Multiple stressor effects on a model soil nematode, Caenorhabditis elegans: combined effects of the pathogen Klebsiella pneumoniae and zinc oxide nanoparticles

Abstract

Research utilizing the model soil nematode Caenorhabditis elegans has revealed that agriculturally relevant nanoparticles (NP), such as zinc oxide NP (ZnONP), cause toxicity at low concentrations and disrupt molecular pathways of pathogen resistance. However, in most nanotoxicity assessments, model organisms are exposed to a single stressor but in nature organisms are affected by multiple sources of stress, including infections, which might exacerbate or mitigate negative effects of NP exposure. Thus, to expand our understanding of the environmental consequences of released NP, this project examined the synergistic/antagonistic effects of ZnONP on C. elegans infected with a common pathogen, Klebsiella pneumoniae. Individual exposures of C. elegans to ZnONP, zinc sulfate (Zn²⁺ ions) or K. pneumoniae significantly decreased nematode reproduction compared to controls. To assess the combined stress of ZnONP and K. pneumoniae, C. elegans were exposed to equitoxic EC₃₀ concentrations of ZnONP (or Zn ions) and K. pneumoniae. After the combined exposure there was no decrease in reproduction. This complete elimination of reproductive toxicity was unexpected because exposures were conducted at EC₃₀ Zn concentrations and reproductive toxicity due to Zn should have occurred. Amelioration of the pathogen effects by Zn are partially explained by the Zn impact on the K. pneumoniae biofilm. Quantitative assessments showed that external biofilm production and estimated colony forming units (CFU) of K. pneumoniae within the nematodes were significantly decreased. Taken together, our results suggest that during the combined exposure of C. elegans to both stressors Zn in ionic or particulate form inhibits K. pneumoniae ability to colonize nematode’s intestine through decreasing pathogen biofilm formation. This highlights the unpredictable nature of combined stressor effects, calling into question the utility of exposures in simplified laboratory media.

Graphical Abstract

1. Introduction

Nanoparticles (NP) are being explored for use in agriculture (Rajput et al., 2021) as fertilizers (Verma et al., 2022), and for delivery of herbicides and pesticides (Pestovsky and Martínez-Antonio, 2017; Wang et al., 2022), which will result in their direct entry into soils (Khan et al., 2022; Mueller and Nowack, 2008). The other route for NP into soils is through their release from consumer products into wastewater, partitioning into soil sludge and via amendment of biosolids to the agricultural soils (Mueller and Nowack, 2008). Nanoparticles have unique properties that distinguish them from bulk materials simply by virtue of their size and large surface area, such as increased chemical reactivity (Murthy, 2007). While these nano-specific properties may make these materials fit for their intended use, they may also increase the risk to human and ecosystem health once released into the environment, by having increased reactivity for example (Tiede et al., 2009). For some materials, the comparatively large reactive NP surface area can facilitate the production of reactive oxygen species (ROS), which may lead to toxicity in non-target organisms (Abdal Dayem et al., 2017). Metal-based NP may dissolve into reactive ions prior or also after uptake by the organisms and initiate a cascade of the stress responses (McCourt et al., 2022; Starnes et al., 2019b; Starnes et al., 2016).

Emerging metal NP used in agriculture have been shown to induce toxic responses at relatively low concentrations in vitro and in vivo (Luo et al., 2015; Murthy, 2007; Paramo et al., 2020; Rossbach et al., 2020; Tiede et al., 2009; Yu et al., 2013). Although there are few studies outside of simplified laboratory exposures at high concentrations, silver NP (AgNP), copper-oxide NP (CuONP), and zinc-oxide NP (ZnONP) negatively affect model soil organisms under environmentally relevant conditions (Starnes et al., 2019a; Starnes et al., 2019b; Velicogna et al., 2021; Yang et al., 2014). Organisms’ exposure to NP in complex environments with multiple species may have different responses than in simple single species under simplified laboratory exposures due to interspecies interactions and other factors (Avellan et al., 2018). Interspecies interactions have been studied for few environmental contaminants in general; however, due to the complex interactions of nanomaterials with organisms, interspecies impacts on toxicity may be important, including interactions that cause additional physiological stress (Bone et al., 2012; Unrine et al., 2012).

