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Impact of Aflatoxin B1 on Caenorhabditis elegans Gut Microbiome and Host Resistance



Nematodes are common detritophagous organisms exposed to a wide variety of microbiotas. Interestingly, different species of nematodes are known to acquire different species of bacteria from their habitats. Microbiomes can have a diverse set of functions, including protection against pathogens, metabolic alteration and xenobiotic detoxification. However. effect of gut microbiome is rarely acknowledged in toxicity studies for nematodes. Exposures of N2 strain of Caenorhabditis elegans inoculated with the bacterial isolates from their native habitat, rotten apple compost, over multiple generations have demonstrated that presence of native microbiome increases host resistance to aflatoxin B1 – a potent DNA-damage inducing agent produced by a fungus Aspergillus flavus. Microbiome deficient worms, grown on laboratory OP50 E. coli strain, have significantly more mtDNA lesions than compost-fed worms, especially evident in the first generation likely due to aflatoxin metabolization by a certain bacterial taxon. There also is a response in mitochondrial DNA copy number with worms on average having greater copy number at some aflatoxin concentrations. Compost-fed worms have more unique taxa but their distribution is skewed significantly more to Proteobacteria than in OP50-fed worms, as shown by alpha-diversity and taxonomic analyses, meaning that just a small portion of environmentally available taxa gain competitive advantage in C. elegans gut. Metagenomic sequencing data has shown that with aflatoxin exposure gut microbiome biodiversity is significantly impacted in OP50-fed worms, while in compost-fed nematodes it stays largely the same. OP50-fed worms also exhibit an increase in Actinomycetales, a taxon with multiple aflatoxin-metabolizing members. On the other hand, compost-fed worms showed a decrease in Actinomycetales along with an increase in Flavobacteriales, another potential aflatoxin-metabolizing taxon. Overall, these data suggest that microbiome composition may affect the adverse effects of toxins in C. elegans which is very important since aflatoxin B1 is a very common mycotoxicant and environmental contaminant.
Impact of Aflatoxin B1 on Caenorhabditis elegans Gut
Microbiome and Host Resistance
Sokolskyi, T. 1, Leuthner, T. 1, Meyer, J. 1
1 Nicholas School of Environment, Duke university, Durham, NC, USA 27708
There is very little research done on diverse functions of C. elegans microbiomes
(Zhang et al., 2017). It is particularly interesting whether toxins affect the nematode
gut microbiome, and if the microbiome increases host resistance to toxins. Great
diversity of its composition implies the possibility that some taxa in C. elegans guts are
able to metabolize certain toxins having a host-protective or sensitizing effect. We
exposed the model organism Caenorhabditis elegans grown on native compost isolate
and laboratory OP50 food to Aflatoxin B1 (AB1)-- a potent DNA-damage inducing agent
produced by the fungus Aspergillus flavus to test these interactions.
Introduction Experimental design
Fig. 1. Scheme of the experiment
Native microbiome decreases AB1-induced mtDNA damage
Fig. 2. Level of mtDNA damage in
C. elegans grown on apple
compost isolate and OP50. This
shows that at high concentrations
of AB1 there is less mtDNA
damage in compost-grown worms.
P<0.05 for both generations.
Fig. 3. Relative mtDNA copy
number. We observe a lower
response in compost-grown
worms at least in the first
generation, implying possibility of
other mechanisms of toxin
protection. P<0.05 for gen. 1 and
P>0.05 for gen. 5.
Assay performed according to
Gonzalez-Hunt et al., 2014.
16S rRNA miSeq data highlights selectivity of C. elegans gut
Main take-aways
OP50-fed worms have diverse gut microbiomes, consisting
not just of E. coli. Aligns with data of Lee et al., 2020.
C. elegans gut selects for certain taxa, as evident by
significantly lower diversity of initial gut samples compared
to apple compost samples.
Fig. 4. Microbiome composition of the samples, results of 16S rRNA
gene sequencing. miSeq assay (Caporaso et al., 2012) was
performed in Duke Center for Genomic and Computational Biology.
Fig. 5. Community evenness (Pielou
index) for OP50 and compost-fed
worms. Alpha diversity in compost-fed
worms, albeit lower in control
samples, stays stable with AB1
Fig. 6. Specific changes in community
composition during exposure to 150
µM AB1. Underlined are potential
AB1-metabolizing taxa.
Our data shows at least two taxa with known AB1-
metabolizing species found in 150 µM treatments :
Actinomycetales (Lapalikar et al., 2012) and Flavobacteriales
(Ciegler et al., 1966). Interestingly, Actinomycetales dominate
in OP50-fed worms while Flavobacteriales are present
exclusively in compost-fed worms. Compost-fed worms, on
the other hand, have a greater amount of unique taxa,
however, their distribution is skewed significantly towards
Proteobacteria. Future directions include adding a bacterial
control for aflatoxin metabolism, analyzing differential
expression of detoxification-related genes, and extending the
length of the experiment to observe community changes
over a greater timespan. Overall, our data shows that
microbiomes significantly alter host response to AFB1, a
common soil and food contaminant, and promote host
Caporaso, J. G., Lauber, C. L., Walters, W. A., Berg-Lyons, D., Huntley, J., Fierer, N., ... & Gormley, N. (2012).
Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms. The ISME
journal,6(8), 1621.
Ciegler, A et al. “Microbial detoxification of aflatoxin” Applied microbiology vol. 14,6 (1966): 934-9.
