3.3 The mechanisms of DA detoxification by the gut microbiota ofA. erythraea
Microorganisms utilize multiple pathways for the biotransformation of DA
(Du et al. 2022; Du et al. 2023; Li et al. 2024a; Li et al. 2024b). In
our results, we observed a significant upregulation of the fatty acid
degradation pathway (map00071) (Fig. 3C; Table S4). Specifically, as the
main process of fatty acid degradation, fatty acid β-oxidation is also
the crucial step in the DA biotransformation pathway (Fig. 4; Pathway 1)proposed by Du et al. (2022). Moreover, the involved genes such
as ACSL , fadI , fadJ , and fadN , were all
significantly upregulated in our findings (p <0.05, Table
S5). To achieve the anaerobic bioconversion of DA to the non-toxic
compound P215-type I (Fig. 4; Pathway 1), additional steps are necessary
beyond β-oxidation, including decarboxylation, dehydrogenation, and
carboxylation (Du et al. 2022). Key genes involved in these processes,
such as panD , adhP , and pyc , were also
significantly upregulated (p <0.05, Table S5). To obtain
these upregulated DEGs taxonomic information, we conducted a BLAST
search of the nucleotide sequences of these DEGs against the Nr database
(Table S5). In this pathway, Neobacillus sp. upregulated thepanD gene, which encodes aspartate 1-decarboxylase and catalyzes
the first decarboxylation reaction in this pathway. In the subsequent
step, Aureispira sp. significantly upregulated the pyc gene, which encodes pyruvate carboxylase and facilitates the
carboxylation of P281. This species is also involved in the β-oxidation
step of P325, upregulating the ACSL , fadI , fadJ ,
and fadN genes, which promote the transformation of P325 to
P215-type I. Additionally, Tenacibaculum sp. ,Pseudoalteromonas sp. , Shewanella sp. , and Vibrio
sp. also participate in the β-oxidation step of this pathway (Fig. 4).
In addition, Li et al. (2024a) proposed that microbial amino acid
metabolism drives the degradation of DA in sediments and suggested
another DA biotransformation pathway (Pathway 2). Our results indicated
that amino acid metabolism pathways, including alanine, aspartate, and
glutamate metabolism (map00250), cysteine and methionine metabolism
(map00270), and valine, leucine, and isoleucine degradation (map00280),
were all significantly induced on the second day. We predict that more
DA will be taken up or accumulated on the second day compared to the
first, and these processes are dose-dependent. Among these pathways, the
genes gadA , fadJ , and fadI , which are involved in
amino acid metabolism, were significantly upregulated
(p <0.05) and also play a role in DA biotransformation.
Furthermore, the genes mqo , putB , and ACSL were
significantly upregulated (p <0.05), contributing
importantly to this DA biotransformation pathway. Ultimately, DA is
converted into P215-type II, a long-chain unsaturated fatty acid, which
is subsequently cleaved into short-chain fatty acids in the TCA cycle
and ultimately mineralized (Li et al. 2024a). In this pathway, along
with the β-oxidation steps involving the genes ACSL , fadJ ,
and fadI , Alteromonas sp. upregulated the gadA gene, which encodes glutamate decarboxylase and facilitates the
decarboxylation of the intermediate product P273.
Lastly, Li et al. (2024b) also described the mechanism of aerobic DA
degradation in Pseudoalteromonas sp. (Pathway 3), identifyingldh , nqrA , and nqrF as key genes, which are
responsible for hydroxylation , decarboxylation and oxidation of
DA and its intermediates. Our results demonstrated a significant
upregulation of nqrA and nqrF (p <0.05). In
this pathway, Pseudoalteromonas sp. , Aureispira sp. ,Vibrio sp. , Tenacibaculum sp. , and other unidentified
bacteria upregulated the nqrA and nqrF genes, which encode
the Na+-translocating NADH-quinone oxidoreductase. The
enzyme catalyzes the production of biogenic
O2·−, facilitating the conversion of
the intermediate P301 to P215-type I.
