Identification of novel immunoreactive proteins of Leishmania martiniquensis and Leishmania orientalis using mass spectrometry-based proteomics

Leishmaniasis, a neglected tropical disease caused by protozoan parasites of the Leishmania genus. This disease remains a global health challenge, particularly among immunocompromised populations. In Thailand, the emergence of autochthonous infections caused by Leishmania (Mundinia) martiniquensis and Leishmania (Mundinia) orientalis underscores the urgent need for specific diagnostic markers. This study presents the first immunoproteomic characterization of these two Leishmania species, employing two-dimensional electrophoresis, Western blotting, and mass spectrometry-based proteomics to identify novel immunoreactive proteins. Pooled sera from HIV-infected patients with confirmed leishmaniasis (n = 10; five L. martiniquensis and five L. orientalis), alongside pooled control sera from Leishmania-negative HIV patients (n = 30) and immunocompetent individuals (n = 30), were analyzed. Using sera from Leishmania-infected HIV patients, both species exhibited species-specific and cross-species antibody recognition patterns. Notably, stronger and more diverse immunoreactivity was observed in L. martiniquensis, while L. orientalis showed fewer and less intense signals. Three shared immunoreactive proteins were identified: tubulin alpha chain from L. martiniquensis, as well as actin and enolase from L. orientalis. In silico analysis predicted actin and enolase to be highly antigenic, while all three proteins were classified as non-allergenic and contained predicted B-cell epitopes. All candidates are also known as components of extracellular vesicles, supporting their immunological relevance. These findings highlight tubulin, actin, and enolase as conserved antigens with strong potential for the development of cross-species serological tools for L. martiniquensis and L. orientalis infections. This study provides valuable insights into their antigenic profiles and supports the development of efficient diagnostic tools to address the increasing clinical cases of these emerging species.