Phenotypical Differences between Leishmania (Leishmania) amazonensis PH8 and LV79 Strains May Impact Survival in Mammal Host and in Phlebotomine Sand Flies.

We previously showed that L. (Leishmania) amazonensis promastigotes and amastigotes of the PH8 strain generated larger lesions in mice than LV79, and that lesion-derived amastigotes from the two strains differ in their proteomes. We recently reported that PH8 promastigotes are more phagocytized by macrophages. Promastigotes' membrane-enriched proteomes showed several differences, and samples of each strain clustered based on proteomes. In this paper, we show phenotypic differences between PH8 and LV79 promastigotes that may explain the higher virulence of PH8. We compared in vitro macrophage infections by day 4 (early) and day 6 (late stationary phase) cultures, resistance to complement, and LPG characteristics. PH8 promastigotes showed a higher infectivity and were more resistant to murine complement. LPG was different between the strains, which may influence the interaction with macrophages and survival to complement. We compared the infection of the permissive vector Lutzomyia longipalpis. PH8 was more abundant in the vector's gut 72 h after feeding, which is a moment where blood digestion is finished and the parasites are exposed to the gut environment. Our results indicate that PH8 promastigotes are more infective, more resistant to complement, and infect the permissive vector more efficiently. These data suggest that PH8 is probably better adapted to the sand fly and more prone to survive in the vertebrate host.

Proteome and morphological analysis show unexpected differences between promastigotes of Leishmania amazonensis PH8 and LV79 strains.

BACKGROUND: Leishmaniases are diseases caused by Leishmania protozoans that affect around 12 million people. Leishmania promastigotes are transmitted to vertebrates by female phlebotomine flies during their blood meal. Parasites attach to phagocytic cells, are phagocytosed and differentiate into amastigotes. We previously showed that PH8 and LV79 strains of Leishmania amazonensis have different virulence in mice and that their amastigotes differ in their proteomes. In this work, we compare promastigotes' infectivity in macrophages, their proteomes and morphologies. METHODS/PRINCIPAL FINDINGS: Phagocytosis assays showed that promastigotes adhesion to and phagocytosis by macrophages is higher in PH8 than LV79. To identify proteins that differ between the two strains and that may eventually contribute for these differences we used a label-free proteomic approach to compare promastigote´s membrane-enriched fractions. Proteomic analysis enabled precise discrimination of PH8 and LV79 protein profiles and the identification of several differentially abundant proteins. The proteins more abundant in LV79 promastigotes participate mainly in translation and amino acid and nucleotide metabolism, while the more abundant in PH8 are involved in carbohydrate metabolism, cytoskeleton composition and vesicle/membrane trafficking. Interestingly, although the virulence factor GP63 was more abundant in the less virulent LV79 strain, zymography suggests a higher protease activity in PH8. Enolase, which may be related to virulence, was more abundant in PH8 promastigotes. Unexpectedly, flow cytometry and morphometric analysis indicate higher abundance of metacyclics in LV79. CONCLUSIONS/SIGNIFICANCE: Proteome comparison of PH8 and LV79 promastigotes generated a list of differential proteins, some of which may be further prospected to affect the infectivity of promastigotes. Although proteomic profile of PH8 includes more proteins characteristic of metacyclics, flow cytometry and morphometric analysis indicate a higher abundance of metacyclics in LV79 cultures. These results shed light to the gaps in our knowledge of metacyclogenesis in L. amazonensis, and to proteins that should be studied in the context of infection by this species.

CD100/Sema4D Increases Macrophage Infection by Leishmania (Leishmania) amazonensis in a CD72 Dependent Manner.

Leishmaniasis is caused by trypanosomatid protozoa of the genus Leishmania, which infect preferentially macrophages. The disease affects 12 million people worldwide, who may present cutaneous, mucocutaneous or visceral forms. Several factors influence the form and severity of the disease, and the main ones are the Leishmania species and the host immune response. CD100 is a membrane bound protein that can also be shed. It was first identified in T lymphocytes and latter shown to be induced in macrophages by inflammatory stimuli. The soluble CD100 (sCD100) reduces migration and expression of inflammatory cytokines in human monocytes and dendritic cells, as well as the intake of oxidized low-density lipoprotein (oxLDL) by human macrophages. Considering the importance of macrophages in Leishmania infection and the potential role of sCD100 in the modulation of macrophage phagocytosis and activation, we analyzed the expression and distribution of CD100 in murine macrophages and the effects of sCD100 on macrophage infection by Leishmania (Leishmania) amazonensis. Here we show that CD100 expression in murine macrophages increases after infection with Leishmania. sCD100 augments infection and phagocytosis of Leishmania (L.) amazonensis promastigotes by macrophages, an effect dependent on macrophage CD72 receptor. Besides, sCD100 enhances phagocytosis of zymosan particles and infection by Trypanosoma cruzi.

