Compositions and methods for preventing adverse effects of methylmercury exposure

Abstract

Provided herein are bacteria comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid; compositions comprising said bacteria; and methods of using said bacteria.

Description

[0001] Attorney Docket No. : UCH-42725

[0002] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0003] COMPOSITIONS AND METHODS FOR PREVENTING ADVERSE EFFECTS OF METHYLMERCURY EXPOSURE

[0004] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No.

[0005] 63 / 739,748, filed December 30, 2024, which is incorporated by reference herein in its entirety.

[0006] BACKGROUND

[0007] Anthropogenic mercury releases have significantly increased the environmental burden of its organic neurotoxic form methylmercury (MeHg). Volatile elemental mercury (Hg°) emitted into the atmosphere from natural sources and human activities oxidizes into divalent mercury (Hg2+), which is deposited in aquatic environments where microbial processes convert it to MeHg. This transformation enables MeHg to enter and accumulate in food webs, biomagnifying to high levels especially in top predators consumed as seafood.

[0008] As a result, human consumption of seafood MeHg poses substantial health risks, particularly for vulnerable groups like pregnant women and children. MeHg accumulates in neural and fetal tissues by binding thiol groups in cysteine and mimicking methionine to cross the blood-brain barrier and placental barrier. MeHg disrupts the cellular redox balance maintained by selenoproteins, promotes oxidative stress, and impairs neurotransmission, ultimately causing long-term neurocognitive and metabolic deficits in adults and children. Despite initiatives to reduce anthropogenic mercury emissions, global exposure to dietary MeHg continues to rise in prevalence, exacerbating risk for neurological and metabolic diseases.

[0009] Current therapies for MeHg toxicity rely on thiol-based chelation agents that have mixed efficacy for lowering tissue MeHg, especially if not administered immediately after exposure. They non-specifically scavenge metal ions, including essential metals such as copper and zinc, potentially eliciting long term physiological and neurocognitive effects. As such, long-term usage of metal chelators in absence of heavy metal poisoning is not recommended, precluding their ability to treat chronic exposures to MeHg. Additionally, strategies utilized to date have not specifically targeted MeHg already present in food, but rather were designed to sequester inorganic mercury, which is poorly absorbed and less toxic than MeHg. Safer, more targeted interventions are needed to reduce health risks caused by chronic exposure to MeHg.Attorney Docket No. : UCH-42725

[0010] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0011] SUMMARY

[0012] In some aspects, provided herein are bacteria comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid. In some embodiments, the bacterium comprises an additional gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid. In some embodiments, the gene or the additional gene encoding a methylmercurydegrading enzyme encodes a mercuric reductase (MerA) (e.g., Pseudomonas aeruginosa MerA).

[0013] In some embodiments, the gene or the additional gene encoding a methylmercurydegrading enzyme encodes an organomercury lyase (MerB) (e.g., Pseudomonas aeruginosa MerB). In some embodiments, the bacterium is of the Bacteroides genus (e.g., Bacteroides thetaiotaomicron). In some embodiments, the mercuric reductase has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to Pseudomonas aeruginosa MerA In some embodiments, the mercuric reductase is Pseudomonas aeruginosa MerA.

[0014] In some embodiments, the gene or the additional gene encoding a methylmercurydegrading enzyme encodes an organomercury lyase (MerB). In some embodiments, the organomercury lyase has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to Pseudomonas aeruginosa MerB. In some embodiments, the bacterium is of the Bacteroides genus. In some embodiments, the bacterium is of the species Bacteroides thetaiotaomicron. In some embodiments, the bacterium is of the strain Bacteroides thetaiotaomicron VPI-5482. In other aspects, provided herein are methods comprising administering to a subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid. In some embodiments, the subject has been exposed to mercury and / or methylmercury (e.g., as a result of diet or water supply).

[0015] In some embodiments, the method comprises detoxifying or demethylating mercury and / or methylmercury in the gastrointestinal tract of the subject. In some embodiments, the method comprises preventing mercury and / or methylmercury accumulation in the subject. In some embodiments, the method comprises preventing mercury and / or methylmercury accumulation in the liver of the subject. In some embodiments, the method comprises reducing mercury and / or methylmercury in the subject. In some embodiments, the method comprises preventing mercury and / or methylmercury accumulation in the liver of a subject.Attorney Docket No. : UCH-42725

