Analysis of the cadmium removal mechanism of human gut bacteria Enterococcus faecalis strain ATCC19433 from genomic perspective

doi:10.21203/rs.3.rs-3615909/v1

Cadmium (Cd) is one of the most well‒known toxic metals capable of entering the human body via the food chain, leading to serious health problems. Human gut microbes play a pivotal role in controlling Cd bioavailability and toxicity within the human gastrointestinal tract, primarily due to their capacity for Cd adsorption and metabolism. In this work, a Cd resistance bacterial strain, Enterococcus faecalis strain ATCC19433 was isolated from human gut microbiota. Cd binding assays and comprehensive characterization analyses were performed, revealing the ability of strain ATCC19433 to remove Cd from solution. Cd adsorption primarily occurred on the bacterial cell walls, which was ascribed to the exciting of functional groups on the bacterial surfaces, containing alkyl, amide II, and phosphate groups; meanwhile, Cd could enter cells, probably through transport channels or via diffusion. These results indicated that Cd removal by the strain was predominantly dependent on biosorption and bioaccumulation. Whole‒genome sequencing analyses further suggested the probable mechanisms of biosorption and bioaccumulation, including Cd transport by transporter proteins, active efflux of Cd by cadmium efflux pumps, and mitigating oxidative stress induced cell damage by DNA − repair proteases. This study evaluated the Cd removal capability and mechanism of Enterococcus faecalis strain ATCC19433 while annotating the genetic functions related to Cd removal, which may facilitate the development of potential human gut strains for the removal of Cd.

Cadmium

Human gut bacteria

Removal

Enterococcus faecalis strain ATCC19433

Genomics

Cadmium has been extensively employed in various industrial processes and products, including rechargeable batteries, coatings, plating, alloys, biomedical sensing and imaging, stabilizers and nuclear fission manufacturing, due to its nondegradability at high temperature and corrosion resistance [1–3]. However, the extensive application of Cd has led to contamination in water, soil, and crops, resulting in significant risks to agricultural production, food safety, and human health [4, 5]. Cd has high biological toxicity, nonbiodegradable, bioavailable and a potential carcinogen for human body. It can enter human biological systems through food chains [6]. In terms of human health, Cd can participate in biological systemic circulation and accumulate in the kidneys and liver, giving rise to skeletal demineralization, reproductive abnormalities, and organ impairments [7, 8].

As the vital metabolic regulatory system of the human body, the human gut contains numerous gut microbes (at least 10¹⁴ microorganisms and more than 1000 species) with important functions, which play crucial roles in human health [9]. According to studies, gut microbes are important for controlling Cd uptake and diffusion in the human intestinal environment, as they can decrease the bioavailability and bioaccessibility of Cd during its intake from crops and soils [10, 11]. Studying the role of gut microbes in Cd absorption and metabolism could be useful for Cd detoxification and bioremediation [12, 13]. Thus, some specific strains with good Cd resistance capability have been isolated from human gut microbiota, such as Lactobacillus, Bifidobacterium, and Enterococcus species, which can adsorb and remove Cd from foods and water, thereby reducing Cd absorption within the human body [14–16]. These characteristics are governed by pertinent functional genes, encompassing those implicated in metal ion binding, transportation, and efflux, which may be directly or indirectly engaged in Cd removal [12, 17]. Despite these advances in our understanding, there is still a lack of in‒depth research at the genetic and molecular levels on Cd removal by human gut bacteria.

The major bacterial phyla in human gut microbiota include Firmicutes, Bacteroidetes, and Proteobacteria [12]. A diverse range of bacterial genera, such as Clostridium, Bacteroides, Lactobacillus, Bifidobacterium, Enterococcus, and Pseudomonas [13]. The genus Enterococcus are common symbiotic bacteria inhabiting the gastrointestinal tract of healthy humans, with numbers ranging from 10⁵ to 10⁷ bacteria per gram of feces [18, 19]. These bacteria have been identified as crucial contributors to human gut health and are linked to beneficial effects against inflammatory bowel disease and cardiovascular diseases [20, 21]. Motta-Romero et al (2023) [16] found that the Cd‒resistant community of human gut bacteria consisted primarily of Enterococcaceae, which are capable of sequestering Cd. Although the human gut strains isolated from this community exhibited excellent Cd resistance, the capacity of individual strains to bind Cd and the specific mechanisms were found to be limited. This limitation could hinder the development and application of human gut strains for the remediation of Cd toxicity. Therefore, elucidation of the mechanisms employed by specific gut bacteria in Cd binding has crucial theoretical and practical significance in leveraging gut bacteria for the mitigation of Cd toxicity in the human body.