In most nanotoxicity assessments, model organisms are exposed to a single stressor. Though single stressor experiments are essential for understanding of 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 impact NP toxicity. For example, separate studies have demonstrated that exposure to ZnONP decreases immune responses in a model nematode, Caenorhabditis elegans (Li et al., 2020) and interferes with quorum sensing mechanisms and biofilm formation in bacteria, which might reduce their ability to infect their host (Khan et al., 2020). Thus, to better predict the environmental consequences of released NP, more research is needed on the combined interactions between NP and pathogen exposures.

Since metal-containing NP will reach the soil, soil biota will be exposed to NP in addition to other stressors. A soil organism selected to study the impacts from the exposure to multiple stressors, should serve as a powerful toxicity model for both stressors, NP and pathogens. A soil-dwelling nematode, Caenorhabditis elegans satisfies such criteria and represents an ideal toxicity model for various environmental contaminants, including nanomaterials (Choi et al., 2014; Li et al., 2021; Starnes et al., 2015). Among the advantages of using C. elegans are their short generation time, prolific reproduction, low maintenance cost, fully mapped and annotated genome, and availability of functional genomic tools (Choi et al., 2014; Handy et al., 2012). Additionally, C. elegans has been used for screening of anti-infectives when examining their biological responses to different pathogens (Peterson and Pukkila-Worley, 2018). Among such pathogenic bacteria is Klebsiella pneumoniae, which is a pervasive, gram-negative, and opportunistic bacterial pathogen found in different environmental niches including soils, sewage, water, and plant surfaces (Liu et al., 2020; Martin and Bachman, 2018; Peterson and Pukkila-Worley, 2018). Zinc oxide NP were shown to reduce C. elegans reproduction, lifespan, increase mortality, and induce transcriptomic responses similarly to those of Zn ions (Gonzalez-Moragas et al., 2017; Gupta et al., 2015; Starnes et al., 2019b; Starnes et al., 2016). The only published study that examined effects of a pathogen, after nematodes were exposed to ZnONP reported enhanced pathogenicity after subsequent exposure to Pseudomonas aeruginosa (Li et al., 2020). It is unknown if similar responses would occur during combined simultaneous exposure of both stressors and in the presence of a pathogen with a differing disease mechanism. Klebsiella pneumoniae is often studied for its potential pathogenicity to human and animal health (Wyres and Holt, 2018). Exposure to K. pneumoniae causes mortality in C. elegans within 48 h exposure, and the molecular mechanisms of K. pneumoniae pathogenicity have recently been elucidated (Kamaladevi and Balamurugan, 2017).

To our knowledge, no other study has examined NP exposure in the context of multiple stressors. So, to expand our understanding of the environmental consequences of released NP, the combined effects of simultaneous exposure of C. elegans to ZnONP and K. pneumoniae were examined in this study. Specifically, the objectives were to determine if the effects of the two combined stressors, ZnONP (or ionic Zn ²⁺) and K. pneumoniae, differ from the response to each individual stressor for C. elegans, and elucidate if ZnONP (or Zn ions) inhibits K. pneumoniae biofilm formation and its internal colonization before and after their uptake by C. elegans. We hypothesized that the combined exposure of C. elegans to ZnONP and K. pneumoniae would exasperate adverse effects compared to the pathogen exposure alone because of the compounding stress of NP (or Zn ion control) exposure.

