Comparative analysis of the membrane proteome of Leishmania (Leishmania) amazonensis promastigotes of PH8 and LV79 strains.

Author(s)

Fabia Tomie Tano

• Document type: Master's Dissertation
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2021-02-26

Examining board members

Beatriz Simonsen Stolf Carboni; Angela Kaysel Cruz; Mauro Javier Cortez Véliz; Patrícia Sampaio Tavares Veras

• Advisor: Beatriz Simonsen Stolf Carboni

Abstract

Human diseases caused by protozoa of the genus Leishmania are classified according to their clinical manifestations into visceral and cutaneous leishmaniasis. Recently, our group compared the PH8 and LV79 strains of Leishmania (Leishmania) amazonensis, one of the main etiologic agents of cutaneous leishmaniasis in Brazil. Parasites of the PH8 strain cause larger lesions with greater parasite load in murine models. In addition, a greater number of PH8 promastigotes adhere to and are internalized by murine macrophages in vitro, indicating a difference in the membrane composition between these strains. In this project, we show that promastigotes of PH8 are more resistant to lysis by murine complement (serum) than LV79. Bearing in mind the differences in infectivity and sensitivity to lysis described, this study aimed to analyze the membrane composition of PH8 and LV79 early stationary phase promastigotes in order to identify proteins potentially involved in the phenotypic differences observed. To accomplish this task, the preliminary biochemical characterization of the lipophosphoglycan (LPG) and the comparative analysis of the membrane proteome were performed, and the results of the latter were confirmed by Western blot and enzymatic activity assays. The preliminary biochemical characterization indicates that LPG from PH8 promastigotes has a smaller number of repetitive units, which are devoid of side chains. In contrast, LPG from LV79 promastigotes is composed by repetitive units without a side chain or with side chains composed of mono- and disaccharides, confirming the existence of polymorphisms between strains. The comparative analysis of the membrane proteome indicates that each strain has a characteristic profile. Of the 1659 proteins identified, 127 are more abundant in PH8 promastigotes and 78 more abundant in LV79. Among the proteins more abundant in LV79, most of them participate in processes such as protein synthesis (47%), amino acid metabolism (21%), and nucleotide metabolism (14%). On the other hand, the proteins more abundant in PH8 are involved in carbohydrate metabolism (21%), cytoskeleton composition (17%), and vesicle and membrane trafficking (13%). Virulence-related proteins such as the putative ABC (ATP-binding cassette) transporter subfamily G, member 1 and enolase were identified as more abundant in PH8, whereas glycoprotein 63 (GP63) was identified as more abundant in LV79. Analysis of the abundance of GP63 and enolase by Western blot confirmed what was observed in the proteomic analysis. However, enzymatic activity assays obtained dissonant results, indicating that the activity of these proteins can be regulated and does not necessarily correspond to their expression. Altogether, our results indicate that membrane composition of the PH8 and LV79 promastigotes differ from each other. Additional experiments must be carried out to determine the implications of these differences on the strains phenotypes. (AU)

• FAPESP's process: 17/26197-6 - Comparative proteomic analysis of promastigote membrane proteins from l. (l.) amazonensis PH8 and LV79 strains
• Grantee: Fabia Tomie Tano
• Support Opportunities: Scholarships in Brazil - Master