In this study, we found that Enterococcus faecalis strain ATCC19433 is a gut strain with excellent Cd resistance that could remove Cd from solution. However, there has been no research into the Cd removal capacity and mechanisms of Enterococcus faecalis strain ATCC19433. Thus, the purpose of this study were met as follows: (1) A Cd binding assay was conducted to determine the effect of initial Cd concentration, pH value, biosorption time, and inoculum amount on binding ability and to analyze the Cd removal capacity of different cellular components. (2) Characterization analyses were conducted, which included spectrometric techniques (FTIR) and microscopic techniques (SEM‒EDS and TEM‒EDS), to identify the primary form and reaction sites involved in Cd binding. (3) Whole‒genome sequencing analyses were used to determine the genes associated with the Cd binding process and to investigate the corresponding mechanisms.

Experimental strain identification and culture conditions

The experimental strain used in this study was Enterococcus faecalis strain ATCC19433, which was isolated from human feces with excellent Cd resistance and was able to resist nearly 100 mg L^− 1 Cd. The GenBank accession number for the 16S rRNA gene sequence of strain ATCC19433 is OP311607, and a phylogenetic tree of this strain was constructed based on 16S rRNA gene sequences (Figure. S1). The pure culture of the strain was activated in De Man, Rogosa, and Sharpe (MRS) broth medium at 37°C for 24 h and subcultured three times on MRS solid plate medium (Xiya, Shandong, China) before use.

Cadmium binding assay

The Cd binding assay for Enterococcus faecalis strain ATCC19433 was conducted following previously established protocols [22, 23] with certain modifications. Unless otherwise specified, the Cd binding assay was performed with an initial Cd content of 20 mg/L, bacterial content of 1 g/L (wet weight), pH of 7, temperature of 37°C, shaking rate of 150 rpm, and incubation for 120 min. The effect of initial Cd content (1, 5, 10, 20, and 40 mg/L) in solution, pH value (5.0, 6.0, 7.0, 8.0, 9.0), incubation time (30, 60, 90, 120, 180, 240 min), and final bacterial content (1, 2, 5, 10, and 20 g/L) on the Cd binding ability was analysed. After incubation, the supernatant was obtained by centrifugation (4°C, 10000 × g, 10 min) and passed through a 0.22 µm filter. Subsequently, the Cd content in the supernatant was determined using inductively coupled plasma‒optical emission spectrometry (ICP‒OES, ICAP 7400, Thermo Scientific, U.S.). The Cd removal rate of Enterococcus faecalis strain ATCC19433 was calculated according to Eq. (1) [24, 25]:

R = [(C₀ − C₁) / C₀] × 100% (1)

In Eq. (1) R is the removal rate (%); C₀ and C₁ are the initial and equilibrium Cd contents in solution (mg/L), respectively.

Assessment of binding in different cellular parts

In this study, we isolated and investigated two cellular components, namely, the cell wall and protoplasts of Enterococcus faecalis strain ATCC19433 to explore their potential involvement in the Cd binding process. The procedures for preparing the cell walls and protoplasts of the strain were consistent with those used in previous study [25]. The binding tests for different cellular components were conducted under the same conditions as those used in the Cd binding assay for whole cells.

Characterization analyses

Following the Cd binding assay, the biomass was collected through centrifugation at 4°C and 10000 rpm for 10 min. It was then cleansed three times with sterile deionized water before being subjected to characterization analyses targeting Enterococcus faecalis strain ATCC19433.

Scanning electron microscopy with energy dispersive X‒ray (SEM‒EDS) analysis

The harvested bacterial cells were fixed in 2.5% glutaraldehyde in phosphate buffer at pH 7.2 for 24 h and then washed three times with 0.1 M phosphate‒buffered saline (PBS). Subsequently, a dehydration process was carried out using an ethyl alcohol gradient (30%, 50%, 70%, 80%, 90%, and 95%). Next, the cells were subjected to two treatments with isoamyl acetate before being subjected to freeze‒drying and vacuum spraying with gold. Finally, scanning electron microscopy coupled with energy dispersive X‒ray spectroscopy (SEM‒EDS, Zeiss Gemini 300, Germany) was employed to analyse the cellular morphology and elemental composition in specific regions.