[0016] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0017] In some embodiments, the method comprises treating the subject, wherein the subject has mercury and / or methylmercury accumulation. In some embodiments, the method comprises treating or preventing brain damage due to mercury and / or methylmercury exposure in the subject. In some embodiments, the method comprises treating or preventing cognitive impairment after mercury and / or methylmercury exposure in the subject. In some embodiments, the method comprises treating or preventing cognitive impairment due to mercury and / or methylmercury exposure in the subject. In some embodiments, the method comprises treating mercury and / or methylmercury poisoning in a subject. In some embodiments, the method comprises treating or preventing brain damage due to mercury and / or methylmercury poisoning in a subject. In some embodiments, the method comprises treating or preventing cognitive impairment due to mercury and / or methylmercury poisoning in a subject. In some embodiments, the method comprises promoting healthy neural development in a fetus carried by the subject. In some embodiments, the subject is an adult. In some embodiments, the subject is gestating a fetus. In some embodiments, the method comprises of preventing impaired neural development in a fetus carried by the subject. In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is a fetus.

[0018] In some embodiments, the method comprises promoting healthy neural development in a fetus. In some embodiments, the method comprises preventing impaired neural development in a fetus. In some embodiments, the subject has been exposed to mercury and / or methylmercury. In some embodiments, the subject has been exposed to mercury and / or methylmercury in their diet (e.g., high-mercury fish). In some embodiments, the subject has been exposed to mercury and / or methylmercury in their environment (e.g., water supply).

[0019] In some embodiments, the composition is formulated for oral delivery. In some embodiments, the composition is a food product. In some embodiments, the food product is a dairy product. In some embodiments, the food product is yogurt. In some embodiments, the composition is formulated for rectal delivery. In some embodiments, the composition is for self-administration. In some embodiments, the subject is a human. In some embodiments, healthy neural development comprises a reduction in anxiety-like behaviors. In some embodiments, healthy neural development comprises prevention of learning and memory deficits.Attorney Docket No. : UCH-42725

[0020] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0021] BRIEF DESCRIPTION OF THE FIGS.

[0022] FIGs. 1A-1G show engineered Bacteroides thetaiotaomicron (BtmerA / B) demethylates MeHg-Cl and dietary methylmercury in vitro and in intestinal contents of monocolonized mice. FIG. 1A shows a schematic of the mechanism of action and cloning strategy for MeHg detoxification enzymes mercuric reductase (MerA) and organomercurial lyase (MerB). MerB 2+

[0023] demethylates MeHg into divalent mercury (Hg ), which is reduced by MerA to poorly absorbed elemental mercury (Hg°). MerB and merA, under control of the strong constitutive promoter pBfPlE6, were integrated into the B. thetaiotaomicron genome between putative genes Bt3575-Bt3576 and NBU2 insertion sites respectively. FIG. IB shows a growth curve showing minimal growth defects of Blmer Awith overexpression of mercury detoxification genes. (n=3 biological replicates / condition). FIG. 1C shows the reduction of MeHg by BtmerA / B compared with wild-type Bt in minimal media supplemented with MeHg-Cl (starting concentration 3.83 ± 0.13 ng Hg / mL). Area under the curve and rate of demethylation was calculated from 0 to 10 hours. (n=3 biological replicates / condition, unpaired / -test). FIG. ID shows the reduction of diet-derived MeHg by Btme'11compared with wild-type Bt in minimal media supplemented with digested bluefin tuna (starting MeHg concentration = 2.21 ± 0.17 ng Hg / mL). Area under the curve and rate of demethylation was calculated from 0 to 12 hours. (n=3 biological replicates / condition, unpaired / -test). FIG. IE shows a schematic of in vivo reduction of MeHg in fecal samples of mice colonized with Blmer Aover 24 hours.

[0024] Germ-free mice were monocolonized with Bt or BerA / Bfor 3 days, and then gavaged once with MeHg-Cl (250 pg / kg). FIG. IF shows the reduction of MeHg in fecal samples from monocolonized mice orally gavaged with MeHg-Cl (250 pg / kg) at time 0. Area under the curve was calculated from 0 to 24 hours. (n=3 mice / condition, unpaired / -test). FIG. 1G shows the bacterial load in fecal samples 3 days after monocolonization with B. thetaiotaomicron strains. (n=6 mice / condition, unpaired / -test). For all, **** = p-value < 0.0001; ns = p-value > 0.20; Data in bar plots represent mean ± s.d.