Quantitative proteomic analysis of amastigotes from Leishmania (L.) amazonensis LV79 and PH8 strains reveals molecular traits associated with the virulence phenotype.

BACKGROUND: Leishmaniasis is an antropozoonosis caused by Leishmania parasites that affects around 12 million people in 98 different countries. The disease has different clinical forms, which depend mainly on the parasite genetics and on the immunologic status of the host. The promastigote form of the parasite is transmitted by an infected female phlebotomine sand fly, is internalized by phagocytic cells, mainly macrophages, and converts into amastigotes which replicate inside these cells. Macrophages are important cells of the immune system, capable of efficiently killing intracellular pathogens. However, Leishmania can evade these mechanisms due to expression of virulence factors. Different strains of the same Leishmania species may have different infectivity and metastatic phenotypes in vivo, and we have previously shown that analysis of amastigote proteome can give important information on parasite infectivity. Differential abundance of virulence factors probably accounts for the higher virulence of PH8 strain parasites shown in this work. In order to test this hypothesis, we have quantitatively compared the proteomes of PH8 and LV79 lesion-derived amastigotes using a label-free proteomic approach. METHODOLOGY/PRINCIPAL FINDINGS: In the present work, we have compared lesion development by L. (L.) amazonensis PH8 and LV79 strains in mice, showing that they have different virulence in vivo. Viability and numbers of lesion-derived amastigotes were accordingly significantly different. Proteome profiles can discriminate parasites from the two strains and several proteins were differentially expressed. CONCLUSIONS/SIGNIFICANCE: This work shows that PH8 strain is more virulent in mice, and that lesion-derived parasites from this strain are more viable and more infective in vitro. Amastigote proteome comparison identified GP63 as highly expressed in PH8 strain, and Superoxide Dismutase, Tryparedoxin Peroxidase and Heat Shock Protein 70 as more abundant in LV79 strain. The expression profile of all proteins and of the differential ones precisely classified PH8 and LV79 samples, indicating that the two strains have proteins with different abundances and that proteome profiles correlate with their phenotypes.

Distinct courses of infection with Leishmania (L.) amazonensis are observed in BALB/c, BALB/c nude and C57BL/6 mice.

Leishmania (L.) amazonensis [L. (L.) amazonensis] is widely distributed in Brazil and its symptomatic infections usually lead to few localized lesions and sometimes to diffuse cutaneous form, with nodules throughout the body, anergy to parasite antigens and poor therapeutic response. The variability of these manifestations draws attention to the need for studies on the pathophysiology of infection by this species. In this study, we analysed the course and immunological aspects of L. (L.) amazonensis infection in BALB/c and C57BL/6 strains, both susceptible, but displaying different clinical courses, and athymic BALB/c nude, to illustrate the role of T cell dependent responses. We analysed footpad thickness and parasite burden by in vivo imaging. Furthermore, we evaluated the cellular profile and cytokine production in lymph nodes and the inflammatory infiltrates of lesions. Nude mice showed delayed lesion development and less inflammatory cells in lesions, but higher parasite burden than BALB/c and C57BL/6. BALB/c and C57BL/6 mice had similar parasite burdens, lesion sizes and infiltrates until 6 weeks after infection, and after that C57BL/6 mice controlled the infection. Small differences in parasite numbers were observed in C57BL/6 macrophages in vitro, indicating that in vivo milieu accounts for most differences in infection. We believe our results shed light on the role of host immune system in the course of L. (L.) amazonensis infection by comparing three mouse strains that differ in parasitaemia and inflammatory cells.

Regulation of Leishmania (L.) amazonensis protein expression by host T cell dependent responses: differential expression of oligopeptidase B, tryparedoxin peroxidase and HSP70 isoforms in amastigotes isolated from BALB/c and BALB/c nude mice.

Leishmaniasis is an important disease that affects 12 million people in 88 countries, with 2 million new cases every year. Leishmania amazonensis is an important agent in Brazil, leading to clinical forms varying from localized (LCL) to diffuse cutaneous leishmaniasis (DCL). One interesting issue rarely analyzed is how host immune response affects Leishmania phenotype and virulence. Aiming to study the effect of host immune system on Leishmania proteins we compared proteomes of amastigotes isolated from BALB/c and BALB/c nude mice. The athymic nude mice may resemble patients with diffuse cutaneous leishmaniasis, considered T-cell hyposensitive or anergic to Leishmania's antigens. This work is the first to compare modifications in amastigotes' proteomes driven by host immune response. Among the 44 differentially expressed spots, there were proteins related to oxidative/nitrosative stress and proteases. Some correspond to known Leishmania virulence factors such as OPB and tryparedoxin peroxidase. Specific isoforms of these two proteins were increased in parasites from nude mice, suggesting that T cells probably restrain their posttranslational modifications in BALB/c mice. On the other hand, an isoform of HSP70 was increased in amastigotes from BALB/c mice. We believe our study may allow identification of potential virulence factors and ways of regulating their expression.