Transmission electron microscopy with energy dispersive X‒ray spectroscopy (TEM‒EDS) analysis

The collected cells were subsequently postfixed in a 2.5% glutaraldehyde solution in phosphate buffer for 24 h, followed by three washes with PBS. Subsequently, the cells were fixed with 1% osmium tetroxide in phosphate buffer for 2 h, dehydrated using ethyl alcohol, and embedded in ERL resin. The samples were subjected to ultrathin sectioning (70‒90 nm) using an ultramicrotome, followed by staining with lead citrate and a 50% uranyl acetate solution in ethanol for 5‒10 min each. Finally, the cellular structures were examined using transmission electron microscopy with energy dispersive X‒ray spectroscopy (TEM‒EDS, HITACHI HT7800, Japan).

Fourier transform infrared (FTIR) spectroscopy

The dried cell samples were combined with KBr at a ratio of 1:100 in an agate mortar [26]. Subsequently, the mixture was ground, and a specific amount was pressed into a thick disk. The samples were then scanned by an FTIR spectrometer (FTIR‒850, Gangdong, China) under the same testing conditions, with a spectral range from 4000‒400 cm^− 1, a spectral resolution of 2 cm^− 1, and 40 scans.

Whole − genome sequencing analysis of Enterococcus faecalis strain ATCC19433

DNA extraction

The genomic DNA of Enterococcus faecalis strain ATCC19433 was extracted utilizing a Tiangen Bacterial Genomic DNA Extraction Kit (Tiangen Biotech) following the manufacturer’s instructions. The purified genomic DNA was quantified with a TBS‒380 fluorometer (Turner BioSystems Inc., Sunnyvale, CA). Only DNA samples meeting high‒quality criteria, including no degradation, OD260/280 = 1.8 ~ 2.0, and a quantity of > 1 µg were utilized for subsequent analyses.

Library construction and genome sequencing

Whole‒genome sequence analysis of Enterococcus faecalis strain ATCC19433 was conducted utilizing the PacBio RS II Single Molecule Real Time (SMRT) and Illumina sequencing platforms, carried out by Majorbio Co. in Shanghai, China. A PacBio library with an average insert size of ~ 10 kb was constructed, followed by high‒throughput sequencing.

Genome assembly and functional annotation

The original sequencing data from PacBio were assembled using HGAP and Canu algorithms [27], resulting in a complete genome with continuous chromosomes and plasmids. For the prediction of bacterial genetics, Glimmer 3.02 software was employed for functional annotation [28]. The coding DNA sequences (CdSs) were subsequently analysed through BLAST against several databases, including the NCBI nonredundant (NR), Gene Ontology (GO), Clusters of Orthologous Groups (COG), and Carbohydrate‒Active enzymes (CAZy) databases to acquire the corresponding functional annotations.

Statistical analysis of data

All assays were conducted in triplicate, and the results are presented as the mean ± standard deviation (SD). Details of the software programs employed for genome sequencing and analysis can be found in Table S1.

Effects of Cd concentration, pH, biomass dosage, and incubation time on the capacity of Enterococcus faecalis strain ATCC19433 to bind Cd

The metal binding ability of microbes is typically associated with the initial concentration of metal ions in liquid [23]. As depicted in Fig. 1A, the Cd²⁺ binding capacity of Enterococcus faecalis strain ATCC19433 increased with increasing initial Cd²⁺ concentration from 5 to 20 mg/L. The peak adsorption rate reached 48.48% ± 1.03% at a Cd²⁺ concentration of 20 mg/L, whereas above 20 mg/L, the decreased rapidly. The results indicate that the active binding sites on the cells may reach saturation at an initial Cd²⁺ concentration of 20 mg/L. The effect of pH on the binding of Cd by Enterococcus faecalis strain ATCC19433 is depicted in Fig. 1B. As the pH increased from 5.0 to 9.0, the adsorption rate initially increased before decreasing, with a notable drop occurring at pH values between 8.0 and 9.0. At low pH, hydrogen and hydronium ions compete with Cd ions for binding sites [29]. As the pH continuously increases, the precipitation of Cd hydroxides and Cd − ligand complexes might occur, resulting in a reduced Cd adsorption rate [30]. Thus, a pH of 8.0 was more suitable for Cd adsorption by Enterococcus faecalis strain ATCC19433.

