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[Leishmania(L.)] amazonensis amastigotes reside in macrophages within spacious parasitophorous vacuoles (PVs) which may contain numerous parasites. After sporadic fusion events were detected by time-lapse cinemicrography, PV fusion was examined in two different models. In single infections, it was inferred from the reduction in PV numbers per cell. In a reinfection model, macrophages infected with unlabeled amastigotes were reinfected with GFP-transfected- or carboxyfluorescein diacetate succinimidyl ester-labeled parasites, and fusion was detected by the colocalization of labeled and unlabeled amastigotes in the same PVs. The main findings were: (1) as expected, fusion frequency increased with the multiplicity of infection; (2) most fusion events took place in the first 24h of infection or reinfection, prior to the multiplication of incoming parasites; (3) resident and incoming parasites multiplied at similar rates in fused PVs. The model should be useful in studies of parasite and host cell factors and mechanisms involved in PV fusogenicity.

We have previously shown that free galactosugars and N-acetylgalactosamine glycosaminoglycans, e.g., chondroitin sulfate (CS), release the 67-kDa elastin binding protein (EBP) from arterial smooth muscle cell (SMC) surfaces. This disrupts cell contact with elastin, impairs assembly of new elastic fibers, and increases fibronectin production, all of which promote SMC migration and intimal thickening. The present study uncovered a mechanism regulating fibronectin production in vascular myocytes related to a functional interplay between EBP and the interleukin-1 receptor type I. We showed that CS-induced shedding of the EBP or internalization of this receptor after saturation with elastin-derived peptides (kappa-elastin, kappa-El) stimulated fibronectin production in cultures of coronary artery SMC to a level observed with recombinant interleukin (IL)-1beta. Upregulation of fibronectin by CS or kappa-El was abolished by a soluble IL-1 receptor antagonist, and synergistic stimulation of fibronectin production occurred when CS or kappa-El was added with IL-1beta. Immunohistochemistry showed that EBP and IL-1 receptor type I codistributed on surfaces of unstimulated coronary artery SMC, while CS- and kappa-El-dependent removal of EBP from the cell surface increased binding of radiolabeled IL-1beta to CA SMC. We propose a unique interaction between both receptors in which unoccupied EBP interferes with IL-1beta binding. Conversely, increased accumulation of N-acetylgalactosamine glycosaminoglycans or elastin-derived peptides in the vascular wall may unmask IL-1 receptor type I and increase binding of the cytokine and consequent upregulation of fibronectin production.

Impaired elastin fiber assembly is observed in the fetal ductus arteriosus (DA), associated with a reduced concentration of elastin binding protein (EBP), a 67-kDa galactolectin. It is also seen in cultured aortic (Ao) smooth muscle cells (SMC) following the release of the EBP by glycosaminoglycans rich in N-acetylgalactosamine, such as chondroitin sulfate (CS). In the DA, impaired elastin fiber assembly is observed in conjunction with intimal thickening associated with increased migration of SMC into the subendothelium, a feature we previously related to increased production of fibronectin. In this report, we determined whether SMC use the EBP to attach to an elastin substrate, whether shedding of the EBP promotes SMC migration through a three-dimensional network of pure elastic laminae prepared from sheep aorta, and whether the latter is associated with increased production of fibronectin. We observed reduced attachment to elastin-coated surfaces of DA SMC deficient in EBP compared to Ao SMC. Addition of CS but not heparan sulfate (a glycosaminoglycan which does not induce EBP shedding) decreased Ao SMC attachment to elastin, as did preincubation with VGVAPG elastin-derived peptides which saturate the EBP. The immunolocalization of cell surface EBP suggested that cells can quickly replace EBP released from their surfaces by CS treatment. The magnitude of CS-induced impaired attachment of SMC to elastin was dose dependent and could be further increased by the administration of cyclohexamide and sodium azide. Also, the reversibility of CS-induced detachment was prevented by monensin. This suggests that a process of new synthesis and intracellular transport of the EBP was necessary to replace the EBP molecules released from the cell surface by CS treatment. In the migration assay, both DA and Ao SMC attached to the top of an elastin membrane, but only DA SMC deficient in EBP migrated through the laminae. Addition of CS, which induced shedding of EBP, resulted in Ao SMC migration associated with increased synthesis of fibronectin. We postulate that CS-induced release of EBP from SMC surfaces causes cell detachment from elastin and an increase in fibronectin synthesis, processes which may be critical in promoting SMC migration associated with intimal thickening developmentally in the DA and perhaps also in vascular disease.

