Illuminating Host-Parasite Interaction at the Cellular and Subcellular Levels with Infrared Microspectroscopy

Abstract

Toxoplasma gondii (T. gondii) is an opportunistic protozoan that can cause brain infection and other serious health consequences in immuno-compromised individuals. This parasite has a remarkable ability to cross biological barriers and exploit the host cell microenvironment to support its own survival and growth. Recent advances in label-free spectroscopic imaging techniques have made it possible to study biological systems at a high spatial resolution. In this study, we used conventional Fourier-transform infrared (FTIR) microspectroscopy and synchrotron-based FTIR microspectroscopy to analyze the chemical changes that are associated with infection of human brain microvascular endothelial cells (hBMECs) by T. gondii (RH) tachyzoites. Both FTIR microspectroscopic methods showed utility in revealing the chemical alterations in the infected hBMECs. Using a ZnS hemisphere device, to increase the numerical aperture, and the synchrotron source to increase the brightness, we obtained spatially resolved spectra from within a single cell. The spectra extracted from the nucleus and cytosol containing the tachyzoites were clearly distinguished. RNA sequencing analysis of T. gondii-infected and uninfected hBMECs revealed significant changes in the expression of host cell genes and pathways in response to T. gondii infection. These FTIR spectroscopic and transcriptomic findings provide significant insight into the molecular changes that occur in hBMECs during T. gondii infection.

Assessment of Toxoplasma gondii lytic cycle and the impact of a gene deletion using 3D label-free optical diffraction holotomography

“…However, with improved machine learning approaches millions of cells, including pathogens, could be analysed in real time providing valuable information regarding pathogenicity, drug responses and pathobiological effects. Indeed, high content imaging-based techniques have emerged as essential tools in many areas of scientific research, including elucidating the pathophysiology of infectious disease ( Firdaus et al., 2020 ; Elsheikha et al., 2022 ). Their power lies in the amount of quantitative information that can be derived in real time without labelling.…”

“…Conventional light microscopy techniques are incapable of measuring physical properties, whilst fluorescence techniques such as scanning confocal microscopy require the use of fluorescent tagging probes or proteins which have numerous limitations in analysing dynamic alterations in cells mainly due to photobleaching and phototoxicity ( Kim et al., 2021 ). However, recent advances in label-free imaging technologies have made it possible to study biological systems at a high spatial resolution, in the Toxoplasma field infrared microspectroscopy has recently been employed to analyse chemical changes induced by infection of human brain microvascular endothelial cells ( Elsheikha et al., 2022 ), and the utility of optical diffraction holotomography (ODH) has also been demonstrated ( Firdaus et al., 2020 ). ODH exploits the intrinsic optical properties of a sample and allows the direct calculation of the optical phase delay introduced by refractive index (RI) alterations in live biological samples over relatively long time spans ( Kim et al., 2021 ).…”