The effect of biomass dosage on the Cd binding capacity of Enterococcus faecalis strain ATCC19433 is illustrated in Fig. 1C. As the biomass dose increased from 1 g/L to 50 g/L, the Cd adsorption rate of strain ATCC19433 increased from 52.91–73.46%. The surface area of adsorption increased along with the amount of biomass, which led to the binding sites also increasing and benefiting Cd adsorption by bacterial cells [25]. As shown in Fig. 1D, the Cd binding capacity of Enterococcus faecalis strain ATCC19433 initially increased and then exhibited a slight decrease with the adsorption time extending from 0 min to 240 min. Because of the availability of unsaturated active sites, the cells adsorbed Cd quickly within the first 120 min. The Cd adsorption rate declined when the adsorption time was above 120 min, which might be due to the saturation of adsorption sites or the cells entering decline phases and beginning to autolyze with prolonged incubation time [23, 31]. Therefore, the adsorption equilibrium time for Cd by Enterococcus faecalis strain ATCC19433 was 120 min.

Cd binding capacities of different cell parts

The distribution of adsorbed Cd in different cellular components is shown in Fig. 2. The Cd adsorption rate of cell walls (33.29 ± 1.08%) was significantly higher (P < 0.05) than that of protoplasts (8.24 ± 0.46%) constituting 72.35% of the total cellular Cd adsorption (46.06 ± 1.43%) when cells were exposed to Cd. This result indicated that Cd is primarily adsorbed by the cell walls, with only small amounts of Cd binding in the protoplasts. Similar accumulation of Cd in cells was also reported in other bacterial strains, such as Lactobacillus plantarum CCFM8661, W.viridescens ZY‒6, and Pseudomonas fluorescens H2 [25, 32, 33]. On the one hand, the cell walls represent the primary interface where bacterial cells directly interact with the extracellular environment [34]. They predominantly consist of teichoic acid, peptidoglycans, polysaccharides, and proteins, which contain a substantial amount of active negatively charged groups, such as carboxyl groups, hydroxyl groups, and phosphate groups; therefore, positively charged Cd ions can bind with these negatively charged groups on cell walls through adsorption, electrostatic attraction, complexation, and precipitation reactions [35, 36]. On the other hand, Cd ions bound in bacterial cell walls and cell surfaces can enter cells through transport channels or facilitate diffusion and become bound in bacterial protoplasts [25, 37].

Characterization analyses

SEM‒EDS and TEM‒EDS imaging analyses

SEM‒EDS was employed to examine the detailed morphological information and surface element composition of Enterococcus faecalis strain ATCC19433 before and after Cd exposure (Fig. 3). The SEM images are shown in Fig. 3A, cells incubated without Cd exposure displayed intact cellular morphology, smooth surfaces, and clear contours. However, cells incubated in the presence 20 mg/L Cd exhibited pits, wrinkles, adhesion and were covered with a small amount of tiny white crystals (Fig. 3B). The results of EDS spectrum analysis are shown in Fig. 3C and D, comparative analysis showed that control group had no Cd peak. Two obvious Cd absorption peaks at 3.16 keV and 3.28 keV under 20 mg/L Cd stress, confirmed the binding of Cd ions by Enterococcus faecalis strain ATCC19433. Interestingly, the element absorption peak of Na at 1.04 keV and N at 0.39 keV decreased after Cd binding. This result indicated that Cd binding by Enterococcus faecalis strain ATCC19433 is associated with an ion exchange reaction, where Cd ions interact competitively with negatively charged functional groups that participate in ion exchange processes [38]. Furthermore, the element absorption peaks of O (0.52 keV) and P (2.13 keV) increased after Cd exposure, indicating the potential formation of Cd‒containing oxide and phosphate precipitate on the cell surfaces, which might be another Cd binding process [39, 40]. Hence, the presence of Na, N, O, and P within cell surface structures might play important role in Cd binding.

TEM‒EDS was used to further study the ultrastructure of Enterococcus faecalis strain ATCC19433 cells that were exposure to 20 mg/L Cd. The TEM images are shown in Fig. 4a and b. Many irregular black floccules were observed on the cell surface and intracellular, whereas no such particles were visible in the control group. This observation could be attributed to the accumulation of metal complexes in the form of granules following Cd binding [41]. In the EDS analysis, no Cd signals could be found in untreated group (Fig. 4c); however, two Cd absorption peaks were evident in the Cd‒treated group (Fig. 4d), and the levels of O and P were increased in cells under Cd stress, which was same with the SEM‒EDS results. Moreover, Cd appeared in the elemental mapping results, confirming that some amount of Cd bound intracellularly, which might explain the results of the Cd binding assays of different cell parts of Enterococcus faecalis strain ATCC19433, in which Cd was detected on the protoplasts.

