Proteomic analysis reveals differentially expressed proteins in macrophages infected with Leishmania amazonensis or Leishmania major.

CBA macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. Employing a transcriptomic approach, we previously showed the up-regulation of the genes involved in the classical pathway of macrophage activation in resistant mice. However, microarray analyses do not evaluate changes in gene expression that occur after translation. To circumvent this analytical limitation, we employed a proteomics approach to increase our understanding of the modulations that occur during infection and identify novel targets for the control of Leishmania infection. To identify proteins whose expression changes in CBA macrophages infected with L. major or L. amazonensis, protein extracts were obtained and digested and the peptides were characterized using multi-dimensional liquid chromatography coupled with tandem mass spectrometry analyses. A total of 162 proteins were selected as potentially modulated. Using biological network analyses, these proteins were classified as primarily involved in cellular metabolism and grouped into cellular development biological networks. This study is the first to use a proteomics approach to describe the protein modulations involved in cellular metabolism during the initial events of Leishmania-macrophage interaction. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of infection.

Leishmania amazonensis fails to induce the release of reactive oxygen intermediates by CBA macrophages.

CBA mouse macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. It has been established that some Leishmania species are destroyed by reactive oxygen species (ROS). However, other species of Leishmania exhibit resistance to ROS or even down-modulate ROS production. We hypothesized that L. amazonensis-infected macrophages reduce ROS production soon after parasite-cell interaction. Employing a highly sensitive analysis technique based on chemiluminescence, the production of superoxide (O(·-)(2)) and hydrogen peroxide (H(2)O(2)) by L. major- or L. amazonensis-infected CBA macrophages were measured. L. major induces macrophages to release levels of (O(·-)(2)) 3·5 times higher than in uninfected cells. This (O(·-)(2)) production is partially dependent on NADPH oxidase (NOX) type 2. The level of accumulated H(2)O(2) is 20 times higher in L. major-than in L. amazonensis-infected cells. Furthermore, macrophages stimulated with L. amazonensis release amounts of ROS similar to uninfected cells. These findings support previous studies showing that CBA macrophages are effective in controlling L. major infection by a mechanism dependent on both (O(·-)(2)) production and H(2)O(2) generation. Furthermore, these data reinforce the notion that L. amazonensis survive inside CBA macrophages by reducing ROS production during the phagocytic process.

The scavenger receptor MARCO is involved in Leishmania major infection by CBA/J macrophages.

CBA/J mice are resistant to Leishmania major infection but are permissive to L. amazonensis infection. In addition, CBA/J macrophages control L. major but not L. amazonensis infection in vitro. Phagocytosis by macrophages is known to determine the outcome of Leishmania infection. Pattern recognition receptors (PRR) adorning antigen presenting cell surfaces are known to coordinate the link between innate and adaptive immunity. The macrophage receptor with collagenous structure (MARCO) is a PRR that is preferably expressed by macrophages and is capable of binding Gram-positive and Gram-negative bacteria. No research on the role of MARCO in Leishmania-macrophage interactions has been reported. Here, we demonstrate, for the first time, that MARCO expression by CBA/J macrophages is increased in response to both in vitro and in vivo L. major infections, but not to L. amazonensis infection. In addition, a specific anti-MARCO monoclonal antibody reduced L. major infection of macrophages by 30%-40% in vitro. The draining lymph nodes of anti-MARCO-treated mice displayed a reduced presence of immunolabelled parasite and parasite antigens, as well as a reduced inflammatory response. These results support the hypothesis that MARCO has a role in macrophage infection by L. major in vitro as well as in vivo.

A dhfr-ts- Leishmania major Knockout Mutant Cross-protects against Leishmania amazonensis

E10-5A3 is a dhfr-ts- Leishmania major double knockout auxotrophic shown previously to induce substantial protection against virulent L. major infection in both genetically susceptible and resistant mice. We investigated the capacity of dhfr-ts- to protect against heterologous infection by L. amazonensis. The degree of protection was evaluated by immunization of BALB/c or C57BL/6 mice with E10-5A3, followed by L. amazonensis challenge. Whether immunized by subcutaneous (SC) or intravenous (IV) inoculation, susceptible and resistant mice displayed a partial degree of protection against challenge with virulent L. amazonensis. SC-immunized BALB/c mice developed lesions 40 to 65 percent smaller than non immunized mice, while IV immunization led to protection ranging from 40 to 75 percent in four out of six experiments compared to non immunized animals. The resistant C57BL/6 mice displayed comparable degrees of protection, 57 percent by SC and 49 percent by IV immunization. Results are encouraging as it has been previously difficult to obtain protection by SC vaccination against Leishmania, the preferred route for human immunization

Fusion of Leishmania amazonensis parasitophorous vacuoles with phagosomes containing zymosan particles: cinemicrographic and ultrastructural observations.

Studies on fixed preparations have shown that vacuoles containing zymosan (Z) particles internalized by infected macrophages can selectively fuse with the large parasitophorous vacuoles (PVs) that shelter Leishmania amazonensis. To examine the kinetics of vacuolar fusion in individual cells, particles were followed by time-lapse cinemicrography from their uptake to their entry in a PV. Newly formed Z-containing vacuoles moved centripetally and, if they contacted a PV, the two vacuoles remained closely apposed for variable, often extended, periods of time before they eventually fused. Transmission electron microscopy confirmed that the cytoplasm separating the partner vacuoles could be reduced to a very thin layer. Initiation of fusion was indicated by reduced refractility of the boundary between Z vacuoles and target PVs. Within a few minutes the PV enlarged and encompassed the Z particles, which remained immobile throughout. The interval between phagocytosis and fusion, 50 +/- 7.4 min (N = 17; range, 4 to 108 min), suggests that most but not all Z vacuoles underwent significant maturation by the time of fusion. Some particles were transferred singly, others entered PVs in groups of 2 or more, and additional clustered transfers to the same vacuole were also observed. These observations provide a baseline for studies of the biochemical mechanisms and the pharmacological control of the fusion of Leishmania PVs, and for the comparison of the fusion behavior of the PVs with that of other phagocytically derived vacuoles.