[0025] FIGs. 2A-2H show BlmerA Areduces MeHg in colon content of monocolonized mice exposed to oral MeHg-Cl or fed diets formulated with MeHg-rich fish. FIG. 2A shows a schematic of acute oral exposure to MeHg-Cl. Germ-free mice were monocolonized with Bt or Btme'11for 3 days, and then gavaged once with MeHg-Cl (250 pg / kg). Colon content and tissues were collected 4 days after MeHg gavage. FIG. 2B shows the reduction of MeHg in fecal samples from monocolonized mice orally gavaged with MeHg-Cl (250 pg / kg) at time 0. Statistical comparison was made with area under the curve was from 1 to 4 days post-gavageAttorney Docket No. : UCH-42725

[0026] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0027] (n=3 mice / condition, unpaired t-test). FIG. 2C shows MeHg concentration, total Hg concentration, and MeHg percentage out of total Hg in colon content of mice orally gavaged with MeHg. (n=6 mice / condition, unpaired t-test). FIG. 2D shows MeHg levels in colon, liver, and brain in monocolonized mice 4 days after oral gavage of MeHg. (n=3-6 mice / condition, unpaired t-test). FIG. 2E shows a schematic of chronic dietary exposure to MeHg. Germ -free mice were monocolonized with Bt or Bf"erA / Band fed a custom diet containing 20% bluefin tuna powder (107.14 ng Hg / g hydrated diet) ad libitum for 10 days. Tissues were collected on day 10. FIG. 2F shows the reduction of MeHg in fecal samples from monocolonized mice fed with MeHg-rich diet for 10 days. Statistical comparison was made with area under the curve calculated from 3 to 10 days post-diet start (n=3 mice / condition, unpaired / -test). FIG. 2G shows MeHg concentration, total Hg concentration, and MeHg percentage out of total Hg in colon content of monocolonized mice fed with MeHg-rich diet for 10 days. (n=6 mice / condition, unpaired t-test). FIG. 2H shows MeHg levels in colon, liver, and brain of monocolonized mice fed MeHg-rich diet for 10 days. (n=3-6 mice / condition, unpaired t-test). For all, *** = p-value < 0.001; ** = p-value < 0.01; * = p-value < 0.05; ns = p-value > 0.20; Data in bar plots represent mean ± s.d.

[0028] FIGs. 3A-3F show BfnerA / Breduces tissue bioaccumulation of MeHg in pregnant dams exposed to dietary MeHg and their fetuses. FIG. 3A shows a schematic of chronic dietary exposure to MeHg-rich diet during pregnancy. Germ-free mice were monocolonized with Bt or Bii,,a'AB, time-mated, and fed a custom diet containing 20% bluefin tuna powder (107.14 ng Hg / g hydrated diet) ad libitum from E0.5 to E18.5. FIG. 3B shows MeHg levels in maternal colon content, maternal liver, placental decidua (maternal compartment), placental labyrinth (fetal compartment), and fetal brain from monocolonized pregnant mice (El 8.5) fed MeHg-rich bluefin tuna diet during gestation. Placental decidua, placental labyrinth, and fetal brain MeHg values were averaged from 3 offsprings per dam. (n=6-7 dams / condition, unpaired t-test). FIG. 3C shows the correlation between fetal brain and placental labyrinth MeHg concentration in mice exposed to MeHg-rich diet. (n=6-7 dams / condition, p-value refers to slope non-zero test). FIG. 3D shows a schematic of chronic dietary exposure to MeHg-modest diet during pregnancy. Germ-free mice were monocolonized with Bt or BlmerA Itime-mated, and fed a custom diet containing 20% salmon powder (2.39 ng Hg / g hydrated diet) ad libitum from E0.5 to E18.5. FIG. 3E shows MeHg levels in maternal colon content, maternal liver, placental decidua (maternal compartment), placental labyrinth (fetal compartment), and fetal brain from monocolonized pregnant mice (El 8.5) fed MeHg-containing salmon dietAttorney Docket No. : UCH-42725

[0029] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0030] during gestation. Placental decidua, placental labyrinth, and fetal brain MeHg values were averaged from 3 offsprings per dam. (n=9-10 dams / condition, unpaired t-test). FIG. 3F shows the correlation between fetal brain and placental labyrinth MeHg concentration in mice exposed to MeHg-modest diet. (n=9-10 dams / condition, p-value refers to slope non-zero test). For all, **** = p-value < 0.0001; *** = p-value < 0.001; ** = p-value < 0.01; * = p-value < 0.05; ns = p-value > 0.20; Data in bar plots represent mean ± s.d.; Line in scatter plot represents linear regression line with 95% confidence interval.