2. Materials and Methods

2.1. Strains and exposure conditions

Klebsiella pneumoniae (ATCC 10231) was purchased from the American Type Culture Collection (ATCC, Manassas, Virginia). Before each experiment, K. pneumoniae, from a frozen glycerol stock, was incubated in Luria-Bertani broth (LB, 0.5% yeast extract, 1.0% tryptone, 0.5% NaCl) supplemented with 100 ug/mL ampicillin at 37 °C for 24 h. Escherichia coli OP50 and Caenorhabditis elegans wild type N2 were obtained from Caenorhabditis Genetic Center (CGC, Minneapolis, Minnesota). Nematode maintenance and age-synchronization were conducted following established protocols (Starnes et al., 2016; Starnes et al., 2015; Tsyusko et al., 2012). Before each test, nematodes were age-synchronized using a NaClO/NaOH solution to isolate the eggs. The eggs were grown at 20 °C on K-agar plates with a E. coli OP50 bacterial lawn until needed (Williams, 1988). Unless otherwise stated, all exposures were conducted in moderately hard reconstituted water (MHRW), a low ionic media with a similar chemical composition to soil pore water (USEPA, 2002), supplemented with 5 μL glucose (20%)/mL of MHRW. For nonpathogenic controls, 10 μL of E. coli OP50 stock solution per mL of exposure solution (OD₆₀₀ = 1 stock solution) was added; for pathogenic exposure treatments, 100 μL of K. pneumoniae (OD₆₀₀ = 0.1 stock solution) was added. Before each experiment, nematodes soaked in MHRW supplemented with 100 ug ampicillin/mL, to remove any external bacteria. Afterwards, nematodes were acclimatized to MHRW. Zinc sulfate (ZnSO₄) was used as an ionic control.

2.2. Synthesis and characterization of ZnO Nanoparticles

Zinc-oxide nanoparticles were synthesized following a procedure developed by Becheri et al. (2007). Briefly, a 0.2 M solution of zinc chloride (ZnCl₂) was stirred in a 90 °C water bath for 10 minutes. After, a 5.0 M solution of sodium hydroxide (NaOH) was added dropwise until a white precipitate formed. After cooling the solution to room-temperature, particles were centrifuged at 3220 × g, the supernatant was removed, and the pellet was resuspended in 18 MΩ deionized (DI) water. This process was repeated twice.

Dynamic light scattering (DLS, Malvern ZetaSizer Nano-ZS, Malvern, United Kingdom) was used to measure the mean intensity hydrodynamic diameter (z-average) in the exposure media at 100 mg ZnONP/L. The ζ-potential of the particles were calculated using Hückel approximation from electrophoretic mobilities measured with phase analysis light scattering (PALS, Malvern Zetasizer Nano-ZS). To measure NP size, 10 μL of 50 ZnONP mg/L was plated onto lacey carbon films on copper grids. A Talos F200X transmission electron microscope (TEM, Thermo Fisher Scientific, Waltham, USA) was used to assess particle size. To determine the primary particle size distribution, the diameters of approximately 100 individual particles were measured using ImageJ software (https://imagej.nih.gov/ij/).

2.3. Nematode reproduction assay

We aimed to expose C. elegans at sub-lethal equitoxic Zn concentrations. Previous research has shown that higher than EC₃₀ exposure for Zn ions or ZnONP would result in nematode’s mortality (Starnes et al., 2015). As such, EC₃₀ was used to assess combined effects of ZnONP (or Zn⁺²) and the pathogen on nematode’s reproduction. We also used EC₃₀ in our previous study to examine transcriptomic signatures induced by exposures to ZnONP and Zn ions in C. elegans. This would also allow us to make an adequate comparison between previous and this study. To elucidate EC₃₀ of Zn⁺² and ZnONP, approximately 100 age-synchronized L3 stage nematodes were exposed to different Zn⁺² or ZnONP concentrations, ranging from 1 mg/L to 21 mg/L. For age-synchronization, we used a previously established NaClO/NaOH protocol (Stiernagle, 2006). The number of the nematodes were very similar in the control and each treatment group with about 100 nematodes corresponding to the volume of 100 μl after gently centrifuging nematodes for 1min. After 24 h exposure, six nematodes were transferred to six separate 3-cm K-agar plates with E. coli OP50 bacterial lawns. Plates were incubated for 48 h to allow hatching. Afterwards, plates were stained with rose Bengal (0.5 mg/L) and heated at 50 °C for 55 minutes. Fully hatched nematodes were scored under a microscope. To assess antagonistic/synergistic effects to pathogen exposure, nematodes were exposed to Zn⁺² or ZnONP at their determined EC₃₀ (6.5 mg/L and 7.5 mg/L, respectively). In addition, nematodes were fed K. pneumoniae or non-pathogenic E. coli OP50 during the exposure period. Nematodes were transferred, stained, and counted as described above. After each experiment, Zn concentrations were verified by acidifying exposure solutions with 1% HNO₃ and analyzed with an Inductively Coupled Plasma Mass Spectrometry (ICP-MS). The Zn EC₃₀ ranged from 6.6 mg/L to 7.8 mg/L.