Cohabitation of Leishmania amazonensis and Coxiella burnetii.

Intracellular pathogens customize the composition and function of the vacuoles they occupy, and can arrest or distort vacuolar maturation. In doubly infected cells, vacuoles that contain two different parasites can be used to test for exclusionary mechanisms, for expression of vacuolar phenotypes that permit or restrict fusion, and for the survival of pathogens targeted to an unusual cellular compartment.

Entry and survival of Leishmania amazonensis amastigotes within phagolysosome-like vacuoles that shelter Coxiella burnetii in Chinese hamster ovary cells.

Coxiella burnetii, a rickettsia, and Leishmania amazonensis, a protozoan flagellate, lodge in their host cells within large phagolysosome-like vacuoles. In the present study, C. burnetii-infected Vero or CHO cells were superinfected with L. amazonensis amastigotes to determine if these parasites can home to and survive within heterologous vacuoles. Six hours after superinfection, Leishmania amastigotes were located almost exclusively within large Coxiella-containing vacuoles. Thereafter, the numbers of parasites in the vacuoles increased at the same rate as those in cells infected with L. amazonensis alone. Furthermore, in cultures shifted to 25 degrees C, some of the amastigotes transformed into promastigote-like forms that moved their flagella within the adoptive vacuoles. Thus, L. amazonensis amastigotes not only entered Coxiella vacuoles, most likely by fusion of donor and recipient vacuoles, but temporarily survived, differentiated, and replicated therein. This appears to be the first account of the temporary cohabitation of two living pathogens within the same vacuole in a mammalian cell.

Fusion between large phagocytic vesicles: targeting of yeast and other particulates to phagolysosomes that shelter the bacterium Coxiella burnetii or the protozoan Leishmania amazonensis in Chinese hamster ovary cells.

This report examines the fusion of phagocytic vesicles with the large phagolysosome-like vacuoles induced in Chinese hamster ovary cells by the bacterium Coxiella burnetti or the Protozoan flagellate Leishmania amazonensis. Infection by these organisms is compatible with cell survival and multiplication. Fusion was inferred from the transfer of microscopically identifiable particles from donor to target vesicles. Donor vesicles contained heat-killed yeast, zymosan, beta-glucan or latex beads taken up by the host cells. Yeast and zymosan were also coated with Concanavalin A to increase their uptake by the cells (Goldman, R., Exp. Cell Res. 104, 325-334, 1977). Particle localization, routinely ascertained by phase-contrast microscopy, was confirmed by confocal laser fluorescence and by transmission electron microscopy. Coxiella vacuoles admitted all the particles tested and transfer took place whether the particles were given to the cells prior to or after infection. Transfer of uncoated or Concanavalin-A-coated yeast or zymosan was dependent on the number of particles ingested and on the incubation period (between 2 and 24 hours). Furthermore, the transfer step was quite efficient, since over 85% of the particles ingested entered Coxiella vacuoles at all particle to cell ratios examined. The fraction of uncoated or Concanavalin-A-coated yeast or zymosan transferred to Leishmania vacuoles was consistently lower and diminished at higher particle loads. In addition, only rarely did latex beads enter these vacuoles. The models proposed may be useful for the delineation of biochemical and molecular mechanisms involved in the fusion of large phagocytic vesicles and the modulation of the latter by cellular and pathogen-derived signals.

Transfer of zymosan (yeast cell walls) to the parasitophorous vacuoles of macrophages infected with Leishmania amazonensis.

Leishmania are flagellated protozoan parasites which, in their amastigote stages, survive and multiply within phagolysosome-like parasitophorous vacuoles (PV) of mammalian macrophages (MO). This study develops an earlier ultrastructural, incidental observation that zymosan particles (Z) were transferred to the PV of macrophages infected with Leishmania amazonensis. In the present report, a pulse-chase light microscopic assay was used to delineate several features of the Z transfer. The assay reflects both the movement of internalized particles to a position adjoining a PV, and their delivery to the vacuoles. Transfer was selective, in the sense that Z, beta-glucan or heat-killed yeast particles were transferred, whereas latex beads, aldehyde-fixed, or immunoglobulin G-coated erythrocytes were not. This selectivity may be related to the high density of carbohydrate ligands displayed on the surface of yeast-derived particles, to ligand resistance to lysosomal degradation or to signals encoded in the cytosolic tails of the receptors involved in particle recognition. A few Z particles could be found within PV after 1 h of incubation with infected MO, but chase periods of several hours at 34 degrees C were required for particle transfer to the PV in a substantial proportion of the MO. Ammonium chloride, chloroquine, or monensin, compounds which increase the pH in acidified compartments, substantially enhanced the transfer of Z particles. Finally, transfer was inhibited by cytochalasin D, but was unaffected by the antitubulin nocodazole. Although it is not yet known if particle transfer occurs by fusion of donor vesicles with PV or by interiorization of the former into the latter, the model described should be useful in the study of the interactions between large phagocytic vesicles and the modulation of those interactions by cellular, parasitic, and environmental signals.