[0031] FIGs. 4A-4E show maternal colonization with BfnerA / Battenuates dietary MeHg-induced transcriptomic and cellular alterations in the fetal brain. FIG. 4A shows differential expression of Reactome, Biocarta, and Wikipathway gene sets in El 8.5 fetal brains from dams monocolonized with Bt or BfnerA / Band fed MeHg-rich diet (20% bluefin tuna powder, 107.14 ng Hg / g), MeHg-modest diet (20% salmon powder, 2.39 ng Hg / g), or standard diet (20% casein, 0 ng Hg / g), where effects of MeHg-containing diets are attenuated by BerA / B. NES= normalized enrichment score derived from GSEA statistics. (n=5 mice / condition, adjusted p-value from GSEA). FIG. 4B shows differential expression of Tabula Muris Senis and Descartes single-cell sequencing gene sets in E18.5 fetal brains from dams monocolonized with Bt or BtmerA / Band fed MeHg-rich diet (20% bluefin tuna powder, 107.14 ng Hg / g), MeHg-modest diet (20% salmon powder, 2.39 ng Hg / g), or standard diet (20% casein, 0 ng Hg / g), where effects of MeHg-containing diets are attenuated by BerA / B. NES= normalized enrichment score derived from GSEA statistics. (n=5 mice / condition, adjusted p-value from GSEA). FIG. 4C shows quantification of TUNEL and Ki67+cells in E18.5 fetal hippocampus from dams monocolonized with Bt or BerA / Band fed MeHg-rich diet containing 20% bluefin tuna powder (107.14 ng Hg / g). (n=3-7 mice / condition, unpaired t-test) FIG. 4D shows quantification of Ibal+cells in El 8.5 fetal hippocampus from dams monocolonized with Bt or Blme,A Band fed MeHg-rich diet containing 20% bluefin tuna powder (107.14 ng Hg / g). (n=6-7 mice / condition, unpaired t-test). FIG. 4E shows the correlation between Ibal+cells in hippocampus and fetal brain MeHg concentration. (n=6-7 mice / condition, p-value refers to slope non-zero test). For all, **** = p-value < 0.0001; *** = p-value < 0.001; ** = p-value < 0.01; * = p-value < 0.05 ; ns = p-value > 0.20; Data in bar plots represent mean ± s.d. Line in scatter plot represents linear regression line with 95% confidence interval.

[0032] FIGs. 5A-5C show there are no significant effects of BerA / Bon food consumption or gross liver histology. FIG. 5A shows a schematic of chronic dietary exposure to MeHg.Attorney Docket No. : UCH-42725

[0033] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0034] Germ-free mice were monocolonized with Bt or lmer Band fed a custom diet containing 20% bluefin tuna powder (107.14 ng Hg / g hydrated diet) ad libitum for 10 days. FIG. 5B shows total intake of MeHg-rich diet for 10 days of ad libitum administration (left) and mice weight between groups before and after administration of MeHg-rich diet (right). (n=6 mice / condition, unpaired t-test and unpaired t-test with Bonferroni correction) (n=6 mice / condition, unpaired / -test). FIG. 5C shows representative liver H&E staining and Suzuki scoring of liver damage. (n=6 mice / condition, Mann-Whitney rank test). For all, ** = p-value < 0.01; ns = p-value > 0.20; Data in bar plots represent mean ± s.d.

[0035] FIGs. 6A-6F show there are no significant effects of lmer Bon fetal viability or weight. FIG. 6A shows a schematic of chronic dietary exposure to MeHg-rich diet during pregnancy. Germ-free mice were monocolonized with 71 / o BerA / B, time-mated, and fed a custom diet containing 20% bluefin tuna powder (107.14 ng Hg / g hydrated diet) ad libitum from E0.5 to E18.5. FIG. 6B shows gross measures of fetal outcomes in pregnant mice fed with MeHg-rich diet. Measurements include litter size, percentage of resorbed fetus, percentage of deformed fetus, mean fetal and placental weight. Mean fetal and placental weight are calculated individually for each litter. (n=6-7 litters / condition, unpaired t-test). Far right: Correlation plot between fetal brain and maternal liver MeHg in MeHg-rich diet fed mice, showing lower maternal MeHg burden leads to lower fetal MeHg burden. (n=6-7 dams / condition, p-value refers to slope non-zero test). FIG. 6C shows a schematic of chronic dietary exposure to MeHg-modest diet during pregnancy. Germ-free mice were monocolonized with Bt or BerA'B, time-mated, and fed a custom diet containing 20% salmon powder (2.39 ng Hg / g hydrated diet) ad libitum from E0.5 to E18.5. FIG. 6D shows gross measures of fetal outcomes in pregnant mice fed with MeHg-modest diet. Measurements include litter size, percentage of resorbed fetus, percentage of deformed fetus, mean fetal and placental weight. Mean fetal and placental weight are calculated individually for each litter. (n=9-10 litters / condition, unpaired t-test). Far right: Correlation plot between fetal brain and maternal liver MeHg in MeHg-rich diet fed mice, showing lower maternal MeHg burden leads to lower fetal MeHg burden (n=6-7 dams / condition, p-value refers to slope non-zero test). FIG. 6E shows a schematic of dietary exposure to standard 20% casein (no Hg) diet during pregnancy. Germ -free mice were monocolonized with Bt or BlmerAtime-mated, and fed standard diet ad libitum from E0.5 to E18.5. FIG. 6F shows the gross measure of fetal outcome in pregnant mice fed with standard diet. Measurements are proportion of viable fetus, percent of resorbed fetus, and percent of deformed fetus. Proportion of viable fetus andAttomey Docket No.: UCH-42725

