Date Available

3-1-2025

Year of Publication

2024

Degree Name

Doctor of Philosophy (PhD)

Document Type

Doctoral Dissertation

College

Agriculture, Food and Environment

Department/School/Program

Plant and Soil Sciences

First Advisor

Dr. Olga V. Tsyusko

Abstract

Metal and metal oxide nanoparticles (NP), which have received interest for use in agriculture, show in vitro and in vivo toxicity at relatively low concentrations. Silver nanoparticles (Ag NP) and zinc oxide nanoparticles (ZnO NP) cause adverse effects in model soil organisms under environmentally relevant conditions. In most toxicity assessments of nanomaterials, model organisms are exposed to a single stressor. Though single stressor experiments are essential to understanding the fate and effects of released NP, in nature, organisms are affected by multiple stressors, including pathogens, which might exacerbate or mitigate negative effects of NP exposure. There is limited research on how NP exposure of organisms influences their interaction with pathogens, or conversely how presence of the pathogens affect NP toxicity. Interspecies interactions have been studied for few environmental contaminants in general, but due to the complex interactions NP have with organisms, interspecies impacts on toxicity may be important, including interactions that cause additional physiological stress. Prior research has not examined NP toxicity in the context of multiple stressors. Thus, to expand our understanding of the environmental consequences of released NP, this project examined the synergistic/antagonistic effects of two commonly used NP (i.e., Ag NP and ZnO NP) on a model soil nematode Caenorhabditis elegans infected with a model, gram-negative pathogen, Klebsiella pneumoniae.

Zinc oxide NP cause toxicity at low concentrations and disrupt molecular pathways of pathogen resistance. Nematodes exposed to ZnO NP, zinc sulfate (ionic control) or K. pneumoniae showed significantly decreased reproduction compared to controls. To assess combined stress of ZnO NP and K. pneumoniae, C. elegans were exposed to EC30 concentrations of ZnO NP (or Zn ions) and K. pneumoniae. The combined exposure unexpectedly restored nematode reproduction to control levels compared to the individual exposures. Amelioration of pathogenicity by Zn is partially explained by the Zn impact on the K. pneumoniae biofilm, a necessary component of infection. External biofilm production was significantly decreased after Zn exposure. Within exposed nematodes, Zn in particulate or ionic form significantly decreased K. pneumoniae colony forming units (CFU), which functioned as an assessment of viable pathogens inside infected nematodes. Taken together, our results suggest that combined exposure of C. elegans to both stressors Zn in ionic or particulate form inhibits K. pneumoniae colonization of nematode intestine through decreasing pathogen biofilm formation.

Unlike Zn, Ag lacks biological functions inside C. elegans, and therefore, homeostatic mechanisms are not as extensive for Ag compared to Zn. Thus, I hypothesized that reproduction would not return to control levels after exposure to both Ag NP and K. pneumoniae. Additionally, I tested sulfidized-Ag NP (sAg NP) to assess real world implications of Ag NP release. Individual exposures to all stressors (i.e., Ag NP, sAg NP, or K. pneumoniae) significantly decreased nematode reproduction compared to controls. To assess the combined stress of Ag NP/sAg NP and K. pneumoniae, nematodes were exposed to equitoxic EC30 concentrations of ionic, particulate pristine, or sulfidized Ag alongside K. pneumoniae. Combined exposures resulted in reproduction decline that was not significantly different compared to single exposures. Silver exposure decreased K. pneumoniae biofilm production outside the nematode and significantly reduced viable pathogens inside the host, mirroring observations from Zn exposures. My results indicate that by hindering K. pneumoniae ability to colonize nematode's intestine, Ag reduces K. pneumoniae pathogenicity regardless of form. However, the toxic effects from Ag are not mitigated when exposed to both stressors, illustrating an antagonistic relationship between the two stressors.

To elucidate the molecular mechanisms behind pathogen resistance and recovery, I examined the transcriptomic responses of nematodes exposed to ZnO NP and K. pneumoniae. Individual and combined exposures at the EC30 for reproduction yielded 7,967 significantly differentially expressed genes (DEGs) at fold change of ±1.5 compared to controls. There are only 10 unique DEGs in response to ZnSO4 while the other DEGs are shared with the individual or combined ZnO NP treatments, indicating some responses are due to NP dissolution. The gene expression profile was much stronger in response to ZnO NP compared to ZnSO4 with 1154 unique DEGs. Among the genes induced at ten-fold in Zn exposed treatments are metal response genes (mtl-1, mtl-2, numr-1, numr-2, hizr-1 and ttm-1). The decrease in reproduction from Zn exposure can partially be explained by drastic reduction in expression of vitellogenin associated genes (vit-1, vit-3, vit-5, and vit-6). Interestingly, vit-1 and vit-3 are overly expressed only in K. pneumoniae treatment. Additionally, the pathogen seems to affect nematodes’ ability to recover from Zn exposure. Excess Zn is stored inside the intestine in lysosomal related organelles (LROs). Pathogen exposure increased LROs in nematodes exposed to both stressors, suggesting that C. elegans infection with K. pneumoniae might elicit increased Zn sequestration, partially explaining how reproduction might have recovered.

My results highlight the unpredictable nature of combined stressor effects. Unexpectedly, combined stressors resulted in antagonistic responses, calling into question the utility of single stressor exposures when assessing real world implications to released NP.

Digital Object Identifier (DOI)

https://doi.org/10.13023/etd.2024.34

Funding Information

  • Caenorhabditis elegans strains were provided by the Caenorhabditis Genetics Center, which is funded by the NIH Office of Research Infrastructure Programs (P40 OD010440).
  • A portion of this project was funded by USDA NIFA multistate project NC1194 and Hatch Project KY006133.
  • Chemical analyses were supported by UK-CARES through NIEHS Grant P30 ES026529.

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