Label-Free Proteomic Analysis Reveals Parasite-Specific Protein Alterations in Macrophages Following Leishmania amazonensis, Leishmania major, or Leishmania infantum Infection

Abstract

Leishmania is an obligate intracellular parasite known to modulate the host cell to survive and proliferate. However, the complexity of host-parasite interactions remains unclear. Also, the outcome of the disease has been recognized to be species-specific and dependent on the host's immune responses. Proteomics has emerged as a powerful tool to investigate the host−pathogen interface, allowing us to deepen our knowledge about infectious diseases. Quantification of the relative amount of proteins in a sample can be achieved using label-free proteomics, and for the first time, we have used it to quantify Leishmania-specific protein alterations in macrophages. Protein extracts were obtained and digested, and peptides were identified and quantified using nano-LC coupled with tandem mass spectrometry analyses. Protein expression was validated by Western blot analysis. Integrated Proteomics Pipeline was used for peptide/protein identification and for quantification and data processing. Ingenuity Pathway Analysis was used for network analysis. In this work, we investigated how this intracellular parasite modulates protein expression on a host macrophage by comparing three different Leishmania speciesL. amazonensis, one of the causative agents of cutaneous disease in the Amazon region; L. major, another causative agent of cutaneous leishmaniasis in Africa, the Middle East, China, and India; L. infantum, the causative agent of visceral leishmaniasis affecting humans and dogs in Latin Americaand lipopolysaccharide stimulated macrophages as an in vitro inflammation model. Our results revealed that Leishmania infection downregulates apoptosis pathways while upregulating the activation of phagocytes/ leukocytes and lipid accumulation.

Differentially modulated proteins associated with Leishmaniasis—a systematic review of in-vivo and in-vitro studies

“…Despite the different experimental conditions and setup, heat-shock and Tryparedoxin related proteins were differentially regulated in 17 studies, indicating that those proteins are often modulated in the parasite. Moreover, several proteins were consistently identifiable and also to be similarly modulating in studies with similar experimental conditions [ 42 , 40 , 41 ]. Only three studies explored the host response (human subjects) during Leishmania infection.…”

“…Also, the study reported U6 snRNA-associated Sm-like protein and heat repeat-containing protein 3 to be precisely up-regulated in infection with L. major , while protein tweety homolog 3, phosphatidate cytidylyltransferase, and macrophage metalloelastase to be precisely up-regulated in infection with L. amazonensis . Overall, the studies by Negrão et al [ 40 , 42 , 41 ] demonstrated the species-specific protein modulation in Leishmania infection as well as differentiated the host response through LPS inoculation and Leishmania infection. To observe the immune response effects of malnutrition in Leishmania infection, Losada-Barragán et al [ 43 , 44 ] conducted the differential proteomic studies involving malnourished mice.…”

“…major , and L . infantum Western blot; Nano LC–MS/MS; LTQ-OrbitrapVelos MS Infected vs healthy 107 42 2019 [ 40 ] 6 L . amazonensis and L .…”

“…The four up-regulated proteins of infected mice include phospholipase D1, RAS-related protein, glucuronidase β, and phosphotransferase regulation domain. Negrão et al [ 40 ] performed a proteomic study among mice infected with L. amazonensis , L. major , L. infantum , mice inoculated with lipopolysaccharide (as positive control), and healthy mice (as negative control). The study reported 31 proteins to be differentially regulated only in L. amazonensis , 25 proteins to be differentially regulated only in L. major , and 27 proteins to be differentially regulated only in L. infantum .…”

“…Among the studies with the different hosts, one could observe consensus as well as contrasting regulation of the same protein. For instance, the transferrin protein down-regulated in dogs [ 47 ] was up-regulating in mice [ 43 , 40 ]. On the other hand, the amyloid protein is up-regulating in both dogs and humans [ 35 , 46 ].…”