[0036] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0037] percent of resorbed fetus was calculated with respect to total litter size (viable and resorbed). Percent of deformed fetus was calculated with respect to total viable fetus, (n = 5-10 litters / condition, 2-way ANOVA with Dunnet’s post-hoc test). For all, ns = p-value > 0.20; Data in bar plots represent mean ± s.d.

[0038] FIGs. 7A-7K show differentially expressed genes in fetal brains from dams exposed to dietary MeHg and representative images of immunohistochemical staining of fetal hippocampus. FIG. 7A shows a schematic of chronic dietary exposure to MeHg-rich diet during pregnancy. Germ-free mice were monocolonized with Bt or Blmertime-mated, and fed a custom diet containing 20% bluefin tuna powder (107.14 ng Hg / g hydrated diet) ad libitum from E0.5 to E18.5. FIG. 7B shows a Venn diagram of differentially expressed genes (p-value<0.05) in El 8.5 fetal brains from dams monocolonized with Bt QV Blmer'Band fed MeHg-rich diet (20% bluefin tuna powder, 107.14 ng Hg / g hydrated diet) and dams monocolonized with Bt and fed either MeHg-rich diet or standard diet. Out of 160 shared DEGs, 64 genes were upregulated with MeHg exposure and a concurrent downregulation with BerA / Bmonocolonization; 87 genes were downregulated with MeHg exposure and a concurrent upregulation with BerA / Bmonocolonization. FIG. 7C shows a schematic of chronic dietary exposure to MeHg during pregnancy. Germ-free mice were monocolonized with 71 / or BtmerA / B, time-mated, and fed a custom diet containing 20% salmon powder (2.39 ng Hg / g hydrated diet) ad libitum from E0.5 to E18.5. FIG. 7D shows a Venn diagram of differentially expressed genes (p-value<0.05) in El 8.5 fetal brains from dams monocolonized with Bt or Bt"'"'A Band fed MeHg-modest diet (20% salmon powder, 2.39 ng Hg / g hydrated diet) and dams monocolonized with Bt and fed either MeHg-modest diet or standard diet. Out of 124 shared DEGs, 63 genes were upregulated with MeHg exposure and a concurrent downregulation with BerA / Bmonocolonization; 60 genes were downregulated with MeHg exposure and a concurrent upregulation with BerA / Bmonocolonization. FIG. 7E Top differentially expressed genes in El 8.5 fetal brains from dams monocolonized with Bt or ^^merA / Ban(j fecj MeHg-rich diet (20% bluefin tuna powder, 107.14 ng Hg / g), MeHg-modest diet (20% salmon powder, 2.39 ng Hg / g), or standard diet (20% casein, 0 ng Hg / g). Selected genes are ones with adjusted p-value < 0.1 and log2(fold change) > 0.5 in any of the comparisons. Highlighted genes in red are expressed by microglia based on CellMarker2.0 and MSigDB M8 Microglial Cell Aging. (n=5 mice / condition). FIG. 7F shows GSEAof Reactome, Biocarta, and Wikipathways pathway in El 8.5 fetal brains from dams monocolonized with Bt or BtmerA / Band fed MeHg-rich diet (20% bluefin tuna powder, 107.14Attorney Docket No. : UCH-42725

