The genus Leishmania comprises protozoan parasites that infect phagocytic cells of mammals, causing a wide spectrum of diseases referred to as leishmaniasis. At least 21 Leishmania species are pathogenic to humans and their distribution spans 98 countries in Europe, Africa, Asia, and the Americas (Akhoundi et al., 2016). The interaction between the infecting Leishmania species and the host immune response is a key factor for disease outcome. Cutaneous leishmaniasis presents with a wide spectrum of clinical manifestations. The localized form may cause singular ulcerative or nodular lesions in the skin, whereas in the diffuse form, parasites grow uncontrollably in multiple non-ulcerative lesions across the skin. The mucocutaneous manifestation is a disfiguring infection characterized by partial or total destruction of nasopharyngeal tissues following parasite dissemination. Several species are causative agents of cutaneous leishmaniasis; in Brazil, the most relevant species—because of their wide geographical distribution—are Leishmania braziliensis and L. amazonensis (Silveira et al., 2004). L. amazonensis is the only species, however, associated with the rare diffuse form (Silveira et al., 2004; Barral et al., 1991). Visceral leishmaniasis refers to systemic infections affecting internal organs such as the spleen, liver, and bone marrow, and it has a high mortality rate if not treated. Leishmania infantum is the causative agent of visceral leishmaniasis in the Mediterranean Basin, Middle East, and Latin America (Wilson et al., 2005). It is well documented that L. amazonensis, however, can also be associated with visceral disease in humans, dogs, and animal models in the Americas (Aleixo et al., 2006; Celeste et al., 2017).

In the mammalian host, Leishmania parasites are obligatory intracellular organisms that reside in macrophage phagolysosomes, wherein promastigotes transform into and replicate as amastigotes (Chang and Dwyer, 1976; Neuber H. 2008). Macrophages are specialized immune cells that play an essential role in the defense against pathogens through phagocytosis and antigen presentation to T lymphocytes (Bogdan and Röllinghoff, 1999; Duque and Descoteaux, 2015). Therefore, to survive and multiply, Leishmania has developed mechanisms to subvert macrophage microbicide activity. For example, L. donovani has been shown to significantly increase MIP1β expression levels in murine BALB/c macrophages, which could potentially result in recruitment of additional macrophages to the infection site (Buates and Matlashewski, 2001). Leishmania is also able to induce changes in the nucleus and impact nucleocytoplasmic transport of infected macrophages through the zinc-metalloprotease GP63, affecting the protective functions of these cells (Isnard A, Shio MT, Olivier M , 2012).

Proteomics-based strategies have been applied extensively to elucidate the biological mechanisms of Leishmania parasites and aspects of their host interactions, drug resistance, and pathogenesis (Sundar and Singh, 2018). The quantitative proteomics approach relies on mass spectrometry to precisely detect changes in protein abundances in samples following proteolytic digestion and peptide fractionation. Some quantitative label-based methods involve the incorporation of stable isotopes in peptide samples, which are subsequently pooled and subjected to mass spectrometry analysis and relative quantification (Anand et al., 2017). Disparities in sample preparation are minimized by label-based methods, and they generate more accurate quantitative data in comparison to label-free approaches (Megger et al., 2014).

Several studies have performed quantitative proteomics analysis to investigate differences between Leishmania life stages (Lynn et al., 2013; Singh et al., 2015; Pires et al., 2014), host responses to infection (Hassani and Olivier, 2013; Menezes et al., 2013; Negrão et al., 2019), post-translational modifications (Tsigankov et al., 2014), and pathogenesis caused by different species (Negrão et al., 2019). To date, however, only one study has used a label-based approach (isobaric tags for relative and absolute quantitation; iTRAQ) to verify differential protein modulation in macrophages infected with Leishmania (Singh et al., 2015). Considering the advantages of the label-based approach, we performed a comparative proteomics analysis using DIGE (difference gel electrophoresis) and TMT (tandem mass tags) labeling to assess how Leishmania species that cause different disease outcomes alter the protein abundance in BALB/c macrophages after in vitro infection. Differentially abundant proteins identified in this study may play an important role in the biology of Leishmania species and in the macrophage immune response. They may also contribute to a better understanding of the factors that determine the course of infection. Our results suggest several possible targets for vaccines, drugs, and diagnosis of leishmaniasis.