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.

Keywords: elements; metals; scanning transmission electron microscopy; tuberculosis.

Figure 1

Polyphosphate granules in Mycobacteria. Mycobacterial isolates were fixed as whole-cell mounted, and imaged using an FEI Titan 80-200 STEM. Selected images show representative samples from at least two biological replicates, with multiple bacteria imaged within each replicate. Nutrient-starved M. tuberculosis was incubated in water for 21 days, and all other images are from bacteria grown to late-log phase in 7H9 liquid media. Scale bars (lower right) represent 500 nm.

Figure 2

EDS analysis of polyphosphate granules. (A) A representative image of a polyphosphate granule with EDS analysis from wild-type M. tuberculosis. Scale bar (lower right of EM) represents 50 nm. (B) Spectra on the left and right represent EDS spectra on and off the granule, respectively. Data were collected from a minimum of two bacteria from at least two separate biological replicates, and data shown are representative. Elements presented in the spectra include C, for carbon, O, for oxygen, Ca for calcium, Na for sodium, Mg for magnesium, P for phosphorus, and K for potassium.

Figure 3

STEM imaging of a variety of bacterial strains. Bacteria were fixed; whole-cell mounted, and imaged using an FEI Titan 80-200 STEM. Selected images are representative samples from one biological replicate, with multiple bacteria imaged within each replicate. Scale bars (lower right) represent 500 nm.