[0039] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0040] ng Hg / g hydrated diet) (adjusted p-value <0.2; n=5 mice / condition). FIG. 7G shows GSEA of Reactome, Biocarta, and Wikipathways pathway in El 8.5 fetal brains from dams monocolonized with Bt or BerA / Band fed MeHg-modest diet (20% salmon powder, 2.39 ng Hg / g hydrated diet) (adjusted p-value <0.2; n=5 mice / condition). FIG. 7H shows a volcano plot of differentially expressed genes (DESeq2) in El 8.5 fetal brains from dams monocolonized with Bt or BtmerA / Band fed MeHg-rich diet (20% bluefin tuna powder, 107.14 ng Hg / g hydrated diet). Colored points signify genes with adjusted p-value < 0.1 and log2(fold change) > 0.5. (n=5 mice / condition). FIG. 71 shows a volcano plot of differentially expressed genes (DESeq2) in El 8.5 fetal brains from dams monocolonized with / / / or BtmerA / Band fed MeHg-modest diet (20% salmon powder, 2.39 ng Hg / g hydrated diet). Colored points signify genes with adjusted p-value < 0.1 and log2(fold change) > 0.5. (n=5 mice / condition), FIG. 7J shows representative images of fetal brain hippocampus staining from pregnant dams fed with fed MeHg-rich diet (20% bluefin tuna powder, 107.14 ng Hg / g hydrated diet) and monocolonized with Bt orjgmerA / B. ROI denotes E18.5 fetal brain hippocampus based on Allen Brain Atlas for developing 18.5 mouse brain (Sagittal, MPall, images #8-11). Scale bar represents 200 pm. FIG. 7K shows quantification of Nestin integrated density in E18.5 fetal hippocampus from dams monocolonized with Bt or Bt merA / B and fed MeHg-rich diet containing 20% bluefin tuna powder (107.14 ng Hg / g). (n=6-7 mice / condition, unpaired t-test).

[0041] FIGs. 8A-8G shows Blm r Areduces MeHg in livers of SPF mice fed diets formulated with MeHg-rich fish and can persist when co-colonized with wild-type Bt. FIG. 8A shows a schematic of chronic dietary exposure to MeHg with daily oral gavage of live engineered bacteria. Specific pathogen free (SPF) mice were fed a custom diet containing 20% bluefin tuna powder (107.14 ng Hg / g hydrated diet) ad libitum for 10 days. Throughout the dietary exposure, mice were orally gavaged once a day with a fresh inoculum of either BtmerA / Bo Bt in the evening to align with the timing of their active feeding period. FIG. 8B shows the concentration of MeHg in fecal samples from SPF mice exposed to MeHg-rich diet and administered daily oral gavage of / ? / nicrA Bor / / / . Statistical comparison was made with area under the curve from day 3 to 10 post-start of dietary exposure (n=3 mice / condition, unpaired t-test) FIG. 8C shows Log 10 gene copies from fecal sample qPCR of merB and pan-Bt for SPF mice exposed to MeHg-rich diet and administered daily oral gavage of Btm^Aor Bt. Gene copies were normalized with template DNA concentration and were calculated using standard curve of pure Btm^Aculture. Dashed line denotes the assay limit of detectionAttorney Docket No. : UCH-42725

[0042] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0043] (LOD) based on lowest concentration which 90% of replicates are detected. Statistical comparison was made with area under the curve from day 3 to 10 post-diet start. (n=3-4 mice / condition) FIG. 8D shows MeHg concentration, total Hg concentration, and MeHg percentage out of total Hg in colon content and MeHg concentration in liver of SPF mice fed with MeHg-rich diet for 10 days and administered daily oral gavage of BPerA / Bor Bt. (n=4-5 mice / condition, unpaired t-test) FIG. 8E shows a schematic of various colonization paradigm of either only ?tmerA / B, BP^A B+ Bt (1 : 1, v / v, OD600 normalized), and 2?merA / B+ SPF mice fecal slurry (1 :20, v / v). Germ-free mice were inoculated once with the various bacterial mixtures and fecal samples were collected from day 1 - day 10 post inoculation. FIG. 8F shows Log 10 gene copies of merB - as signature of BPerMB- in fecal samples of colonized germ-free mice. Gene copy numbers were normalized against template DNA concentration. Standard curve to derive gene copy number was made from extracted DNA of BPerMBovernight culture. Dashed line denotes the assay limit of detection (LOD) based on lowest concentration which 90% of replicates are detected. Statistical comparison was made with area under the curve from day 1 to 10 post-colonization. (n=3-4 mice / condition, unpaired t-test) FIG. 8G shows LoglO gene copies of merB and 16S across the gastrointestinal tract of colonized germ-free mice. Gene copy numbers were normalized against template DNA concentration. Standard curve to derive gene copy number was made from extracted DNA ofjg / merA / Bovernight culture. Dashed line denotes the assay limit of detection (LOD) based on lowest concentration which 90% of replicates are detected. (n=3-4 mice / condition, 2-way ANOVA with Tukey multiple testing correction). * = p-value < 0.05; ** = p-value < 0.01; *** = p-value < 0.001; ns = p-value > 0.20; Data in bar plots represent mean ± s.d.

