Shedding light on host niches: label-freein situdetection ofMycobacterium gordonaevia carotenoids in macrophages by Raman microspectroscopy

Abstract

SummaryMacrophages are the primary habitat of pathogenic mycobacteria during infections. Current research about the host-pathogen interaction on the cellular level is still going on. The present study proves the potential of Raman microspectroscopy as a label-free and non-invasive method to investigate intracellular mycobacteria in situ. Therefore, macrophages were infected with Mycobacterium gordonae, a mycobacterium known to cause inflammation linked to intracellular survival in macrophages. Here, we show that Raman maps provided spatial and spectral information about the position of bacteria within determined cell margins of macrophages in twodimensional scans and in three-dimensional image stacks. Simultaneously, the relative intracellular concentration and distributions of cellular constituents such as DNA, proteins and lipids provided phenotypic information about the infected macrophages. Locations of bacteria outside or close to the outer membrane of the macrophages were notably different in their spectral pattern compared with intracellular once. Furthermore, accumulations of bacteria inside of macrophages exhibit distinct spectral/molecular information because of the chemical composition of the intracellular microenvironment. The data show that the connection of microscopically and chemically gained information provided by Raman microspectroscopy offers a new analytical way to detect and to characterize the mycobacterial infection of macrophages.

The application of Raman spectroscopy for the detection and identification of microorganisms

“…Several studies using laboratory strains demonstrated the successful detection and identification of medically relevant bacteria using Raman spectroscopy in combination with statistical data analysis . Based on the Raman spectral fingerprint, the discrimination of various pathogens and even between closely related strains was achieved, including S. aureus , S. epidermidis ,pathogenic and non‐pathogenic E. coli , Salmonella , Pseudomonas , Chlamydia , Enterococcus strains, mycobacteria, L. monocytogenes , and many more. Also, viral pathogens were objects of investigation in a clinical context, as it was reported for rotavirus, cytomegalovirus, influenza strains, human papillomavirus (HPV), Varicella zoster virus, and Porcine teschovirus , as well as echovirus 1 .…”

“…Intracellular accumulated bacteria possessed different molecular information than extracellular ones. Also, here, fluorescence imaging was used as a reference method to verify the Raman results . Raman microspectroscopy could also contribute to the investigation of intracellular and extracellular metabolic activity of Protochlamydia amoebophila , a model organism for symbiotic chlamydiae and the only non‐pathogenic chlamydia for which a genome sequence is available .…”

Vibrational Spectroscopy for Imaging Single Microbial Cells in Complex Biological Samples

“…By reducing spectral acquisition times to milliseconds, resonance Raman spectroscopy – a method in which the excitation wavelength matches the electronic transition of a selected molecule – was key to enabling the rapid imaging of these samples. It is also possible to visualize selected strains and their locations within habitats including human endothelial cells (Große et al, 2015), macrophages (Silge et al, 2015) and other environments, even when the taxa of interest are present at low abundances (Kalasinsky et al, 2007). Through combining imaging of Staphylococcus aureus cells with a multivariate classification model [based on principal component analysis (PCA) and linear discriminant analysis (LDA)], Große et al (2015) were further able to detect small differences in the spectral profiles that allowed the authors to discern between intra- and extracellular cells, due to shifts in the physiological state of the bacteria that occur upon host invasion.…”

The interaction of an amino-modified ZrO2 nanomaterial with macrophages—an in situ investigation by Raman microspectroscopy

“…In order to apply an image-based technique to investigate the NM-cell interaction in situ, Raman microspectroscopy was the method of choice. The chemical information of a cell can be spatial resolved in the subcellular range to visualize the distribution of molecular composites [14][15][16][17][18][19][20][21]. The Raman-based analysis of cells is non-invasive, and no labeling technique is necessary.…”

“…The backscattered light of each map point is collected, dispersed on a grating, and detected by a CCD [20]. The resulting dataset of single Raman spectra is analyzed by chemometrical methods and combined with the spatial information to reconstruct the chemical landscape of the cell [15,17,18,20,21].…”