Trypanosoma cruzi: Involvement of proteolytic activity during cell fusion induced by epimastigote form

Human erythrocytes were fused by Trypanosoma cruzi from 7 and 14 day old culture (stationary and declination phases, respectively) while only lysis was induced by "4 day old culture parasite (exponential phase). Lysis and erythrocyte fusion were studied by phase contrast microscopy, measuring of hemolysis and gel electrophoresis. The fusogenicity is Ca²⁺-dependent while lysis is delayed in the absence of exogenous Ca²⁺. The proteolysis of erythrocyte protein bands 1, 2, 2.1, 2.3 and 3 are common features of both fusion and lysis processes. Nevertheless the breakdown rate of ankyrin (band 2.1) and band 3 are different in fused or in lysed cells. The lysis process is associated with a faster degradation of band 2.1 and increase of band 2.3 than in the case of the fusion process. By contrast, degradation of band 3 occurs faster in the fusion than in the lytic event. Treatment of fusogenic parasites but not erythrocytes with TPCK, soybean trypsin inhibitor or FCS inhibited to some extent the fusion process and the decrease of bands 1, 2, 2.1, 2.3 and 3. The results suggest that proteases from fusogenic parasites may be directly or indirectly involved in the proteolysis of band 2.1 in a way related to induction of fusion.Abbreviations TPCK Tos-Phe-CH₂Cl,1-chloro-4 phenyl-3-L-tosylamidobutan-2-one - SDS-PAGE Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis - FCS Fetal Calf Serum     

Trypanosoma musculi with Trichinella spiralis or Heligmosomoides polygyrus: concomitant infections in the mouse

Inbred mice infected with Trypanosoma musculi displayed wide variations in peak blood parasitemia. The most susceptible mice were C3H and A strain, while Balb/c, C57B1/6, and the related congenic B10 strains were the most resistant. The effect of an intestinal infection with either Trichinella spiralis or Heligmosomoides polygyrus on proliferation of T. musculi was investigated. T. spiralis infections given at the same time or up to 45 days before a T. musculi infection always caused an increase in blood parasitemia in C3H mice. Maximum increases were observed when T. spiralis infections preceded T. musculi by 5-10 days. In all mouse strains examined, dual infections increased maximum parasitemia by two- to four-fold, regardless of the degree of resistance of that mouse strain to either T. musculi or T. spiralis. This suggested that the immunological "cost" of a T. spiralis infection was the same for strains that were strong or weak responders to a primary infection with T. spiralis. In contrast, infection with H. polygyrus did not promote T. musculi parasitemia over the level of a single infection. The increase in blood parasitemia in T. spiralis-infected mice was largely due to the intestinal adult worm, but migratory larvae and mature muscle larvae also stimulated increased parasitemias. The increase in parasitemia was proportionate to the dose of T. spiralis, and the sex of the host did not affect the blood trypanosome level.     

Trypanosoma musculi and Trichinella spirails: Concomitant infections and selection for resistance genotypes in mice

Trypanosoma musculi infections were given to mice of different strains before, at the same time, and after an infection with 400 Trichinella spiralis. Examined parameters of the host response to T. spiralis were worm rejection, antifecundity responses, development of immunological memory, and muscle larvae burden. After dual infection, each mouse strain showed characteristic effects on resistance to T. spiralis. This was due to a dynamic interaction between the genes controlling rejection of T. spiralis and those influencing T. musculi growth. C3H mice develop high trypanosome parasitemias. This impairs worm expulsion and the development of memory to T. spiralis when Trypanosoma infections take place on the same day or 7 days before. The C57B1/6 mouse develops low parasitemias and T. musculi infections on the same day, or 7 days before T. spiralis, delaying worm rejection only slightly despite the overall weak capacity of B6 mice to expel worms. NFR-strain mice are strong responders to T. spiralis and also develop low parasitemias. Trypanosome infections on the same day, or after T. spiralis, produce a delay in worm rejection; the former is comparable to C3H mice. However, NFR mice alone showed enhanced rejection of worm when T. musculi infections preceded T. spiralis by 7 days. An unusual feature of C3H mice was that T. musculi infections 7 days before T. spiralis increased antifecundity responses at the same time that worm expulsion was inhibited. Trypanosome infections can therefore modulate distinct antihelminth immune responses in different directions simultaneously. The different outcomes of dual infections compared with single infections provides another selective mechanism by which genetic polymorphisms can be established and maintained in the vertebrate host.     

