Nanoscale reorganizations of histone-like nucleoid structuring proteins in Escherichia coli are caused by silver nanoparticles

Abstract

Silver nanoparticles (AgNPs) and ions (Ag+) have recently gained broad attention due to their antimicrobial effects against bacteria and other microbes. In this work, we demonstrate the use of super-resolution fluorescence microscopy for investigating and quantifying the antimicrobial effect of AgNPs at the molecular level. We found that subjecting Escherichia coli (E. coli) bacteria to AgNPs led to nanoscale reorganization of histone-like nucleoid structuring (H-NS) proteins, an essential nucleoid associated protein in bacteria. We observed that H-NS proteins formed denser and larger clusters at the center of the bacteria after exposure to AgNPs. We quantified the spatial reorganizations of H-NS proteins by examining the changes of various spatial parameters, including the inter-molecular distances and molecular densities. Clustering analysis based on Voronoi-tessellation were also performed to characterize the change of H-NS proteins’ clustering behavior. We found that AgNP-treatment led to an increase in the fraction of H-NS proteins forming clusters. Similar effects were observed for bacteria exposed to Ag+ ions, suggesting that the release of Ag+ ions plays an important role in the toxicity of AgNPs. On the other hand, we observed that AgNPs with two surface coatings showed difference in the nanoscale reorganization of H-NS proteins, indicating that particle-specific effects also contribute to the antimicrobial activities of AgNPs. Our results suggested that H-NS proteins were significantly affected by AgNPs and Ag+ ions, which has been overlooked previously. In addition, we examined the dynamic motion of AgNPs that were attached to the surface of bacteria. We expect that the current methodology can be readily applied to broadly and quantitatively study the spatial reorganization of biological macromolecules at the scale of nanometers caused by metal nanoparticles, which are expected to shed new light on the antimicrobial mechanism of metal nanoparticles.

Faster diffusive dynamics of histone-like nucleoid structuring proteins in live bacteria caused by silver ions

“…Second, the H-NS protein is tightly associated with various biological processes in the bacteria that respond to damages due to Ag, such as modulating the synthesis and stability of RpoS -a central protein/regulator for general stress responses [37][38][39] , compacting DNA and cause DNA condensation [40,41] , modulating the production of deoxyribonucleotides and synthesis of DNA [42] , and enhancing the cellular defenses against ROS [37] . Third, nanoscale spatial reorganization of H-NS proteins has been previously observed to form denser and larger clusters in bacteria subjected to Ag-treatment [43] .…”

“…To quantify the faster diffusive dynamics caused by Ag + ions, we fitted the eMSD data using ⟨Δ# $ ⟩ = 43& 4 and obtained the fitted generalized diffusion coefficients for the untreated bacteria and bacteria treated with 10 µM Ag + ions for 2, 4, 6, and 8 hr. It is noted that our previous results showed that treating E. coli with Ag + ions at 10 µM extended the lag time of the bacterial culture, while the growth rate remained the same [43,44] . As shown in Fig.…”

“…The resultant strain expresses H-NS proteins fused to mEos3.2 photoswitchable fluorescent proteins [99,100] and carries resistance against kanamycin and chloramphenicol [32,99] . The same strain has been used in our previous studies [32,43] .…”

“…The bacteria were grown overnight in a defined M9 minimal medium, supplemented with 1% glucose, 0.1% casamino acids, 0.01% thiamine and appropriate antibiotics (kanamycin + chloramphenicol) at 37ºC in a shaking incubator with a speed of 250 rpm [32,43,101,102] . On the next day, the overnight culture was diluted 50 to 100 times into a fresh medium such that OD600 = 0.05.…”

“…The control (untreated) and treated samples were left in dark at room temperature for 20-30 minutes on the agarose pad to allow the bacteria to be absorbed and mounted. The agarose pad was then flipped and attached firmly and gently to a clean coverslip, which was glued to a rubber O-ring and a clean microscope slide to form a chamber [32,43] .…”