Intracellular Parasites Toxoplasma gondii and Besnoitia besnoiti, Unveiled in Single Host Cells Using AP-SMALDI MS Imaging

Abstract

The obligate intracellular apicomplexan parasites Toxoplasma gondii and Besnoitia besnoiti are important causes of disease in both humans and cattle. To date, effective specific treatments are lacking for both infections. To counteract severe symptoms leading to, e.g., disabilities and even abortion in the case of human toxoplasmosis and bovine besnoitiosis, novel targets are required for development of drugs and vaccines. A promising emerging technique for molecular characterization of organisms is high-resolution atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI) which enables semiquantitative visualization of metabolite distributions. MSI was here used to trace and characterize lipid metabolites in primary bovine umbilical vein endothelial cells (BUVECs) upon infection with tachyzoites, an early and pathogenic fast-replicating life stage of T. gondii and B. besnoiti. A cell bulk, derived from noninfected controls and parasite-infected cell pellets, was analyzed by AP-SMALDI MSI in technical and biological triplicates. Multivariate statistical analysis including hierarchical clustering and principle component analysis revealed infection-specific metabolites in both positive- and negative-ion mode, identified by combining database search and LC-MS2 experiments. MSI analyses of host cell monolayers were conducted at 5 μm lateral resolution, allowing single apicomplexan-infected cells to be allocated. This is the first mass spectrometry imaging study on intracellular T. gondii and B. besnoiti infections and the first detailed metabolomic characterization of B. besnoiti tachyzoites. MSI was used here as an efficient tool to discriminate infected from noninfected cells at the single-cell level in vitro.

Advances in MALDI Mass Spectrometry Imaging Single Cell and Tissues

“…Scanning microprobe matrixassisted laser desorption ionization (SMALDI) mass was invented by Hubert, M. in 2002 (Spengler andHubert, 2002). It was further improved as atmospheric pressure scanning microprobe matrix-assisted laser desorption/ionization mass spectrometry (AP-SMALDI MS) for imaging (Vegvari et al, 2017;Bhandari et al, 2018;Bredehoeft et al, 2019;Kadesch et al, 2020;Mokosch et al, 2021;Mueller et al, 2021;Righetti et al, 2021), while atmospheric pressure MALDI ion sources AP-MALDI have also been developed for MSI (Guenther et al, 2011;Jackson et al, 2018;Keller et al, 2018) and achieved a lateral resolution at 1.4 μm (Kompauer et al, 2017).…”

“…Furthermore, MALDI ion resources can be easily coupled with high-resolution (resolving power) mass analyzers, such as TOF, FT-ICR, and Orbitrap, which provide high mass accuracy for MSI targets. During the recent years, with the developments of some new MALDI ion sources, such as atmosphere pressure AP-MALDI (Kompauer et al, 2017), AP-SMALDI (Vegvari et al, 2017;Bhandari et al, 2018;Bredehoeft et al, 2019;Kadesch et al, 2020;Mokosch et al, 2021;Mueller et al, 2021;Righetti et al, 2021), MALDI-2 (Soltwisch et al, 2015), transmission MALDI-2 (Niehaus et al, 2019), and nano laser probed-based laser desorption ionization (Meng et al, 2020), the lateral resolution of MALDI-MSI will possibly achieve nm or µm level for single cell and even subcellular scale imaging. Finally, the scanning speed of MALDI-MSI largely depends on the speed of MS detection, spectra recording and data processing.…”

Next-Generation Technologies and Systems Biology for the Design of Novel Vaccines Against Apicomplexan Parasites

“…In addition to proteomics (39), lipidomics analysis in T. gondii has been addressed in the last decade, revealing parasite-specific proteins and lipids, unraveling parasite-host interactions (45). Recently, Kadesch and collaborators (2020) (46) carried out a mass spectrometry imaging in Besnoitia besnoiti and T. gondii infection in primary bovine umbilical vein endothelial cells using atmospheric-pressure scanning microprobe matrix-assisted laser desorption/ionization (AP-SMALDI) mass spectrometry imaging (MSI), an emerging technique that provides high resolution and allows analysis of single cells, allowing a metabolomic characterization. This study has revealed biomolecular markers of infection in both parasites and has shown striking differences in the metabolites during infection between both parasites, despite their closer phylogenic relationship, related in particular to lipid classes such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, cardiolipins, phosphatidic acids, and phosphatidylinositol (46).…”

Atmospheric‐pressure scanning microprobe matrix‐assisted laser desorption/ionization mass spectrometry imaging of Neospora caninum‐infected cell monolayers

“…Fixed pellets were immediately frozen in liquid nitrogen and stored at −80 • C until further use. 33 Also, BUVEC were seeded on glass coverslips (15 mm; Thermo Fisher Scientific) and allowed to grow until confluency. Then, cell layers were infected as described above.…”