Living Leishmania amazonensis amastigotes were incubated with radioiodinated N-benzyloxycarbonyl-L-tyrosyl-L-alanyl diazomethane (Z-Tyr-AlaCHN2), an irreversible inhibitor of mammalian cathepsins B and L. Parasite lysates were subjected to electrophoresis in gelatin-containing sodium dodecyl sulfate-acrylamide gels to detect regions of proteolytic activity, and the distribution of the inhibitor was ascertained by autoradiography. Of the three main bands of proteolysis associated with cysteine proteinases, two, with apparent molecular weights of 28 and 31 kDa, were shown to be labeled. The third enzyme activity, detected at the 35-kDa region in substrate gels, was only faintly labeled. The distribution of labeled bands was similar when lysates of untreated parasites were electrophoresed and the gels incubated with the radioiodinated inhibitor. Under reducing conditions, the inhibitor bound to polypeptides of 29, 31, 32, and 34 kDa, of which the first and the last were the most intensely labeled. Polypeptides with the same apparent molecular weights were labeled when amastigote lysates were incubated with the 125I inhibitor. Uptake of radioactivity by the parasites was time and concentration-dependent and more than 80% of the total counts could be precipitated with trichloroacetic acid. Radioactivity associated with the amastigotes was quite stable after they were pulsed with labeled inhibitor and chased for up to 24 hr in inhibitor-free medium. Both total uptake and labeling of cysteine proteinases were markedly reduced in parasites preincubated with Z-Phe-AlaCHN2 prior to exposure to Z-Tyr(125I)-AlaCHN2. However, more radioiodinated inhibitor was taken up by parasites preincubated with cold inhibitor and chased in inhibitor-free medium, suggesting de novo synthesis or processing of inactive enzyme precursors.

Bone marrow-derived mouse macrophage cultures infected with Leishmania mexicana amazonensis amastigotes were given a 2-hr pulse with 10 microM phenazine methosulfate (PMS), a cationic electron carrier which destroys the intracellular parasites. Cultures were fixed at different times after the PMS pulse and processed for the detection of acid phosphatase (AcP) activity at the electron microscopic level. Only a small proportion of nontreated, infected macrophages stained for AcP. In contrast, 2 to 6 hr after exposure to PMS, many infected cells displayed AcP-positive lysosomes and parasitophorous vacuoles. This increased AcP reactivity paralleled the reduction in the percentage of morphologically intact parasites. In addition, qualitative observations indicated that while nontreated infected cells contained only few recognizable lysosomes, the lysosomal complement noticeably increased a few hours after exposure to PMS. Most intact intracellular amastigotes were not stained, but damaged parasites were often positive for AcP. Twenty hours after the PMS pulse, the percentage of AcP-positive macrophages dropped to the levels initially present in noninfected cultures and all of the parasites were destroyed. Exposure of noninfected macrophages to PMS did not affect their AcP reactivity.