2.4. Nematode growth assay

Age-synchronized L3 stage nematodes were divided into five treatment groups: E. coli OP50, Zn⁺²:: E. coli OP50, Zn⁺²:: K. pneumoniae, ZnONP:: E. coli OP50, and ZnONP:: K. pneumoniae. Zinc concentrations corresponded to their reproductive EC₃₀ values (6.5 mg/L and 7.5 mg/L for Zn⁺² and ZnONP, respectively). Nematodes were placed into 4 mL of exposure solution in separate 15 mL centrifuge tubes and incubated at 20 °C for 24 h. Afterwards, at least 20 nematodes were removed from exposures, paralyzed with sodium azide (3 μL of 170 mM), and imaged using a light microscope. Measurements of surface area of the nematodes in two dimensional images were then made using ImageJ software (https://imagej.nih.gov/ij/).

2.5. Biofilm formation assays

Biofilm formation by K. pneumoniae was assessed qualitatively using procedures described by Liu et al. (2020. Biofilm formation was indicated by darkened colonies after 24 h of growth at 37 °C. To quantitatively assess biofilm growth, 5 mL of each exposure solution described above were added to individual 6-cm, sterile petri dishes. Cultures were incubated statically at 37 °C for 24 h and then discarded. Each dish was washed three times with phosphate buffered saline (PBS) and stained with crystal violet (0.1% in ethanol) for 20 min. Plates were washed three times with DI water, allowed to air dry overnight, and the absorbed dye was later dissolved with 5 mL of ethanol. From each plate, 200 μL of sample was taken and the absorbance at 570 nm was measured.

2.6. SEM micrographs

Biofilm micrographs were prepared by a modified protocol of Brossard and Campagnari (2012. Briefly, three treatments (e.g., K. pneumoniae, zinc ion plus K. pneumoniae, and ZnONP plus K. pneumoniae) of exposure solutions were prepared as described above. For each treatment, three cover slips were placed into three separate 10-cm petri dishes and just enough solution (i.e., ~8 mL) was added to submerge the slips. The cultures were statically incubated at 37 °C for 24 h. Afterwards, the cover slips were removed, gently washed with PBS, and fixed for 1 h with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 0.075% ruthenium red and 0.075 M lysine acetate at pH 7.2. Samples were then rinsed three times with 0.2 M sodium cacodylate buffer containing 0.075% ruthenium red and then dehydrated through graded incubations in 30%, 50%, 75%, 95%, and 100% ethanol. Coverslips were then analyzed by SEM (FEI Helios Nanolab, UK, Lexington).

2.7. Quantification of internal colonization

Intestinal colonization by K. pneumoniae in nematodes was determined by following a method outlined by Kamaladevi and Balamurugan (2015 with some modifications. After exposure for 8 h, nematodes were centrifuged at 1000 g for 1 min and the supernatant discarded. The nematodes were then rinsed in MHRW twice, placed in 4 mL of MHRW supplemented with E. coli OP50, and incubated for 16 h. Before grinding, nematodes were chilled to prevent peristalsis and then washed with 0.1% Triton X-100 in MHRW twice. From each treatment, 10 individual nematodes were selected and then ground in a microcentrifuge tube with a plastic pestle. Lysates were incubated on LB agar plates supplemented with 100 ug mL⁻¹ ampicillin at 37 °C for 24 h. Colony forming units (CFU) were recorded the following day.

2.8. Determination of zinc size fractionation

To determine if combined effects of Zn⁺² and ZnONP exposures were due to Zn partitioning mostly to K. pneumoniae and C. elegans biomass, Zn size fractionation in exposure solutions was determined. After performing exposure experiments outlined above, exposure media were separated into three fractions: unfiltered, filtered through a 0.2 μm filter, and filtered through a 0.9 nm filter. The 0.2 μm filters removed bacteria, nematodes and large ZnONP aggregates, while the 0.9 nm filters removed ZnONP and Zn bound to ligands (>3 kDa) suspended in the media. Zinc concentrations in the solutions were determined using Inductively Coupled Plasma Mass Spectrometry (ICP-MS; Agilent 7900, Santa Clara, CA, USA) following U.S. EPA method 200.8 (USEPA, 1994).