Restriction map, partial cloning and localization of 9S and 12S kinetoplast RNA genes on the maxicircle component of the kinetoplast DNA of Leishmania tarentolae.

We have constructed a restriction map of the maxicircle component of the kinetoplast DNA of Leishmania tarentolae for the enzymes EcoRI, Bam HI, HaeIII, HpaII, SalI, BglII and HindIII. The 9 and 12S kinetoplast RNAs were localized on this map. Two fragments of this maxicircle molecule were cloned in the bacterial plasmid, pBR322, including a 4.4 . 10(6) dalton EcoRI/BamHI fragment which contains the 9 and 12S RNA genes.     

Ultrastructural distribution of poly (A)+ RNA during Trypanosoma cruzi-cardiomyocyte interaction in vitro: a quantitative analysis of the total mRNA content by in situ hybridization

Ultrastructural in situ hybridization was used to visualize the spatial distribution of poly (A)+ RNA and quantitate its relative amount within different cellular compartments of cardiomyocytes after T. cruzi infection. The amount of poly (A)+ RNA remained about the same up to 24 h post-infection. In contrast, its content was reduced 65% after 72 h of interaction, showing a marked decrease in the cell cytoplasm. This decline in poly (A)+ RNA level in host cell cytoplasm was concomitant with intracellular proliferation of T. cruzi amastigotes. Thus, T. cruzi may affect host cell cytoplasmic mRNA stability, associated with the parasite's intracellular multiplication.     

The human pathogen Leishmania donovani secretes a histidine acid phosphatase activity that is resistant to proteolytic degradation

Promastigotes of all pathogenic Leishmania species secrete acid phosphatase (SAcP) activity during their growth in vitro. It has been suggested that this enzyme may play a role in the survival of the parasite within its sandfly-vector host. To carry out such functions, SAcP would have to be relatively resistant to endogenous sandfly gut-proteases. Therefore, the current study was undertaken to ascertain whether L. donovani SAcP activity was affected by treatment with various proteases. Native L. donovani SAcP was treated with a variety of serine-, thiol-, metallo- and mixed-proteases and subsequently assayed for enzymatic activity. Of the eleven proteases tested, only bromelain and subtilisin treatments caused a pronounced reduction in SAcP activity. Treatment of SAcP with seven out of the remaining nine proteases, resulted in an overall enhancement in SAcP enzymatic activity ranging from approximately 10% (e.g. with trypsin) to > or = 90% (e.g. with ficin). The resistance of the Leishmania SAcP to various proteases may prolong its functional life within the sandfly gut and help to facilitate parasite infection in this host.     

Distribution of Macromolecular Methylations in Promastigotes of Leishmania donovani and Impact of Sinefungin

ABSTRACT. Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent both in vitro and in vivo. This molecule, structurally related to S-adenosylmethionine, is a good competitive inhibitor of methyltransferases in vitro. The aim of this report is to analyze the impact of sinefungin on methylation pattern and the subcellular localisation of methyl groups and various methylases in promastigotes of Leishmania donovani . We have shown the presence of various methylated macromolecules in different subcellular fractions, with somewhat higher concentration in membrane fraction. In vitro, sinefungin inhibits the three main protein methylases, but in cells cultured in its presence the protein carboxylmethylations are specifically inhibited.     

Effects of Mesoionic Xanthine Analogs On Trypanosoma Musculi Development In Mice

ABSTRACT. Two derivatives of the mesoionic thiazolo[3,2-a]pyrimidine-5,7-diones 1 were prepared and examined for in vivo antiprotozoan activity to study structure-activity relationships that might lead us to more active derivatives. Mesoionic compounds 1A and 1B were inoculated into Swiss Webster male mice with Trypanosoma musculi infection. the effects were measured by studying parasite populations during the course of patent period (days 9 through 15 post-infection).
The injection of 200 μg of compound 1A along with 5 times 10⁴ trypanosomes affected the level of parasitemia at both the peak and during days 9 to 13 post infection. Experimental animals that received drug 1A prior to and after infection with T. musculi developed significantly lower parasitemia as compared to the control animals at the height of parasite populations (day 11 of observation). the group that received the drug simultaneously with trypanosome infection had significantly lower parasitemias on day 11 and 13 of infection compared to the controls.