Single-Cell Elemental Analysis of Bacteria: Quantitative Analysis of Polyphosphates in Mycobacterium tuberculosis

Abstract

More than 1.8 million people die annually from infection with Mycobacterium tuberculosis, the causative agent of tuberculosis. The ability of M. tuberculosis to obtain and distribute micronutrients, including biometals, is known to play a role in its intracellular survival and virulence within a host. Techniques to detect elemental distributions within M. tuberculosis cells have previously been limited to bulk detection methods or low-resolution analyses. Here, we present a method for determining the elemental distribution within M. tuberculosis on a single-cell level, at high (individual nanometer) resolution, using scanning transmission electron microscopy (STEM) in concert with energy-dispersive X-ray spectroscopy (EDS). Results revealed the presence of large polyphosphate granules in all strains of Mycobacteria tested. These persisted even through starvation conditions, and might play a role connected to elemental homeostasis in M. tuberculosis. Associated with the polyphosphate granules were micronutrients such as calcium and magnesium. In addition, we expanded the technique beyond Mycobacteria to show that STEM and EDS could be used as a simple screen to detect the presence or absence of concentrated elements on a single-cell level within all six other bacterial types tested, with minimal processing to the bacteria. Overall, we believe that this technique represents a first step in developing a better understanding of the role that components of the intracellular milieu, including polyphosphates and biometals, play in the pathogenesis of M. tuberculosis, with potential future applications for in vivo analysis.

Colistin-Functionalized Nanoparticles for the Rapid Capture of Acinetobacter baumannii

“…Figure 3.6B, verified that gold (Au) and nitrogen (N) were present in the EDS spectrum (see Fig. S2 of Appendix), thereby confirming the presence of Col-PEG-AuNPs and bacteria [75], [76]. In the HAADF-EDS image shown in Figure 3.6B, contrast is directly related to atomic number.…”

The Polyphosphate Kinase Gene ppk2 Is Required for Mycobacterium tuberculosis Inorganic Polyphosphate Regulation and Virulence

“…The regulatory molecule poly P has been implicated in the bacterial stringent response (16). The presence of poly P in M. tuberculosis was reported several decades ago (17), and recent studies have used scanning transmission electron microscopy together with energy-dispersive X-ray spectroscopy to visualize poly P granules in individual M. tuberculosis bacilli during nutrient starvation (18). In addition to its role in inducing the stringent response (11, 19), poly P levels have been shown to influence susceptibility of M. tuberculosis to certain TB drugs and M .…”

Nanoparticles as Drug Delivery System against Tuberculosis in Zebrafish Embryos: Direct Visualization and Treatment

“…25,26 (7) Finally, we also observed electron-dense granules in the bacterial F cytoplasm ( Figure 4J), reminiscent of polyphosphate granules that have been identified in Mtb. 27 The osmium tetroxide necessary for contrast was not compatible with X-ray microanalysis in these preparations. However, using stationary phase-grown Mm that were prepared for cryo-substitution and embedded in Lowicryl resin, we could identify these granules as being enriched in phosphate in sections using X-ray microanalysis ( Figure 4K).…”