[0044] DETAILED DESCRIPTION

[0045] Despite efforts to decrease mercury emissions, chronic exposure to the neurotoxicant methylmercury (MeHg) continues to be a global problem that contributes to disparities in risk for neurological and metabolic diseases. The present disclosure provides engineered human commensal gut bacteria (e.g., Bacteroides thetaiotaomicrori) that detoxify MeHg by heterologous expression certain genes derived from a resistant bacterium isolated from Hg-polluted mines (e.g., organomercury lyase (MerB) and / or mercuric reductase (MerA)). The engineered bacteria demethylate MeHg both in vitro and within the intestines of a subject orally exposed to MeHg or diets containing MeHg-rich fish. In pregnant subjects exposed to dietary MeHg, the engineered bacteria decrease MeHg accumulation in the maternal liver,Attorney Docket No. : UCH-42725

[0046] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0047] brain, placenta, and fetal brain, and attenuates the expression of cellular stress and apoptosis genes in the fetal brain. The engineered bacteria thus limit MeHg bioaccumulation and reduce adverse effects of chronic MeHg exposure.

[0048] Provided herein are compositions and methods for preventing adverse effects of methylmercury exposure and / or methylmercury poisoning. In some embodiments, the compositions comprise engineered bacteria that can specifically detoxify MeHg. In some embodiments, the administration of such engineered bacteria treats or prevents adverse physiological outcomes associated with MeHg-containing diets. In some embodiments, the administration treats or prevents adverse physiological outcomes associated with MeHg-exposure during adulthood and pregnancy.

[0049] In some aspects, provided herein are bacteria comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid. In some aspects, provided herein are bacteria comprising a gene encoding a methylmercury-reducing enzyme, wherein the gene is encoded by an exogenous nucleic acid. In some embodiments, the bacterium comprises an additional gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0050] In some embodiments, the bacteria comprise at least one gene encoding a methylmercury-degrading enzyme that encodes a mercuric reductase. In some embodiments, the bacteria comprise at least one gene encoding a methylmercury-degrading enzyme that encodes an organomercury lyase.

[0051] In some embodiments, the gene or the additional gene encoding a methylmercurydegrading enzyme encodes a mercuric reductase (MerA). In some embodiments, the mercuric reductase has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to Pseudomonas aeruginosa MerA In some embodiments, the mercuric reductase is Pseudomonas aeruginosa MerA

[0052] In some embodiments, the gene or the additional gene encoding a methylmercurydegrading enzyme encodes an organomercury lyase (MerB). In some embodiments, the organomercury lyase has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to Pseudomonas aeruginosa MerB. In some embodiments, the organomercury lyase is Pseudomonas aeruginosa MerB.

[0053] In some embodiments, the bacterium further comprises one or more genes from mercury resistance (mer) systems. In some embodiments, the bacterium further comprisesAttorney Docket No. : UCH-42725

[0054] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0055] one or more genes encoding proteins that bind MeHg, Hg°, and / or Hg2+. In some embodiments, the bacterium comprises a gene encoding MerR. In some embodiments, the bacterium further comprises a gene encoding a mercury transporter. In some embodiments, the mercury transporter is MerT. In some embodiments, the mercury transporter is MerP. In some embodiments, the mercury transporter is MerE.

[0056] In some embodiments, the genes described herein are encoded by exogenous nucleic acids. In some embodiments, the genes described herein are derived from genes found in a microbe or bacterium isolated from a mercury-rich or methylmercury -rich environment. In some embodiments, the genes described herein are derived from genes found in Pseudomonas species. In some embodiments, the gene described herein are derived from genes found in Pseudomonas aeruginosa.

[0057] In some embodiments, the bacterium is of the Bacteroides genus. In some embodiments, the bacterium is of the species Bacteroides thetaiotaomicron. In some embodiments, the bacterium is of the strain Bacteroides thetaiotaomicron VPI-5482.