2.9. Statistical analysis

All experiments were conducted in triplicate. Statistically significant differences (i.e., p ≤ 0.05) compared to controls were assessed via Dunnett’s test performed after one-way analysis of variance (ANOVA). Normal distributions of variance were checked with Q-Q plots. EC₃₀ values of Zn ions and ZnONP were interpolated from parameters determined with linear regression. Statistical analyses were conducted with R (4.1.2).

3. Results and Discussion

The main finding of this study is that the combined exposure of Zn in ionic or nanoparticulate form with the pathogen K. pneumoniae resulted in antagonistic effect with complete return of C. elegans reproduction back to the control levels. Our results indicate that mitigation of the pathogen effects by Zn is partially explained by decreases in biofilm formation and colony forming units of K. pneumoniae. Though ZnONP are toxic to K. pneumoniae on their own (Kudaer et al., 2022) and thus decrease pathogenicity may be expected, C. elegans in this study were exposed to concentrations of ZnONP that are higher than therapeutic levels, i.e., at levels that also decrease nematode reproduction. As such, the return of reproduction to control levels is unexpected, as nematodes should be affected by Zn toxicity. Below we present and discuss our results in more detail.

3.1. Particle characterization

ZnONP had a primary particle size ranging from 15 to 60 nm, with a median size of 33 nm (SD = 12 nm; Fig. S1). Nanoparticles were mostly hexagonal in shape (Fig. S2). In MHRW at pH 7.2, particles had a z-average hydrodynamic diameter of 168 nm and ζ-potential of −14.6 mV (SD = 5.5 mV). The dissolution experiments were performed by filtration and ultrafiltration in the presence of nematodes and bacteria - non-pathogenic E. coli OP50 or pathogenic K. pneumoniae (Fig 1). In the treatments with K. pneumoniae, the percent Zn found in the 0.9 nm filtrate increased in comparison to ZnONP with OP50 treatment (from 72% to 95%). Therefore, our results do not indicate an increase in Zn partitioning into biomass or ligands secreted by the organisms larger than 3 kDa in K. pneumoniae treatments. Previous studies have shown that release of macromolecules either as a detoxification mechanism or through release of intracellular components can reduce the toxicity of metal-based NP (Bone et al., 2012; Unrine et al., 2012). It is also important to note that the total biomass of the nematodes and bacteria was extremely low relative to the volume of the exposure solution, thus even high tissue/cell Zn concentrations will have a small impact on dissolved Zn concentrations in the bulk solution.

Figure 1.

Zinc partitioning in exposure media after 24 h C. elegans exposure experiment. Treatments include Zn ions + OP50, ZnONP + OP50, Zn ions + K. pneumoniae, and ZnONP + K. pneumoniae. After exposure, each treatment was separated into unfiltered, filtered with 0.2 μm filter, and filtered with 0.9 nm filter. Data are presented with ± 1 SD.

3.2. Zinc ions and ZnONP reduce K. pneumoniae toxicity

Zinc ions and ZnONP reduced reproduction in C. elegans in a concentration-dependent manner (Fig. 2). Average reproduction for controls was 77 offspring per nematode. Linear regression revealed that the EC₃₀ for Zn ions and ZnONP were 6.5 and 7.5 mg/L, respectively. The ZnONP EC₅₀ was 11.6 mg/L, which is higher than reported by Starnes et al.(Starnes et al., 2019b) (i.e., 1.19 mg/L) and by Gupta et al. (Gupta et al., 2015)(i.e., 0.7 – 1 mg/L, depending on ZnO NP size). However, the differences are likely due to the exposure media (e.g., MHRW supplemented with glucose for this study versus simulated soil pore water or K-medium for other studies), developmental stages (L3 in this study vs L1) and exposure period (24 h in this study vs 48 h).

Figure 2.

Mean total number of offspring produced per adult Caenorhabditis elegans after 24 h exposure to (A) Zn ions or (B) ZnONP in MHRW in the presence of food E. coli OP50 (± 1 SD). Asterisk indicates concentrations significantly different compared to the controls.