González-Hunt, C. P., Leung, M. C., Bodhicharla, R. K., McKeever, M. G., Arrant, A. E., Margillo, K. M., ... &
Meyer, J. N. (2014). Exposure to mitochondrial genotoxins and dopaminergic neurodegeneration in
Caenorhabditis elegans. PloS one,9(12)
Lapalikar, G. V., Taylor, M. C., Warden, A. C., Scott, C., Russell, R. J., & Oakeshott, J. G. (2012). F420H2-
dependent degradation of aflatoxin and other furanocoumarins is widespread throughout the
Actinomycetales. PLoS One,7(2).
Lee, S., Kim, Y., & Choi, J. (2020). Effect of soil microbial feeding on gut microbiome and cadmium toxicity in
Caenorhabditis elegans. Ecotoxicology and environmental safety,187, 109777.
Zhang, F., Berg, M., Dierking, K., Félix, M. A., Shapira, M., Samuel, B. S., & Schulenburg, H. (2017).
Caenorhabditis elegans as a model for microbiome research. Frontiers in Microbiology,8, 485.
Discussion & Future directions
Possible selection for AB1 metabolism within the gut
Fig. 4
Fig. 5
Fig. 6
Fig. 2
Fig. 3
Fig. 1
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Full-text available
Neurodegeneration has been correlated with mitochondrial DNA (mtDNA) damage and exposure to environmental toxins, but causation is unclear. We investigated the ability of several known environmental genotoxins and neurotoxins to cause mtDNA damage, mtDNA depletion, and neurodegeneration in Caenorhabditis elegans. We found that paraquat, cadmium chloride and aflatoxin B1 caused more mitochondrial than nuclear DNA damage, and paraquat and aflatoxin B1 also caused dopaminergic neurodegeneration. 6-hydroxydopamine (6-OHDA) caused similar levels of mitochondrial and nuclear DNA damage. To further test whether the neurodegeneration could be attributed to the observed mtDNA damage, C. elegans were exposed to repeated low-dose ultraviolet C radiation (UVC) that resulted in persistent mtDNA damage; this exposure also resulted in dopaminergic neurodegeneration. Damage to GABAergic neurons and pharyngeal muscle cells was not detected. We also found that fasting at the first larval stage was protective in dopaminergic neurons against 6-OHDA-induced neurodegeneration. Finally, we found that dopaminergic neurons in C. elegans are capable of regeneration after laser surgery. Our findings are consistent with a causal role for mitochondrial DNA damage in neurodegeneration, but also support non mtDNA-mediated mechanisms.
Full-text available
Two classes of F(420)-dependent reductases (FDR-A and FDR-B) that can reduce aflatoxins and thereby degrade them have previously been isolated from Mycobacterium smegmatis. One class, the FDR-A enzymes, has up to 100 times more activity than the other. F(420) is a cofactor with a low reduction potential that is largely confined to the Actinomycetales and some Archaea and Proteobacteria. We have heterologously expressed ten FDR-A enzymes from diverse Actinomycetales, finding that nine can also use F(420)H(2) to reduce aflatoxin. Thus FDR-As may be responsible for the previously observed degradation of aflatoxin in other Actinomycetales. The one FDR-A enzyme that we found not to reduce aflatoxin belonged to a distinct clade (herein denoted FDR-AA), and our subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates was an α,β-unsaturated lactone moiety. This moiety occurs in a wide variety of otherwise recalcitrant xenobiotics and antibiotics, so the FDR-As and FDR-Bs may have evolved to harness the reducing power of F(420) to metabolise such compounds. Mass spectrometry on the products of the FDR-catalyzed reduction of coumarin and the other furanocoumarins shows their spontaneous hydrolysis to multiple products.
Microbial community of an organism plays an important role on its fitness, including stress responses. In this study, we investigated the effect of the culturable subset of soil microbial community (SMB) on the stress response of the soil nematode Caenorhabditis elegans, upon exposure to one of the major soil contaminants, cadmium (Cd). Life history traits and the stress responses to Cd exposure were compared between SMB- and Escherichia coli strain OP50-fed worms. SMB-fed worms showed higher reproduction rates and longer lifespans. Also, the SMB-fed worms showed more tolerant response to Cd exposure. Gene expression profiling suggested that the chemical stress and immune response of worms were boosted upon SMB feeding. Finally, we investigated C. elegans gut microbial communities in the presence and absence of Cd in OP50- and SMB-fed C. elegans. In the OP50-fed worms, changes in microbial community by Cd exposure was severe, whereas in the SMB-fed worms, it was comparatively weak. Our results suggest that the SMB affects the response of C. elegans to Cd exposure and highlight the importance of the gut microbiome in host stress response.
Yeasts, molds, bacteria, actinomycetes, algae, and fungal spores were screened for their ability to degrade aflatoxin. Some molds and mold spores partially transformed aflatoxin B(1) to new fluorescing compounds. Only one of the bacteria, Flavobacterium (aurantiacum?) NRRL B-184, removed aflatoxin from solution. Both growing and resting cells of B-184 took up toxin irreversibly. Toxin-contaminated milk, oil, peanut butter, peanuts, and corn were completely detoxified, and contaminated soybean was partially detoxified by addition of B-184. Duckling assays showed that detoxification of aflatoxin solutions by B-184 was complete, with no new toxic products being formed.