Human intraerythrocytic malarial parasites (Plasmodium falciparum) induce permeability changes in the membrane of their host cells. The differential permeability of infected erythrocytes at various stages of parasite growth, in combination with density gradient centrifugation, was used to fractionate parasitized cells according to their developmental stage. By this method it was possible to obtain cell fractions consisting essentially of erythrocytes infected with the youngest parasite stage (i.e., rings). These preparations were used for the measurement of transport of various solutes. It is shown that permeabilization of host erythrocyte membrane appears as early as 6 h after parasite invasion of the erythrocyte and increases gradually with parasite maturation. Since the selectivity for several different solutes and the enthalpy of activation of transport remain unaltered with maturation-related increase of permeability, it is concluded that the number of transport agencies in the host cell membrane increases with parasite maturation. Evidence is presented to indicate the need for parasite protein synthesis as an essential factor for the generation of the new permeability pathways.
The development of the malaria parasite Plasmodium falciparum in human red blood cells induces parasite-dependent perturbations in the permselectivity properties of the host cell membrane. The changes appear as parasites develop from ring to the trophozoite stage and persist during schizogony. In the present work we assessed the permeability changes of the infected cells to anionic substances by the use of radioactive and fluorescent probes. Our data show that i) covalent binding probes, such as diisothiocyano ditritiostilbene disulfonic acid [3H]H2DIDS, which are virtually impermeant to normal red blood cells, became markedly permeant to trophozoites and schizonts, as evidenced by high labeling of intracellular hemoglobin; ii) permeation of the fluorescent anion transport substrate NBD-taurine, measured in the efflux mode, was very rapid and substantially enhanced in parasitized erythrocytes, as compared with noninfected cells; iii) this efflux could not be blocked by H2DIDS, which is a specific inhibitor of anion transport in normal red blood cells; iv) permeation of anionic probes was temperature dependent (Ea:11 +/- 1 kcal/mole); and v) could be blocked by nonspecific transport inhibitors that are known to interact with membrane lipids. The appearance of a new permeation pathway in the host cell membrane of trophozoites is associated with structural modification of the host cell membrane matrix.
Several fibrinolytic activities of Treponema denticola, an oral spirochete associated with gingivitis and periodontal disease, were identified and characterized following phase partitioning with the nonionic detergent Triton X-114. The apparent molecular masses of the proteases ranged from 91 to 228 kDa when analyzed in sodium dodecyl sulfate-polyacrylamide gels containing fibrinogen as the protease substrate. A qualitative analysis of zymograms showed that the proteases were highly enriched in the detergent phase, although the 91-, 173-, and 228-kDa proteases were also found in the aqueous phase. Zymograms of crude outer sheaths prepared by repeated freezing-thawing revealed that the proteases may be associated with this subcellular compartment. The proteases displayed substrate specificity towards fibrinogen, were susceptible to sulfhydryl group reagents, and had a pH optimum between 7 and 8. The similarities in their sensitivity to inhibitors, temperature stability, pH optimum, and laddered protein profiles suggest that these hydrolytic enzymes may be part of a family of oligomeric proteases that may play an important role in the invasiveness of and tissue damage caused by the spirochete.
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