Circumsporozoite protein of Plasmodium berghei: gene cloning and identification of the immunodominant epitopes.

The gene encoding the circumsporozoite (CS) protein of the rodent malaria parasite Plasmodium berghei was cloned and characterized. A cDNA library made from P. berghei sporozoite RNA was screened with a monoclonal antibody for expression of CS protein epitopes. The resulting cDNA clone was used to isolate the CS protein gene from a lambda library containing parasite blood-stage DNA. The CS protein gene contains a central region encoding two types of tandemly repeated amino acid units, flanked by nonrepeated regions encoding amino- and carboxy-terminal signal and anchorlike sequences, respectively. One of the central repeated amino acid unit types contains the immunodominant epitopes.     

Decay-accelerating factor (DAF) shares a common carbohydrate determinant with the variant surface glycoprotein (VSG) of the African Trypanosoma brucei

Decay-accelerating factor (DAF) is an integral membrane protein that inhibits amplification of the complement cascade on the cell surface. We and other investigators have shown that DAF is part of a newly characterized family of proteins that are anchored to the cell membrane by phosphatidylinositol (PI). The group includes the variant surface glycoprotein (VSG) of African trypanosomes, the p63 protein of Leishmania, acetylcholinesterase (AChE), alkaline phosphatase, Thy-1, 5'-nucleotidase, and RT6.2--an alloantigen from rat T cells. The structure of the membrane anchor has been best characterized for VSG, but chemical studies of the membrane anchors of AChE and Thy-1 suggest that similar glycolipid moieties anchor these proteins to the cell surface. In the VSG, the membrane anchor consists of an ethanolamine linked covalently to an oligosaccharide and glucosamine; the entire complex is anchored to the cell membrane by PI. Immunologically, this glycolipid defines an epitope, the cross-reacting determinant (CRD), that is only revealed after removal of the diacyl glycerol anchor by a phospholipase C. By Western blotting, we show here that DAF-S (DAF released from the membrane by PI-specific phospholipase C [PIPLC]) also contains CRD. Using a newly developed immunoradiometric assay (IRMA) in which the solid-phase capturing antibody is a monoclonal antibody to DAF and the second antibody is anti-CRD, we have been able to quantitate DAF-S. By IRMA, we show that the reaction between anti-CRD and DAF-S is specific, since the binding is competitively inhibited only by the soluble form of the VSG. These observations further support the concept that the glycolipid anchors of this new family of proteins have similar structures. DAF is also found as a soluble protein in various tissue fluids as well as in Hela cell supernatants. No evidence for the presence of the CRD epitope was found on these proteins, suggesting that these forms of DAF are not released from the surface of cells by endogenous phospholipases.     

Trypanosoma cruzi invade a mammalian epithelial cell in a polarized manner

We have determined that parasite entry into host cells can be influenced by cell polarity using a DNA probe to quantitate the infection of cultured Madin-Darby canine kidney (MDCK) epithelial cells by Trypanosoma cruzi, the agent of Chagas' disease. Confluent MDCK cells are polarized, with their plasma membrane separated by tight junctions into two domains, apical and basolateral. We show that T. cruzi forms corresponding to the insect infective stages (metacyclics) and the vertebrate blood stages (trypomastigotes) enter confluent MDCK cells preferentially through their basolateral domains. Sparsely plated MDCK cells are less polarized and are better infected than confluent cells. Scanning electron microscopy showed that 92% +/- 4% of the parasites entered at the edges of cells.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Resialylation of sialidase-treated sheep and human erythrocytes by Trypanosoma cruzi trans-sialidase: restoration of complement resistance of desialylated sheep erythrocytes.

Trypanosoma cruzi trans-sialidase (TS) is a recently described enzyme which transfers alpha(2-3)-linked sialic acid from host-derived sialylated glycoconjugates to parasite surface molecules [Schenkman et al. (1991) Cell, 65, 1117]. We report here on the ability of TS to transfer sialic acid from donor sialyl-alpha(2-3)lactose to sialidase-treated sheep and human erythrocytes. Up to approximately 50% resialylation of both desialylated red cells could be attained. Resialylation of desialylated sheep erythrocytes restores their resistance to lysis by human complement. This ascribes a possible biological role for T. cruzi TS and demonstrates directly that sialic acid is solely responsible for preventing alternative pathway activation of human complement by sheep erythrocytes.     

