Actin-rich structures formed during the invasion of cultured cells by infective forms of Trypanosoma cruzi.

Previous work has shown that Trypanosoma cruzi extracellular amastigotes as well as metacyclic trypomastigotes infect cultured cells in a highly specific parasite form-cell type interaction. In this work we have investigated the mode of interaction of both forms with HeLa and Vero cells using scanning electron and confocal fluorescence microscopy. We examined the distribution of several host cell components as well as extracellular matrix elements during cell invasion by both T. cruzi infective forms. Scanning electron microscopy showed that membrane expansions formed during the invasion of cells by extracellular amastigotes. These expansions correspond to small cup-like structures in HeLa cells and are comparatively larger "crater"-like in Vero cells. We detected by confocal microscopy actin-rich structures associated with the internalisation of both infective forms of the parasite that correspond to the membrane expansions. Confocal fluorescence microscopy combining DIC images of cells labelled with monoclonal antibodies to phosphotyrosine, cytoskeletal elements, integrins, and extracellular matrix components revealed that some of the components like gelsolin and alpha-actinin accumulate in actin-rich structures formed in the invasion of amastigotes of both cell types. Others, like vinculin and alpha2 integrin may be present in these structures without evident accumulation. And finally, some actin-rich processes may be devoid of components like fibronectin or alphaV integrin. These studies provide evidence that the repertoire of host cell/extracellular matrix components that engage in the invasion process of T. cruzi forms is cell type- and parasite form-dependent.

Features of host cell invasion by different infective forms of Trypanosoma cruzi.

Through its life cycle from the insect vector to mammalian hosts Trypanosoma cruzi has developed clever strategies to reach the intracellular milieu where it grows sheltered from the hosts' immune system. We have been interested in several aspects of in vitro interactions of different infective forms of the parasite with cultured mammalian cells. We have observed that not only the classically infective trypomastigotes but also amastigotes, originated from the extracellular differentiation of trypomastigotes, can infect cultured cells. Interestingly, the process of invasion of different parasite infective forms is remarkably distinct and also highly dependent on the host cell type.

Performance and host preference of Ascia monuste (Lepidoptera, Pieridae).

Experiments were conducted to determine the performance and host preference of Ascia monuste using kale (Brassica oleraceae, var. Acephala) and mustard (B. juncea). These plants differ significantly in water and nitrogen content, with mustard having larger amounts of water and kale larger amounts of nitrogen. The performance results confirmed that kale is a better food source than mustard for the species, even when eggs were collected on mustard leaves in the field. However, when eggs were collected on mustard, the kale nutritive value was lower than the nutritive value obtained when eggs were collected on kale leaves. Furthermore, the results of oviposition preference obtained in the field and in the laboratory have shown a preference for kale, indicating the presence of a positive correlation with performance. In contrast to the data about oviposition preference, there was no immature feeding preference. These results indicate that host selection occurs during the oviposition process. Furthermore, it is possible that the high abundance of kale cultivated in the region studied and the nutritional quality of this plant are two factors that influence the positive relationship between oviposition preference and performance for A. monuste.

Distribution of epitopes of Trypanosoma cruzi amastigotes during the intracellular life cycle within mammalian cells.

In this study we have examined the distribution of epitopes defined by monoclonal antibodies raised against Trypanosoma cruzi amastigotes during the intracellular life cycle of the parasite. We have raised monoclonal antibodies towards amastigote forms and performed preliminary immunochemical characterization of their reactivities. MAB 1D9, 3G8, 2B7, 3B9, and 4B9 react with carbohydrate epitopes of the parasite major surface glycoprotein--Ssp-4 defined by MAB 2C2 [5]; MAB 4B5 reacts with a noncarbohydrate epitope in all developmental stages of the parasite, and MAB 3B2 also detects a noncarbohydrate epitope preferentially in T. cruzi flagellated forms. Vero cells infected with tissue culture-derived trypomastigotes of clone D11 (G strain) were fixed at different times during the intracellular proliferation of parasites, and processed for immuno-electron microscopy and confocal immunofluorescence with the different monoclonal antibodies. We observed that while the surface distribution of MAB 2C2 and 4B9 epitopes was uniform throughout the cycle, MAB 1D9, 3G8, and 2B7 reacted with cytoplasmic membrane-bound compartments of the amastigotes. MAB 3B9 displayed a unique surface dentate pattern in some amastigotes. MAB 4B5 recognized a curved-shaped structure at the flagellar pocket region in some intracellular amastigotes and localized to the membrane in dividing forms. In intracellular trypomastigotes, MAB 4B5 also displayed a punctate pattern near the flagellar pocket.

Release of membrane-bound trails by Trypanosoma cruzi amastigotes onto modified surfaces and mammalian cells.

Upon incubation at 37 degrees C onto glass coverslips coated with Concanavalin A, poly-L-lysine, or a monoclonal antibody (1D9) directed to the parasite major surface glycoprotein Ssp-4, extracellular Trypanosoma cruzi amastigotes release trails of material barely visible by light microscopy. This release is not associated with parasite movements. Immunolabeling studies confirmed that the material is derived from the parasite's membrane since thin section through samples labeled with 1D9 revealed that the trails are membrane-bound structures. Scanning electron microscopy showed that the approximately 0.1-micron(s) thick trails of material emerging from the amastigotes can be uniform or beaded, indicating a tendency to vesiculation. The trails are preferentially released from the flagellar pocket region and/or at the opposite posterior end of the parasite body, and seem to be devoid of microtubules. The release is time and temperature-dependent and fixed parasites do not form trails. All attempts to inhibit trail release using drugs (antimycin A, sodium azide, cytochalasin D, nocodazole, genistein, staurosporine, EGTA) failed. The observation of trails associated with intracellular parasites and amastigotes invading Vero cells suggests that this is probably a physiological process.