Cell morphology characteristics reflect the interactions between heavy metals and microorganisms to a certain extent [42]. Hence, the changes in cell morphology and ultrastructure prior and following exposure to heavy metals may partially clarify the function driving heavy metal binding by bacterial cells [43, 44]. Electron microscopy analysis revealed that Enterococcus faecalis strain ATCC19433 cells were unable to maintain normal morphology and structure under Cd stress. This may be attributed to Cd deposition in cells, which alters membrane charge and permeability, consequently affecting cell morphology and structure [41]. Furthermore, EDS analysis revealed the accumulation of Cd on the cell surface and in the intracellular region, indicating the presence of specific Cd binding sites on both the cell surface and within the cells [45]. Based on these analyses, the Cd binding by the strain can be explained by two main processes: biosorption, a passive non‒metabolism‒independent process in which Cd can be adsorbed onto the bacterial cell surface and cell walls; bio‒accumulation, an active metabolism‒independent process following biosorption in which bound Cd enters and accumulates in bacterial cells during metabolism [38, 46]. Hence, it could be confirmed that Cd binding by Enterococcus faecalis strain ATCC19433 primarily occurs through biosorption and bioaccumulation.

FTIR spectral analysis

To further identify what functional groups were mainly involved in Cd binding, infrared spectroscopic analysis of Enterococcus faecalis strain ATCC19433 was conducted (Fig. 5). Distinct absorption peaks were observed at 3449.09 cm^− 1, 2921.65 cm^− 1, 1637.28 cm^− 1, 1560.14 cm^− 1, 1384.65 cm^− 1, and 1073.20 cm^− 1, indicating that the bacterial cell surface primarily consists of hydroxyl groups, alkyl groups, amide I groups, amide II (N − H/C − N) groups, carboxyl groups, and phosphate groups [47, 48]. Following Cd treatment, the original absorption peaks at 2921.65 cm^− 1 and 1560.14 cm^− 1, signifying alkyl (− CH) groups and amide II (N − H/C − N) groups, were approximately shifted by 13 cm^− 1 and 15 cm^− 1, respectively. This is likely attributed to the binding of − CH, N − H, and C − N groups with Cd through a complexation reaction [25, 26]. The peak for phosphate groups at 1073.20 cm^− 1 shifted right by nearly 9 cm^− 1 after Cd exposure, which indicated the participation of phosphate groups in Cd binding [49]. Moreover, the absorption peaks at 1384.65 cm^− 1, 1637.28 cm^− 1, and 3449.09 cm^− 1 exhibited a slight rightward shift. Therefore, the FTIR analysis confirmed the presence of functional groups on the cell surface of Enterococcus faecalis strain ATCC19433, such as alkyl, amide II, and phosphate groups might participate in Cd binding.

Genome sequencing and analyses of Enterococcus faecalis strain ATCC19433

Genome properties

The genome of Enterococcus faecalis strain ATCC19433 generated 400,842 high − quality PacBio reads, averaging 10,166.29 bp in length, which were assembled into a single scaffold, resulting in a circular chromosome of 2,778,926 bp with a G + C content of 33.56% (Figure. 6). The genome contains 2,522 annotated genes (CdSs) with specific physiological functions, accounting for 88.27% of the entire genome, as annotated in various databases (Table S2). The circular genome map of Enterococcus faecalis strain ATCC19433 provides an overview of key genomic features, including the distribution of genes on both the positive and antisense strands, COG functional annotation of genes, G + C content, homologous genes, and files for rRNA and tRNA, which could support research on the mechanism of Cd resistance and binding [50].