Exposure to K. pneumoniae for 24 h decreased reproduction compared to controls (Fig. 3). Interestingly, Interestingly, when exposed to both Zn ions or ZnONP at their respective EC₃₀ and K. pneumoniae, C. elegans reproduction was not different from the control levels (Fig. 4). This result differed from what was observed by Li et al. (2020) in a study with another pathogen and ZnONP, where they found that early exposure to ZnONP made C. elegans more susceptible to P. aeruginosa. However, in that study, nematodes were not exposed to stressors at the same time, instead the nematodes were treated with ZnONP from L1 to adult and after that were exposed to the pathogen. As such, P. aeruginosa was not exposed to Zn in their experiments. Zinc decreases quorum sensing in bacteria (Khan et al., 2020), which might hinder pathogens from colonizing the host intestine (Begun et al., 2007; Kannappan et al., 2019).

Figure 3.

Mean total number of offspring produced per adult C. elegans after 24 h exposure to (A) Zn ions or (B) ZnONP at EC₃₀ concentrations (6.5 mg/L and 7.5 mg/L, respectively) in MHRW and in the presence of E. coli OP50 or K. pneumoniae (± 1 SD).

Figure 4.

Mean surface area of nematodes exposed to Zn ions or ZnONP at EC₃₀ concentrations with or without K. pneumoniae for 24 h. Data are presented with ± 1 SD. Treatments that are significantly different from the control are indicated with an asterisk.

Individual and combined exposures to K. pneumoniae and Zn significantly decreased growth, as measured by surface area, compared to control (Fig. 4). Average area of controls was 0.039 mm². Thus, the combined exposure to K. pneumoniae and Zn in either particulate or ionic forms did not affect growth inhibition, which was observed in the separate exposures. Despite not showing reproductive toxicity under combined exposures to the two stressors, the nematodes still experienced stress as measured by growth inhibition. The decrease in growth with the recovery of reproduction indicates energy allocation from other biological functions, i.e., growth, during exposure towards reproduction to increase population fitness (Álvarez et al., 2005).

3.3. Zinc ions and ZnONP decrease K. pneumoniae colonization

Scanning Electron Microscope analysis of K. pneumoniae revealed that the pathogen forms biofilms in MHRW supplemented with glucose (Fig. 5). After 24 h of static incubation, K. pneumoniae are at the early phase of biofilm formation (Fig. 5A). Exposure to ZnONP (Fig. 5B) or Zn ions (Fig. 5C) does not completely prevent biofilm formation. However, exposure to Zn²⁺ ions caused morphological changes to K. pneumoniae (Fig. 5C). Quantitative assessments showed that Zn²⁺ ions and ZnONP decreased biofilm formation (Fig. 7). Though the conditions of these assessments are not in vivo within the C. elegans gut, they indicate that Zn adversely affects K. pneumoniae biofilm formation capability, with the potential to decrease pathogen colonization of the host intestine.

Figure 5.

Scanning electron micrographs of K. pneumoniae grown on a glass cover slip for 24 h in (A) only MHRW, (B) MHRW supplemented with ZnONP and (C) Zn ions at EC₃₀ concentrations.

Figure 7.

Colony forming units per nematode exposed to K. pneumoniae alone or in combination with, Zn ions or ZnONP for 8 h. Zinc ions and ZnONP were at EC₃₀ concentrations. Data are presented with ± 1 SD. Treatments that are significantly different from the control are indicated with an asterisk