Inhibition of enteropathogenic Escherichia coli adhesion to HeLa cells by serum of infants with diarrhea and by cord serum.

We have studied the effect of serum from infants with diarrhea and of cord serum on the localized adherence of enteropathogenic Escherichia coli (EPEC) to HeLa cells. Serum samples from 16 infants with diarrhea due to EPEC of serotypes O55:H6, O111: H-, O111:H2, O119:H6 and O142:H6 were used. The adherence ability of EPEC strains belonging to serotypes identical to (homologous) or different from (heterologous) those isolated from the infants' feces was highly inhibited by samples of infant serum collected both during the acute phase of the illness and upon discharge from the hospital. These data confirm the development of antibodies against EPEC adhesins and the cross-reaction between different EPEC serotypes. Cord serum inhibited the localized adherence of EPEC strains at different levels according to the serotype of the strain studied. These results suggest that the placental transfer of adhesin-related antibodies does not protect the newborn against EPEC infections, since half of our patients were less than 30 days old.

Inhibition of enteropathogenic Escherichia coli adhesion to HeLa cells by serum of infants with diarrhea and by cord serum

We have studied the effect of serum from infants with diarrhea and of cord serum on the localized adherence of enteropathogenic Escherichia coli (EPEC) to HeLa cells. Serum samples from 16 infants with diarrhea due to EPEC of serotypes O55:H6, O111: H-, O111:H2, O119:H6 and O142:H6 were used. The adherence ability of EPEC strains belonging to serotypes identical to (homologous) or different from (heterologous) those isolated from the infants' feces was highly inhibited by samples of infant serum collected both during the acute phase of the illness and upon discharge from the hospital. These data confirm the development of antibodies against EPEC adhesins and the cross-reaction between different EPEC serotypes. Cord serum inhibited the localized adherence of EPEC strains at different levels according to the serotype of the strain studied. These results suggest that the placental transfer of adhesin-related antibodies does not protect the newborn against EPEC infections, since half of our patients were less than 30 days old

Inhibition of enteropathogenic Escherichia coli adherence to HeLa cells by immune rabbit sera.

We have studied the effect of immune rabbit sera on the localized (LA) and diffuse (DA) adherence to HeLa cells of 10 enteropathogenic Escherichia coli (EPEC) strains belonging to serogroups O55, O86, O111, O119, and O142. Anti-La1 serum, obtained by rabbit immunization with an E. coli strain harboring a cloned DNA fragment from an EPEC LA plasmid, strongly inhibited the adherence of all serogroups but one (O142). Similar results were obtained with anti-LA2 serum, which is anti-O111 serum absorbed with a non-adherent O111:H- EPEC strain. In contrast, non-absorbed anti-O55 and anti-O111 sera showed an inhibitory effect mainly on the adherence of homologous strains. Except for one experiment diffuse adherence was not inhibited by any antiserum used. The inhibitory effect of immune sera on localized adherence does not seem to be correlated with plasmid curing since adherence plasmid pMS49 proved to be stable after treatment with anti-O55 and anti-O111 sera. The cross-inhibition of adherence by anti-LA sera suggests that localized adherence-related adhesins of the O55, O86, O111, and O119 strains share similar antigens.

Inhibition of enteropathogenic Escherichia coli adherence to Hela cells by immune rabbit sera

We have studied the effect of immune rabbit sera on the localized (LA) and diffuse (DA) adherence to Hela cells of 10 enteropathogenic Escherichia coli (EPEC) strains belonging to serogroups 055, 086, 0111, 0119, and 0142. Anti-La1 serum, obtained by rabbit immunization with an E. coli strain harboring a cloned DNA fragment from an EPEC LA plasmid, strongly inhibited the adherence of all serogroups but one (0142). Similar results were obtained with anti-LA2 serum, which is anti-0111 serum absorbed with a non-adherence 0111:H-EPEC strain. In contrast, non-absorbed anti-055 and anti-0111 sera showed an inhibitory effect mainly on the adherence of homologous strains. Except for one experiment diffuse adherence was not inhibited by any antiserum used. The inhibitory effect of immune sera on localized adhere3nce does not seem to be correlated with plasmid curing sinceadherence plasmid pMS49 proved to be stable a after treatment with anti-055 and anti-0111 sera. The cross-inhibition of adherence by anti-LA sera suggests that localized adherence-related adhesions of the 0.55, 0.86, 0111, and 0119 strains share similar antigens

Aggregation of membrane-associated actin filaments following localized adherence of enteropathogenic Escherichia coli to HeLa cells.

We have previously observed that enteropathogenic Escherichia coli (EPEC) adhere to HeLa cells in a localized manner, which we designated localized adherence as opposed to the diffuse pattern of adhesion. In this paper we have examined the effects of localized adherence of EPEC on the actin microfilament system of host HeLa cells. Centrifugation of bacteria onto HeLa cells improved the localized adherence and rapid rearrangements of actin filaments were detected by immunofluorescence and electron microscopy. Aggregation of microfilaments is consistently observed at the sites of localized adherence, and is abolished by cytochalasin D and low temperatures. Scanning electron microscopy indicates that these aggregates are surface microvilli entangled with attached EPEC.