A quantitative mass spectrometry approach to identify novel proteins involved in macrophage infections by Leishmania parasites

Abstract text

Leishmania are single-cell parasites with an intracellular life cycle stage in phagocytic cells of their vertebrate hosts. Transcriptomic analyses of host cell defense mechanisms and how the parasites respond to these processes are already available. Due to the prevalence of post-transcriptional regulation in kinetoplastids however, transcriptomes do not reflect the protein composition very well.

Here we present a systematic comparative in vitro study of the infection process using a quantitative proteomics approach to uncover novel virulence factors.

Murine bone marrow-derived macrophages (BMDMs) were infected with 3 different species: L. mexicana, L. major, L. infantum. Whole cell protein extracts of parasites and BMDMs were prepared at 7 timepoints post-infection (0.5-72h) and were analysed using quantitative mass spectrometry. Candidate proteins were knocked out in L. mexicana and infection experiments were carried out to discover novel mechanisms involved in parasite adaptation during macrophage infections. Evaluation of infection experiments were carried out using fluorescence microscopy. An ImageJ macro was scripted to process images and automatically determine the number of infected macrophages.

We quantified the expression of approximately 2000 murine proteins in all 3 infection experiments, while 1500, 1000 and 1400 proteins were quantified for infections with L. mexicana, L. major and L. infantum, respectively. The number of differentially-expressed proteins during the time course of infection varied for each species (349, 133, and 287 proteins for L. mexicana, L. major and L. infantum, respectively). Our experiments identified many proteins putatively involved in the infection process, which were not found in previously published transcriptome analyses. For example, gene ontology enrichment showed 41 L. mexicana protein IDs involved in oxidoreductase activity. Additionally, we found 24 differentially-expressed proteins without any functional or structural annotation in the TriTryp database. Automated evaluation of infection experiments with two knockout cell lines showed reduced infectivity.

Our mass spectrometry approach identified novel virulence factors in Leishmania infections, that were not detected in transcriptomic screens. Automated evaluation of infection experiments successfully showed strong phenotypes for two knockout cell lines. Candidate proteins are being further investigated to decipher their role during the infections processes.