To quantify K. pneumoniae colonization of C. elegans intestine, nematodes were fed K. pneumoniae for 8 h and switched to E. coli OP50 for 16 h. The CFU of nematodes exposed to K. pneumoniae was 80 CFU per nematode (Fig. 7). When C. elegans were exposed to the pathogen and Zn ions or ZnONP at reproductive EC₃₀, CFU per nematode significantly decreased by more than 50%, with the CFU being 29 and 35 respectively. This is an additional line of evidence that adverse effects on K. pneumoniae by Zn treatments occurs externally and internally, after the pathogen is ingested. Elimination of the adverse reproductive effects after the combined exposures to both stressors can be partially attributed to inhibition of K. pneumoniae colonization of the C. elegans intestine. To successfully infect an organism, a pathogen often forms a biofilm. This helps the pathogen to attach to the organism’s intestine and avoid immune responses (Begun et al., 2007; Kannappan et al., 2019). By disrupting this process, Zn prevents K. pneumoniae’s ability to avoid nematode’s immune responses and reduces their pathogenicity. However, the lack of effect of Zn treatments on C. elegans remains to be explained given that there is no difference in dissolution or fractionation of Zn in the exposure solutions. Previous studies showed that, in separate exposures, the effects of ZnONP and K. pneumoniae can be partially explained by their disturbance of the p38 Mitogen–Activated Protein Kinase (MAPK) pathway, a highly evolutionary conserved molecular pathway of the innate immune system (Irazoqui et al., 2010; Kamaladevi and Balamurugan, 2017). The MAPK pathway has been also shown to respond to environmental stress and plays an import role in defense against pathogens in C. elegans (Irazoqui et al., 2010).

Thus, the antagonistic response of the combined exposure of C. elegans to Zn and K. pneumoniae can be also explained by Zn activation of the nematode’s innate immunity. For example, NP can impact the p38 MAPK pathway, a key to host immune functions in C. elegans (Li et al., 2020; Roh et al., 2012) and initiate the nematode’s innate immune response, while decreasing K. pneumoniae’s ability to colonize C. elegans. However, there are some contradictory reports on the effects of ZnONP on MAPK pathway in C. elegans. For instance, Li et all (Li et al., 2020) showed that the mRNA levels of the key genes from this pathway (pmk-1, sek-1, and nsy-1) were significantly decreased by the exposure to ZnONP. This also inhibited activation of the transcription factor SKN-1/Nrf, which was required for the defense against the pathogen in their study, P. aeruginosa. The opposite response was documented in vitro in a study with astrocytes, where activation of phosphatidylinositol 3-kinase (PI3K)/ MAPK and JNK pathways was observed after exposure to ZnONP (Song et al., 2019; Wang et al., 2014). In our previous C. elegans transcriptomic study, there was no effect of ZnONP exposure on suppression or activation of MAPK pathway (Starnes et al., 2019b). In that study, among induced pathways identified were the pathway with the main role in protein synthesis (Aminoacyl-tRNA biosynthesis) and metal detoxification pathway (ABC transporters). The increase in protein misfolding mediated by PI3Kinase/AKT-1/mTOR pathway was detected in C. elegans after K. pneumoniae infection (Kamaladevi and Balamurugan, 2017). The p38 MAPK-dependent activation of the ABC transporter P-glycoprotein pgp-5 has been shown in C. elegans after exposure to a pathogen (Kurz et al., 2007). Thus, complex interplay of different pathways activated in response to Zn might play a role in activation of the defense against K. pneumoniae. The Zn exposure might trigger energy allocation from other biological function, e.g., growth, to reproduction and immune responses, hence why in this study there is a decrease in C. elegans growth compared to controls. Future transcriptomic analyses of these treatments are planned to determine which molecular pathways are involved in the observed antagonistic effects.

Given that all soil organisms will encounter NP alongside other stressors, this study examined interaction of NP and pathogen to determine the potential antagonistic/synergistic effects. This is one of the first studies examining these combined effects which, as we demonstrate, can lead to the antagonistic responses. Further research into these antagonistic effects and their mechanisms might shed light on the interactions of organisms’ innate immunity and toxicant defenses and how those interactions influence population-level impacts in real world environments.

Figure 6.

Inhibition of biofilm formation by zinc ion and ZnONP. Data are presented with ± 1 SD. Treatments that are significantly different from the control are indicated with an asterisk.

Highlights.

• Combined effects of two environmental stressors on a model nematode were assessed

• Individual exposure to ZnONP or K. pneumoniae inhibits C. elegans reproduction

• Combined exposure results in antagonistic effect and restored reproduction

• ZnONP (or Zn ions) inhibit external biofilm formation by K. pneumoniae

• ZnONP (or Zn ions) prevent the ability of the pathogen to colonize C. elegans