Annotation and analysis of functional genes in the genome of Enterococcus faecalis strain ATCC19433

Following the sequencing the complete genome of Enterococcus faecalis strain ATCC19433, the gene numbers were annotated in different databases as follows: NR (2520), COGs (2135), GO (1782), and CAZy (92). According to comparison with NR database (Table S3), the genes Gene1233 and Gene2013 were annotated as a “cadmium‒translocating P‒type ATPase”, indicating the presence of specific Cd ion transporters in Enterococcus faecalis strain ATCC19433 [51]. Additionally, several genes were annotated as “heavy metal‒binding domain‒containing protein”, “metal ABC transporter permease”, “metal ABC transporter ATP‒binding protein”, “heavy metal translocating P‒type ATPase”, and “metal − dependent transcriptional regulator”. These annotations suggest that Enterococcus faecalis strain ATCC19433 may respond to metal ions to bind and regulate transcription, providing crucial factors for its ability to remove metal ions from liquids [52–54].

According to the GO database, genes in Enterococcus faecalis strain ATCC19433 were categorized into three primary groups: “Biological Process (913 genes)”, “Cellular Component (923 genes)”, and “Molecular Function (1432 genes)” (Fig. 7). The top subcategories within each major category in the GO database can be divided into three sections. (Ⅰ) Among these, “regulation of transcription, DNA‒templated” (GO: 0006355, 52 genes, 2.06% representation) and “transmembrane transport” (GO: 0055085, 43 genes, 1.7% representation) were ranked third and fourth in the biological process category, respectively, which might be related to the regulation of metal ion adsorption and expulsion [48]. This might explain the results of characterization analyses showing the presence of Cd on the cell wall and intracellular in Enterococcus faecalis strain ATCC19433. (ⅠⅠ) In the category of cellular component, genes with high values were observed in “integral component of membrane” (GO: 0016021, 521 genes, 20.66% representation), “cytoplasm” (GO: 0005737, 246 genes, 9.75% representation), and “plasma membrane” (GO: 0005886, 210 genes, 8.33% representation), which suggested the involvement of various cellular components in biofilm formation in strain ATCC19433. It has been documented that biofilm formation plays a pivotal role in mitigating the cellular damage caused by heavy metal stress [17, 48]. This observation might explain why strain ATCC19433 can form biofilms to resist Cd ions when exposed to Cd. (ⅠⅠⅠ) In the molecular function category, genes exhibiting high values were observed in “ATP binding” (GO: 0005524, 259 genes, 10.27% representation), indicating that Enterococcus faecalis strain ATCC19433 possesses an adequate energy source for various syntheses and metabolic reactions. Furthermore, “Metal ion binding” (GO: 0046872, 120 genes, 4.76% representation) was ranked third in the molecular function category, which suggested that Enterococcus faecalis strain ATCC19433 has molecular mechanisms for binding metal ions.

The genome of Enterococcus faecalis strain ATCC19433 was subjected to functional annotation analysis by comparison with the COG database, as shown in Figure. 8. Among the annotated genes, the highest number was observed in Category G, which is related to carbohydrate transport and metabolism. There were 259 genes in this category, accounting for 11.04% of the total annotated genes. These genes play a crucial role in maintaining stable energy metabolism and enabling the strain to adapt to varying environmental conditions [17]. Within the COG type functional annotation category P, “inorganic ion transport and metabolism” (5.38%), there were 115 genes associated with metal ion transport and metabolism (Table S4), such as COG0053 encoding “Divalent metal cation efflux pump” and COG2217 encoding “Cation‒transporting P‒type ATPase”. In addition, numerous genes were identified encoding proteins associated with the “ABC‒type ions transporter” in Enterococcus faecalis strain ATCC19433, capable of utilizing energy produced through ATP hydrolysis to facilitate the translocation of metal ions (in free and/or complex forms) or other substrates across the bacterial plasma membrane via conformational alterations [55–57].

Analysis of Cd binding‒related genes

Through the genome annotation analyses detailed above for Enterococcus faecalis strain ATCC19433, we identified numerous functional genes associated with heavy metals that were considered to play both direct and indirect roles in Cd binding (Table 1). Transport proteins, as membrane proteins, facilitate chemical conversion and signal exchange within and outside biofilms, including Cd adsorption and expulsion from cells [58]. In Enterococcus faecalis strain ATCC19433, the existence of two cadmium‒translocating P‒type ATPases indicates that the strain possesses specific Cd transporters capable of facilitating the translocation of Cd ions across the cellular plasma membrane [51]. Efflux systems serve as a well‒established mechanism for resisting Cd, as they facilitate the extracellular transport of Cd from both the cytoplasm and cell membrane through divalent metal cation efflux pumps, thereby reducing its accumulation in the cell and enhancing bacterial Cd resistance [17, 58]. The presence of two divalent metal cation (Fe/Co/Zn/Cd) efflux pump genes were found in Enterococcus faecalis strain ATCC19433, which could be triggered when metal Cd ions enter cells [59].

