Tandem Mass Tag Proteomic Analysis of in Vitro and in Vivo Models of Cutaneous Leishmaniasis Reveals Parasite-Specific and Nonspecific Modulation of Proteins in the Host

Abstract

Cutaneous leishmaniasis, the most common form of leishmaniasis, is endemic in several regions of the world, and if not treated properly, it can cause disfiguring scars on the skin. Leishmania spp. infection causes an inflammatory response in its host, and it modulates the host metabolism differently depending on the Leishmania species. Since Leishmania spp. has begun to develop resistance against current therapies, we believe efforts to identify new possibilities for treatment are critical for future control of the disease. Proteomics approaches such as isobaric labeling yield accurate relative quantification of protein abundances and, when combined with chemometrics/statistical analysis, provide robust information about protein modulation across biological conditions. Using a mass spectrometry-based proteomics approach and tandem mass tag labeling, we have investigated protein modulation in murine macrophages (in vitro model) and skin biopsies after exposure to Leishmania spp. (in vivo murine model). Infections induced by L. amazonensis (endemic in the New World) and L. major (endemic in the Old World) were compared to an inflammation model to search for Leishmania-specific and nonspecific protein modulation in the host. After protein extracts obtained from in vitro and in vivo experiments were digested, the resulting peptides were labeled with isobaric tags and analyzed by liquid chromatography-MS (LC-MS). Several proteins that were found to be changed upon infection with Leishmania spp. provide interesting candidates for further investigation into disease mechanism and development of possible immunotherapies.

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

“…The same group [ 41 ] performed a similar experiment with skin biopsies and reported 17 DMP in infected mice. Further, they [ 42 ] adopted the labeled (tandem mass tag) proteomic approach and confirmed their earlier findings. 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 .…”

“…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.…”

“…amazonensis and L . major Tandem Mass Tag; LC–MS/MS LPS stimulated macrophage vs L. major infected macrophage and LPS stimulated macrophage vs L. amazonensis infected macrophage 51 – 2019 [ 42 ] 10 Proteins of Canis lupus Infection based studies Leishmania IFAT nano LC; nano LC–MS/MS High titre immunofluorescent antibody test vs negative IFAT 17 5 2010 [ 46 ] 11 L . infantum ELISA; LC–MS/MS Post infection vs pre infection 19 12 2019 [ 48 ] 12 L .…”

H3 relaxin protects against calcium oxalate crystal‐induced renal inflammatory pyroptosis

“…Briefly, 100 μg of lytic protein per sample was reduced, alkylated, precipitated, resuspended and digested with trypsin before labelling. 35 Subsequently, 20 μL of TMT was added to each sample and mixed, and sodium deoxycholate clearing and peptide desalting were performed. 36 The pooled TMT-labelled peptides were then separated and analysed using a reversed-phase high-performance liquid chromatography and a nano-UPLC (EASY-nLC 1200) coupled to a Q-Exactive mass spectrometer (Thermo Finnigan) as previously described.…”