L-leucine-methyl ester (Leu-OMe) kills Leishmania mexicana amazonensis amastigotes by a mechanism which requires proteolytic cleavage of the ester. N-Benzyloxycarbonyl-phenylalanyl-alanyl diazomethane (Z-Phe-AlaCHN2), a specific and irreversible inhibitor of cysteine proteinases, was used to characterize the enzymes involved in parasite destruction. It was shown that (1) amastigotes preincubated with micromolar concentrations of Z-Phe-AlaCHN2 survived challenge with Leu-OMe concentrations lethal to control parasites; (2) the proteolytic activity of 25- to 33-kDa cysteine proteinases in parasite lysates subjected to electrophoresis in gelatin-containing acrylamide gels was selectively inhibited in parasites pretreated with Z-Phe-AlaCHN2 and chased in inhibitor-free medium; and (3) cysteine proteinase activity was also inhibited in gels incubated with amino acid and dipeptide esters, possibly because the compounds were acting either as substrates (e.g., Leu-Leu-OMe) or as inhibitors (e.g., Ile-OMe) of the enzyme. The results support the involvement of low molecular weight cysteine proteinases in the destruction of amastigotes by Leu-OMe. Characterization of the structure and substrate specificity of the enzymes may permit the rational development of more selectively leishmanicidal amino acid derivatives.

Different temperature requirements for intracellular growth of Leishmania spp. may account for the predominantly peripheral or visceral localization of the parasites in the vertebrate host. We show here that Leishmania mexicana amazonensis, an agent of cutaneous disease, is growth restricted in vitro at 37.5 C and viable at 34 C. This temperature sensitivity has been observed in both infected macrophage cultures and isolated amastigotes. Counts of the percentage of infected macrophages and of the number of amastigotes per infected cell documented that at 34 C the parasites progressively proliferated for at least 11 days. In contrast, no amastigotes were found in infected cells 48 to 72 hr after the cultures were shifted to the restrictive temperature of 37.5 C. This effect was reversible within the first 24 hr of temperature shift. Incorporation of [3H]uracil into parasite RNA was used to monitor the viability of intracellular and of isolated amastigotes. The trichloroacetic acid-precipitable incorporation of [3H]uracil by infected macrophages was markedly reduced as early as 6 hr after the monolayers were placed at 37.5 C. This decrease was also reversible within the first 24 hr at the restrictive temperature. In order to determine whether the restrictive temperature could directly affect the parasites, isolated amastigotes were incubated at either 34 C or 37.5 C and assayed for [3H]uracil incorporation. The results indicated that by 3 hr of incubation at the restrictive temperature the incorporation was significantly decreased and remained low for at least 9 hr.

Intracellular and isolated amastigotes of Leishmania amazonensis can be destroyed by L-amino acid methyl esters known to disrupt mammalian lysosomes. To evaluate the mechanism(s) involved in the leishmanicidal activity, we examined the uptake and hydrolysis of tritiated esters by isolated amastigotes. After incubation with the labeled compounds, parasites were recovered, were washed on filters, and their radioactivity was determined. Alternatively, amastigotes were separated from the medium by centrifugation through oil, and the radioactivity associated with free or esterified amino acids was measured after thin-layer chromatography. The results showed that the methyl esters of Trp, Leu, and Met, which are leishmanicidal, accumulated in and were rapidly hydrolysed by the amastigotes. [3H]Leu derived from [3H]Leu-OMe remained associated with the amastigotes even after a 1-hr chase in label-free medium, but the ester species was rapidly lost upon washing of the parasites. In contrast, the esters of Ile and Ala, which are not leishmanicidal, were only slowly hydrolysed, and most of the radioactivity was lost upon washing. We have previously shown that certain amino acid esters and weak bases protect Leishmania from damage by leucine methyl ester (Leu-OMe). In the present experiments, these compounds reduced, in concentration-dependent fashion, the hydrolysis of [3H]Leu-OMe and the accumulation of [3H]Leu in the amastigotes. Overall, the results indicate that, as in lysosomal disruption, leishmanicidal activity is associated with ester hydrolysis and amino acid accumulation in the parasites. The nature and location of the parasite esterolytic enzymes requires additional investigation.