In addition, the intracellular oxidative stress induced by Cd exposure leads to cellular damage [60], and gene‒encoded enzymes in Enterococcus faecalis strain ATCC19433, including glutathione synthetase, superoxide dismutase, and catalase, may participate in reducing oxidative stress [58, 61, 62]. Moreover, Cd exposure induces oxidative stress and further leads to DNA damage. Under Cd stress, this prompts the activation of genes responsible for DNA and protein repair, which is crucial for sustaining cellular function [58]. In Enterococcus faecalis strain ATCC19433, the DNA mismatch repair proteins MutL and MutS, the DNA repair proteins RecF, RecN, RecO, RadA, and RadC, and the DNA repair exonuclease SbcD may contribute to reducing the lethal effect and increasing Cd resistance [48, 63]. From this, it was inferred that when Enterococcus faecalis strain ATCC19433 is exposed to Cd, relevant functional genes are involved in Cd ion transport and efflux and DNA repair, which can protect bacterial cells and reduce the heavy metal content in the liquid phase simultaneously [51, 64, 65].

A considerable number of genes associated with carbohydrate metabolism were found in Enterococcus faecalis strain ATCC19433. According to the CAZy database, a total of 92 CAZyme coding genes were identified, including glycosyl hydrolases (GHs, 51.09%), glycoside transferases (GTs, 21.73%), carbohydrate esterases (CEs, 19.57%), auxiliary activities (AAs, 0.07%), and polysaccharide lyases (PLs, 0.01%) (Figure. S2), showing that there is enough energy to support the regular growth and metabolism of Enterococcus faecalis strain ATCC19433 to face the Cd exposure [17, 66]. Furthermore, according to other annotation information for Enterococcus faecalis strain ATCC19433 genome, it exhibits excellent resistance not only to Cd but also to other metal ions, including Zn²⁺, Fe²⁺, Mn²⁺, Mg²⁺ and Co²⁺ (Table 1). This suggests its potential application in the bioremediation of heavy metals.

This study demonstrated that human gut bacterium Enterococcus faecalis strain ATCC19433 has the ability to remove Cd from aqueous solution. This capability is affected by the initial Cd content, biomass dosage, incubation time, and pH. Characterization analyses revealed notable changes in the morphology of cells exposed to Cd. Since, Cd accumulates both on the cell surface and within the cell, biosorption on the cell surface and intracellular bioaccumulation were involved. Genomic analysis showed that multiple genes were involved in Cd resistance and binding, and Cd removal was not the result of a single gene. In conclusion, the probable response mechanism to Cd exposure unfolds as follows: the active sites on the cell walls and cell surface, such as alkyl, amide II, and phosphate groups, bind to Cd; several genes encoding metal ion transport proteins may jointly drive enzymatic translocation into the intracellular region; the gene encoding divalent metal cation (Fe/Co/Zn/Cd) efflux pump may cause Cd efflux to the extracellular space, lowing direct damage; and several genes encoding DNA repair proteins likely decrease oxidative damage to genetic material induced by ROS. The results of this study initially revealed the Cd binding mechanism of Enterococcus faecalis strain ATCC19433 at the genetic and molecular levels, which may provide effective strategies for regulating specific human gut bacteria for the treatment of Cd poisoning in humans. How to achieve this in practical applications needs further study.

COMPETING INTERESTS

The authors declare that they have no known competing financial interests or personal relationships.

FUNDING

This work was supported by the National Natural Science Foundation of China (52160015), Guizhou Provincial Science and Technology Development Project (QKZYD [2022]4022), Guizhou Hundred Level Talents Project (Guizhou Kehe Platform Talents [2020]6002) and the Guizhou Provincial Science and Technology Projects, No.[2020]-046.

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Table.1. Genes possibly involved in heavy metals removal in the genome of Enterococcus faecalis strain ATCC19433.

