs, short and long clefts and electron dense material, which are engaged in trafficking of P. brasilianum protein from the parasite to the erythrocyte surface. P. vivax infection induces caveola-vesicle complexes and clefts in the erthrocyte. They are also involved in trafficking of P. vivax protein from the parasite to the erythrocyte membrane. Our studies, therefore, indicate that host cell changes occurring in various species of malarial parasites facilitate the transport of malaria antigens to the host cell membrane.
1. AIKAWA, M. 1988. Am. J. Trop. Med. Hyg. 39: 3-10.
2. AIKAWA, M., L.H. MILLER and I. RABBEGE. 1975. Am. J. Path. 79: 285-300.
3. AIKAWA, M., J.R. RABBEGE, I. UDEINYA and L.H. MILLER. 1983. J. Parasitol. 69: 443-447.
4. AIKAWA, M., S. UNI, A.T. ANDRUTIS and R.J. HOWARD. 1986. Am. J. Trop. Med. Hyg. 35: 30-36.
5. AIKAWA, M., S. UNI, A.T. ANDRUTIS, K.L. KIRK, L.A. COHEN and R. HOWARD. In Host-Parasite Cellular and Molecular Interactions in Protozoal Infections. K.P. Chang and D. Snary, Eds.: 297-306. NATO-ASI volume. Springer, New York.
6. BARNWELL, J.W., C.F. OCKENHOUSE and D.M. KNOWLES. 1985. J. Immunol. 135: 3494-3497.
7. COCHRANE, A.H., Y. MATSUMOTO, K.K. KAMBOJ, M. MARACIC, R.S. NUSSENZWEIG and M. AIKAWA. 1988. Infect. Immun. 56: 2080-2088.
8. HOWARD, R.J., S. UNI, J.A. LYON, D.W. TAYLOR, W. DANIEL and M. AIKAWA. In Host-Parasite Cellular and Molecular Interactions in Protozoal Infections. K.P. Chang, and D. Snary, Eds.: 281-296. NATO-ASI volume. Springer, New York.
9. KILEGIAN, A. 1979. Proc. Natl. Acad. Sci. USA 76: 4650-4653.
10. MATSUMOTO, Y., M. AIKAWA and J.W. BARNWELL. In press. Am. J. Trop. Med. Hyg.
11. OO, M.M., M. AIKAWA, T. THAN, T.O.M. AYE, P.T. MYINT, I. IGARASHI and W.C. SCHOENE. 1987. J. Neuropath. Exptl. Neurol. 46: 223-231.
12. ROBERTS, D.D., J.A. SHERWOOD, S.L. SPITALNIK, L.J. PANTON, R.J. HOWARD, V.M. DIXIT, W. A. FRAZIAR, L.H. MILLER and V. GINSBURG. 1985. Nature 318: 64-66.
13. UDAGAMA, P.V., C.T. ATKINSON, J.S.M. PEIRIS, P. DAVID, K.N. MENDIS and M. AIKAWA. 1988. Am. J. Path. 131: 48-52.
14. UNI, S., A. MASUDA, M.J. STEWART, I. IGARASHI R.S. NUSSENZWEIG and AIKAWA, M. 1987. Am. J. Trop. Med. Hyg. 36: 481-488.