Microelectrode studies of the active Na transport pathway of frog skin

When the outer surface of short-circuited frog skin was penetrated with microelectrodes, stable negative potentials that averaged near -100 mV were recorded consistently, confirming the results of Nagel (W. Nagel. 1975. Abstracts of the 5th International Biophysics Congress, Copenhagen. P-147.). The appearance of these stable potentials, V(O), concurrent with the observations that (a) a high resistance outer barrier R(O) accounting for approximately 75 percent or more of the transcellular resistance of control skins had been penetrated and that (b) 10(-5) M amiloride and reduced [Na] outside caused the values of V(O) to increase towards means value near -130 mV while the values of percent R(O) increased to more than 90 percent. It was of relationships were the same as the values of E(1) observed in studies of the current-voltage relationships were the same as the values of E’(1) defined as the values of voltage at the inner barrier when the V(O) of the outer barrier was reduced to zero by voltage clamping of the skins. Accordingly, these data are interpreted to mean that the values of E(1), approximately 130 mV, represent the E(Na) of the sodium pump at the inner barrier. 2,4-DNP was observed to decrease the values of transepithelial voltage less than E(1) the V(O) was negative. These data can be interpreted with a simple electrical equivalent circuit of the active sodium transport pathway of the frog skin that includes the idea that the outer membrane behaves as an electrical rectifier for ion transport.     

Freeze-fracture study of malaria sporozoites: antibody-induced changes of the pellicular membrane.

Plasmodium cynomolgi, Plasmodium knowlesi, and Plasmodium berghei sporozoites, before and after incubation with immune serum, were studied after freeze-fracture by electron microscopy. There were evenly distributed numerous intramembranous particles (IMP) on the P face of the outer membrane. The E face of the plasma membrane had fewer IMP than its P face. The E face of the intermediate membrane had few IMP and also linear arrays of slightly raised ridges running the length of the parasite. The P face of the intermediate membrane had many IMP aligned along the long axis of the sporozoite. On the P face of the inner membrane, IMP were arranged in very distinct rows conforming to the long axis of the parasite; the E face of this membrane had a few randomly distributed IMP. A prominent change in the sporozoite incubated in immune serum was the appearance of a layer of aggregated particles around the parasite. The P face of the plasma membrane had several clear areas devoid of IMP and IMP aggregates. No changes were seen in the other fractured faces of the pellicle. These observations suggest that immune serum acts only on the P face of the plasma membrane.     

Attachment of Trypanosoma cruzi trypomastigotes to receptors at restricted cell surface domains

We have used glutaraldehyde-fixed target cells to study the attachment phase of cell invasion by live trypomastigotes of Trypanosoma cruzi, and determined that attachment is polarized and receptor-mediated. T. cruzi trypomastigotes bind much less efficiently to confluent epithelial cells, which are polarized, than to sparse epithelial cells. When the tight junctions of confluent epithelial cells are disrupted by removing Ca2+ from the incubation medium before glutaraldehyde fixation, binding of T. cruzi increases. T. cruzi also shows preference for attachment underneath cells or to the edges of cells. The binding occurs within a few minutes, is saturable, and is influenced by the parasite developmental stage. Fab fragment derived from monoclonal antibodies that immunoprecipitate a 160-kDa molecule present only on the surface of trypomastigotes inhibit adhesion to fixed and live cells. Future characterization of the target cell receptors for this molecule and the use of fixed target cells should facilitate studies of the mechanisms involved in the initial interaction of T. cruzi with its host cells.     

Trichinella spiralis: anaphylactic antibody formation and susceptibility in strains of inbred mice.

The anaphylactic antibody response of various strains of inbred mice of different H-2 specificities was investigated using the passive cutaneous anaphylactic technique (PCA) for the detection of the antibody response. Neither IgC₁ nor reaginic antibody were detected in serum samples obtained at the end of the first week of infection with Trichinella spiralis. Subsequently, all animals had detectable IgG₁ antibodies, although in some strains the titers were very low. Reaginic antibody was detected in relatively high titers in C57L, A, and DBA/1 mice. Two other strains were very poor responders (SJL and AKR). In most strains, reagin and IgG₁ remained detectable for 14 wk or longer. The pattern of response of all strains was very reproducible, indicating genetic control of the anaphylactic antibody production to the infection. In F₁ hybrids obtained from crosses between good and poor anaphylactic antibody responders, intermediate levels of both antibody classes were detected.Adult worm recovery rates were established at various points during the intestinal phase of infection, and no correlation between worm numbers and reaginic antibody titers in the various strains of mice could be demonstrated. There were noticeable differences in larval yields obtained after muscle digestion of mice belonging to the different inbred strains. In fact, we generally observed an inverse relationship between the number of larvae recovered from a given strain and their reaginic antibody titer.The intravenous injection of newborn larvae (NBL), obtained upon in vitro incubation of adult worms, produced detectable antibodies only in mice of the DBA/1 strain. These antibodies were consistently of low titer and became detectable only after the administration of two additional injections of NBL. This contrasted with the results observed after “per os” infection of DBA/1 mice, where high titers of these antibodies were always obtained, in spite of comparable ratios of muscle larval yield.