Categorie	Gene ID	Gene name	Annotation information	Reference strain
Metal transporter	gene1233 gene2013	zntA	cadmium-translocating P-type ATPase	Bacillus subtilis subsp. subtilis str. 168
	gene0237 gene0635	-	Divalent metal cation (Fe/Co/Zn/Cd) efflux pump	-
	gene0052	znuA	metal ABC transporter substrate-binding protein	Streptococcus pyogenes MGAS8232
	gene0053	znuC	metal ABC transporter ATP-binding protein	Streptococcus pneumoniae R6
	gene0054	znuB	metal ABC transporter permease	Bacillus subtilis subsp. subtilis str. 168
	gene0098	-	metalloregulator ArsR/SmtB family transcription factor	Methanocaldococcus jannaschii DSM 2661
	gene0174	-	heavy metal-binding domain-containing protein	Enterococcus faecalis V583
	gene0195	ftsH	ATP-dependent zinc metalloprotease FtsH	Streptococcus pneumoniae TIGR4
	gene0255	copA	heavy metal translocating P-type ATPase	Staphylococcus epidermidis ATCC 12228
	gene0536	zntA	heavy metal translocating P-type ATPase	Bacillus subtilis subsp. subtilis str. 168
	gene0773	troR	metal-dependent transcriptional regulator	Mycobacterium tuberculosis H37Rv
	gene1362	cpdA	metallophosphoesterase	Rhodococcus erythropolis PR4
	gene1599	mntH	divalent metal cation transporter	Latilactobacillus sakei subsp. sakei 23K
	gene1693	mntA	metal ABC transporter ATP-binding protein	Listeria monocytogenes EGD-e
	gene1694	mntB	metal ABC transporter permease	Listeria innocua Clip11262
	gene1695	mntC	metal ABC transporter substrate-binding protein	Streptococcus parasanguinis
	gene1761	-	zinc metallopeptidase	Bacillus subtilis subsp. subtilis str. 168
	gene1827	rseP	RIP metalloprotease RseP	Enterococcus faecalis V583
	gene1859	czrA	metalloregulator ArsR/SmtB family transcription factor	Bacillus subtilis subsp. subtilis str. 168
	gene1868	gloB	MBL fold metallo-hydrolase	Streptococcus pyogenes MGAS10394
	gene2478	znuA	ZinT/AdcA family metal-binding protein	Streptococcus pneumoniae R6
DNA repair enzymes	gene2313	-	DinB family glutathione transferase	-
	gene2369	gshA	bifunctional glutamate--cysteine ligase/glutathione synthetase	Enterococcus faecalis V583
	gene2524	gor	Angstrom Resolution Crystal Structure of Glutathione Reductase from Enterococcus faecalis in Complex with FAD	Streptococcus thermophilus
	gene0356	-	superoxide dismutase	Enterococcus faecalis V583
	gene1352	katE	catalase	Bacillus subtilis subsp. subtilis str. 168
	gene2440	MutL	DNA mismatch repair endonuclease MutL	Lactiplantibacillus plantarum WCFS1
	gene2441	MutS	DNA mismatch repair protein MutS	Enterococcus faecalis V583
	gene0004	recF	DNA replication/repair protein RecF	Enterococcus faecalis V583
	gene0040	radA	DNA repair protein RadA	Streptococcus pyogenes MGAS8232
	gene0752	recN	DNA repair protein RecN	Alkalihalobacillus halodurans C-125
	gene1846	recO	DNA repair protein RecO	Enterococcus faecalis V583
	gene2240	radC	DNA repair protein RadC	Enterococcus faecalis V583
	gene0448	sbcD	DNA repair exonuclease	Bacillus subtilis subsp. subtilis str. 168

No competing interests reported.

SupplementaryMaterial.docx

Analysis of the cadmium removal mechanism of human gut bacteria Enterococcus faecalis strain ATCC19433 from genomic perspective

Status:

Journal Publication

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Experimental strain identification and culture conditions

Cadmium binding assay

Assessment of binding in different cellular parts

Characterization analyses

Scanning electron microscopy with energy dispersive X‒ray (SEM‒EDS) analysis

Transmission electron microscopy with energy dispersive X‒ray spectroscopy (TEM‒EDS) analysis

Fourier transform infrared (FTIR) spectroscopy

Whole − genome sequencing analysis of Enterococcus faecalis strain ATCC19433

DNA extraction

Library construction and genome sequencing

Genome assembly and functional annotation

Statistical analysis of data

RESULTS AND DISCUSSION

Cd binding capacities of different cell parts

Characterization analyses

SEM‒EDS and TEM‒EDS imaging analyses

FTIR spectral analysis

Genome properties

Annotation and analysis of functional genes in the genome of Enterococcus faecalis strain ATCC19433

CONCLUSION

Declarations

References

Tables

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1