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

Abstract

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.

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

“…We have already shown that PH8 early stationary phase promastigotes bind more and are more efficiently phagocytized by murine macrophages after 5 min of contact compared to LV79 [ 35 ]. This short initial interaction between the host cell and Leishmania promastigotes is very important and may favor either intracellular survival or parasite elimination depending on the molecules involved [ 37 ].…”

“…To analyze if the higher phagocytosis of PH8 promastigotes by macrophages leads to a higher infection rate after a longer period, we analyzed macrophage infection and parasite loads after 24 h. Infections were performed with promastigotes collected at day 4 and 6 of culture, corresponding to early stationary and late stationary phases, as reported [ 35 ]. Figure 1 shows infection levels from a representative experiment, indicating that PH8 infects a higher percentage of macrophages and leads to higher parasite loads.…”

“…In addition, sand flies show immune responses to Leishmania [ 40 ], and the exposure of different virulence factors might impact the success of vector infection by the parasite. Since PH8 and LV79 strains differ in terms of membrane proteins [ 35 ] and in LPG biochemical composition, we analyzed promastigote persistence in sand fly at different times after artificial infection.…”

“…Bone-marrow-derived macrophages (BMDM) were differentiated from BALB/c mice femur as described in Tano et al, 2022 [ 35 ]. For infections, macrophages were harvested and counted, and 4 × 10 5 were plated over coverslips in 24-well plates and incubated overnight at 37 °C under 5% CO 2 .…”

“…We have previously shown that PH8 promastigotes and amastigotes are more virulent to mice than LV79 [ 5 ]. We have recently reported that promastigotes from PH8 bind more and are more efficiently phagocytized by macrophages than those of LV79 [ 35 ]. The proteomes of membrane-enriched fractions from promastigotes of these strains have several differences, and although LV79 displayed higher amounts of GP63, PH8 showed increased GP63 activity [ 35 ].…”