Notably, rather than a single bacterial group, multiple microorganisms
are involved in all three DA biotransformation pathways. Similarly, Li
et al. (2024a) pointed out that during the DA degradation process in
sediments, the microbial community transitioned from nutritional
competition to metabolic interrelationships, with DA enhancing
cooperation among microbiota. We compared the relative abundance of
these taxa at the genus level based on 16S rRNA analysis. Taxa such asAureispira sp. , Pseudoalteromonas sp. , Tenacibaculum
sp. , Vibrio sp. , and Shewanella sp. exhibited higher
abundance on the DA diet (Fig. 5). This increased relative abundance
suggests that these bacteria have a competitive advantage on the toxic
diet, potentially due to their ability to biotransform DA. Previous
studies have reported that Pseudoalteromonas sp. possesses the
capability to degrade DA (Li et al. 2024b). Additionally, Coral and
Yildirim (2014) found that concentrations of 2.5 µg/ml and 5 µg/ml of DA
induce growth in certain marine Vibrio strains isolated from
Iskenderun Bay and Samandag shore.
These five genera also play significant roles in various steps of both
aerobic and anaerobic DA biotransformation (Fig. 4). Our results
strongly suggest that Aureispira sp. , Tenacibaculum sp. ,Pseudoalteromonas sp. , Shewanella sp. , and Vibrio
sp. are potential detoxifying gut microbiota that interact with one
another to assist A. erythraea in detoxifying DA. This conclusion
is supported by their higher relative abundance in the toxic diet and by
their upregulation of detoxification-related genes.
Besides, we also noted that the drug metabolism - other enzymes pathway
(map00983) was significantly upregulated as a component of the
detoxification process. Within this pathway, three DEGs encoding
glutathione S-transferase (GST) were significantly upregulated. GSTs are
crucial enzymes involved in phase II detoxification of various
xenobiotics, catalyzing the conjugation of reduced glutathione (GSH) to
the electrophilic centers of xenobiotics, thereby probably facilitating
detoxification (Li, Schuler, and Berenbaum 2007). This process may also
contribute to DA detoxification.
During the detoxification of DA, several metabolic processes that
contribute to energy production were significantly enhanced, including
the TCA cycle (map00020), oxidative phosphorylation (map00190),
glycolysis/gluconeogenesis (map00010), and the metabolism of certain
amino acids and fatty acid β-oxidation (Fig. 3C). Key rate-limiting
enzymes of the TCA cycle, such as citrate synthase (K01647), isocitrate
dehydrogenase (K00030 and K00031), and the E1 (K00164) and E2 (K00658)
components of 2-oxoglutarate dehydrogenase, were significantly
upregulated (p<0.05, Table S6). The TCA cycle generates NADH,
which drives ATP production in the oxidative phosphorylation pathway
(Oexle, Gnaiger, and Weiss 1999). Fatty acid β-oxidation is a crucial
metabolic pathway for maintaining energy homeostasis when glucose supply
is limited (Houten and Wanders 2010), and it also provides acetyl-CoA to
the TCA cycle. Additionally, amino acid catabolism can contribute to
energy production, with precursors such as pyruvate and other
intermediates (e.g., acetyl-CoA, succinyl-CoA, and succinate from the
TCA cycle) being replenished through amino acid metabolism. These
results suggest that under the DA diet, microbial energy input is
elevated, likely to meet the demands of DA detoxification or other
defensive mechanisms.
.
Conclusion
This study employed a combined physiological and multi-omics approach,
including amplicon and metatranscriptomic analyses, to explore how gut
microbiota assist copepods in detoxifying DA diet. Our findings indicate
that, although DA diet suppressed the growth of A. erythraea , the
gut microbiota of A. erythraea plays a crucial role in
detoxifying DA, thereby enhancing their survival on a toxic diet. We
observed that the gut microbiota of A. erythraea upregulated
several DA biotransformation genes, including ACSL , fadI ,fadJ , fadN , panD , adhP , pyc ,gadA , mqo , putB , nqrA , and nqrF , when
DA producing PSN was ingested. This upregulation facilitates
three biotransformations of DA, encompassing both anaerobic and aerobic
transformations, ultimately aiding the host copepod in overcoming the
toxic effects of DA. Additionally, we identified five taxa, i.e.,Aureispira sp. , Tenacibaculum sp. , Pseudoalteromonas
sp. , Shewanella sp. , and Vibrio sp. , as potential
detoxification taxa. These taxa not only exhibited higher relative
abundance in the toxic diet but also showed increased expression of DA
biotransformation genes.