M. Rabinovitch

Intracellular differentiation of Leishmania amazonensis promastigotes to amastigotes: presence of megasomes, cysteine proteinase activity and susceptibility to leucine-methyl ester.

Intracellular differentiation of Leishmania promastigotes to amastigotes is a critical step in the establishment of infection. In this report three related features of mexicana subspecies amastigotes were used to follow the differentiation of the parasites within macrophages. Early after infection, (a) parasites did not contain ultrastructurally recognizable megasomes, (b) cysteine proteinase activity of parasite lysates was not detected in gelatin-containing acrylamide gels, and (c) parasites were essentially resistant to L-leucine-methyl ester (Leu-OMe). Typical megasomes were first identified on the 5th day, were more prevalent on day 7, and underwent swelling in macrophages exposed to Leu-OMe. Cysteine proteinase activity was first detected on day 3 and increased thereafter. Susceptibility to Leu-OMe of parasites studied in situ or isolated from infected macrophages increased with time of intracellular residence and by 7 days approached that of amastigotes isolated from mouse lesions. In contrast, parasites derived from either promastigotes or amastigotes were equally susceptible to another leishmanicidal compound, tryptophanamide (Trp-NH2). The results provide additional support for the involvement of megasomes and their cysteine proteinases in parasite killing by Leu-OMe, and highlight the slow pace of the intracellular differentiation of L. amazonensis promastigotes to amastigotes.

Construction of chimeric phagosomes that shelter Mycobacterium avium and Coxiella burnetii (phase II) in doubly infected mouse macrophages: an ultrastructural study

Dual infection of cells may divert pathogens to intracellular compartments different from those occupied in mono-infected cells. In the present studies, mouse bone marrow in vitro-derived macrophages were first infected with virulent Mycobacterium avium, which are normally singly lodged within tight phagosomes. These phagosomes do not mature; they undergo homotypic fusion with early endosomes and do not fuse with lysosomes. Seven days later, the cultures were superinfected with phase II (non-virulent) Coxiella burnetii, organisms sheltered in lysosome- (or prelysosome)-like, multi-occupancy phagosomes. The latter can attain large size and engage in efficient homo- and heterotypic fusion with other phagosomes. Cultures were fixed for transmission electron microscopy 6, 12, 24, and 48 h later. Other M. avium-infected cultures were superinfected with amastigotes of the trypanosomatid flagellate Leishmania amazonensis, which are also sheltered in lysosome- (or prelysosome)-like multi-occupancy vacuoles, and fixed at the same time periods. Chimeric phagosomes containing both M. avium and C. burnetii, were found already at 6 h and the proportion of M. avium that colocalized with C. burnetii in the same phagosomes reached over 90% after 48 h. In such phagosomes, both organisms were ultrastructurally well preserved. In contrast, colocalization of M. avium and L. amazonensis was rarely found. Speculative scenarios that could underlie the formation of chimeric phagosomes could involve delayed maturation of C. burnetii-containing phagosomes in presence of M. avium, which would allow for fusion of C. burnetii- and M. avium-containing phagosomes; the production, by C. burnetii, of molecules that upregulate the fusion of M. avium-containing phagosomes with those that contain C. burnetii; and the secretion of factors that could favour the survival of M. avium within chimeric vacuoles.

Proteinase inhibitors protect Leishmania amazonensis amastigotes from destruction by amino acid esters.

Lysosomotropic amino acid esters and amides kill Leishmania amazonensis amastigotes by a mechanism which probably involves enzymatic hydrolysis of the compounds and rapid accumulation of less permeant amino acid within the parasites. We show here that, in agreement with this model, the proteinase inhibitors antipain and chymostatin prevented the killing of intracellular and isolated parasites by L-leucine methyl ester (Leu-OMe). Survival of Leishmania within macrophages was assessed microscopically, and that of isolated amastigotes was measured by tetrazolium (MTT) reduction. Near maximal protection of intracellular parasites was obtained after 24 h incubation of macrophage cultures with 50 micrograms ml-1 antipain or chymostatin. Incubation for greater than 1 h with chymostatin or greater than 4 h with antipain alone resulted in loss of viability of the parasites. Protective activity was only slightly diminished by 20 h chase of isolated parasites in inhibitor-free medium. Two synthetic chymostatin analogues, Z-Val-Phe-Sc and Z-Ile-Phe-Sc, protected isolated amastigotes at 4 or 10 micrograms ml-1. With the exception of Trp-NH2, the toxicity of which was only minimally inhibited, antipain and chymostatin also prevented parasite destruction by other amino acid derivatives. Finally, in concentration-dependent fashion, the inhibitors reduced the accumulation of [3H]leucine in isolated amastigotes incubated with [3H]Leu-OMe. Since uptake of labelled ester was unaffected, we postulate that protection involves inhibition of the parasite enzymes which hydrolyse the amino acid derivatives.

Leishmania amazonensis: Specific labeling of amastigote cysteine proteinases by radioiodinated N-benzyloxycarbonyl-tyrosyl-alanyl diazomethane

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.

Leishmania mexicana: Acid phosphatase ultrastructural cytochemistry of infected mouse macrophage cultures treated with phenazine methosulfate

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.

The anti-leishmanial activity of dipeptide esters on Leishmania amazonensis amastigotes

L-Amino acid esters, such as L-Leu-OMe, kill Leishmania amazonensis amastigotes by a mechanism which appears to involve ester hydrolysis by cysteine proteinases located in the parasite megasomes. We have examined the killing of isolated amastigotes by L-dipeptide esters and derived some structure-activity correlations. Toxicity of the compounds for the parasites was measured by a tetrazolium (MTT) reduction assay. The results show that active dipeptide esters contained at least 1 hydrophobic amino acid (Leu, Ile, Val, Phe or Trp). The activity of homodipeptide methyl esters depended on the nature of the amino acid, as indicated by the following series: Phe-Phe-OMe greater than Val-Val-OMe greater than Leu-Leu-OMe greater than Trp-Trp-OMe greater than Ile-Ile-OMe. The nature of the amino acids in Leu-X-OMe and X-Leu-OMe was relatively unimportant when X was Phe, Trp or Val. However, when X was Ala or Gly, Leu-X-OMe was several-fold more active than X-Leu-OMe. A similar preference for the more hydrophobic residue in the amino terminal position was also found in esters containing a single phenylalanine or valine. Protection of the amino group by benzyloxycarbonyl (Z) or t-butyloxycarbonyl (BOC) substituents markedly enhanced the activity of the esters. An-mPhe-Gly-OEt, a retro-inverso analogue of Bz-Phe-Gly-OEt, was several-fold more active than the parent compound. Selected esters were assayed on infected macrophages and concentrations that induced minimal toxicity to the host cells were estimated. The ED50s for intracellular parasites were 1.5 to 5-fold higher than those for isolated amastigotes.(ABSTRACT TRUNCATED AT 250 WORDS)

Lysosomes of Leishmania Mexicana Sp. as Targets for Potential Therapeutic Agents

Hydrophobic amino acid esters and peptides disrupt lysosomes in cell-free fractions by a mechanism involving trapping by protonation, enzymatic hydrolysis and accumulation of less permeant products within the organelles (Goldman & Naider 1974; Ransom & Reeves 1983). Damage to lysosomes possibly accounts for the selective toxicity of the compounds for monocytes, NK cells and cytotoxic T cells. This toxicity may be due to ester conversion to membranolytic polymers catalysed by a dipeptidyl-peptidase I (Thiele & Lipsky 1990 a, b).