[0058] In another aspect, provided herein are methods of detoxifying or demethylating mercury and / or methylmercury in the gastrointestinal tract of a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0059] In another aspect, provided herein are methods of detoxifying or demethylating mercury and / or methylmercury in the gastrointestinal tract of a subject with mercury and / or methylmercury poisoning, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0060] In another aspect, provided herein are methods of preventing mercury and / or methylmercury accumulation in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercurydegrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0061] In another aspect, provided herein are methods of treating or preventing mercury and / or methylmercury poisoning in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercurydegrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.Attorney Docket No. : UCH-42725

[0062] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0063] In another aspect, provided herein are methods of reducing mercury and / or methylmercury levels in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0064] In another aspect, provided herein are methods of treating a subject with mercury and / or methylmercury accumulation, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0065] In another aspect, provided herein are methods of treating or preventing brain damage due to mercury and / or methylmercury exposure in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0066] In another aspect, provided herein are methods of treating or preventing brain damage due to mercury and / or methylmercury poisoning in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0067] In another aspect, provided herein are methods of treating or preventing cognitive impairment after mercury and / or methylmercury exposure in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0068] In another aspect, provided herein are methods of treating or preventing cognitive impairment after mercury and / or methylmercury poisoning in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0069] In another aspect, provided herein are methods of treating or preventing cognitive impairment due to mercury and / or methylmercury exposure in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.Attorney Docket No. : UCH-42725

[0070] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0071] In another aspect, provided herein are methods of treating or preventing cognitive impairment due to mercury and / or methylmercury poisoning in a subject, the methods comprising administering to the subject a composition comprising a bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

[0072] In some embodiments, the subject is an adult. In some embodiments, the subject is a pregnant individual. In some embodiments, the subject is a fetus. In some embodiments, the subject is a pediatric subject. In some embodiments, the subject is gestating a fetus.

[0073] In another aspect, provided herein are methods of promoting healthy neural development in a fetus, the methods comprising administering to a subject gestating the fetus a composition comprising a bacterium disclosed herein. In some embodiments, healthy neural development comprises a reduction in anxiety-like behavioral deficits. In some embodiments, healthy neural development comprises prevention of learning and memory deficits.

[0074] In another aspect, provided herein are methods of preventing impaired neural development in a fetus, the methods comprising administering to a subject gestating the fetus a composition comprising a bacterium disclosed herein.

[0075] In some embodiments, administration of a bacterium disclosed herein results in altered gene and protein expression in the subject. In some embodiments, the altered gene and protein expression comprises a change in expression of pathways associated with MeHg exposure. In some embodiments, the altered gene and protein expression comprises a change in expression of one or more pathways in Table 1.

[0076] In some embodiments, cognitive impairment, impaired neural development, and / or brain damage in the subject are associated with altered gene expression in the brain tissue of the subject. In some embodiments, the altered gene expression in the brain tissue of the subject comprises increased expression of gene sets related to aging of microglia, oligodendrocytes, pericytes, and / or endothelial cells. In some embodiments, cognitive impairment, impaired neural development, and / or brain damage in the subject are associated with elevated density of microglia (e.g., IbaU microglia) in the hippocampus.

[0077] Also provided herein are compositions and methods for treating disorders and diseases that result from mercury and / or methylmercury poisoning and / or exposure, such as acrodynia (pink disease) in which the skin becomes pink and peels.Attorney Docket No. : UCH-42725

[0078] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0079] Table 1

[0080]

[0081] In some embodiments, the subject has been exposed to mercury and / or methylmercury. In some embodiments, the subject is or has been exposed to mercury and / or methylmercury in their diet (e.g., high-mercury fish). In some embodiments, the subject is or has been exposed to mercury and / or methylmercury in their environment (e.g., water supply).

[0082] In some embodiments, the composition is formulated for oral delivery. In some embodiments, the composition is a food product. In some embodiments, the food product is a dairy product. In some embodiments, the food product is yogurt.

[0083] In some embodiments, the composition is formulated for rectal delivery.

[0084] In some embodiments, the composition is for self-administration.

[0085] In some embodiments, the subject is a human.

[0086] In some embodiments, the subject is treated with antibiotics before administration of the composition. In some embodiments, the subjects are antibiotic-treated.Attorney Docket No. : UCH-42725

[0087] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0088] In some embodiments, the bacterium or composition is for conjoint administration with a probiotic and / or prebiotic.

[0089] Definitions

[0090] As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claim(s), when used in conjunction with the word "comprising", the words "a" or "an" may mean one or more than one. As used herein “another” may mean at least a second or more.

[0091] As used herein, the term “about” is defined as being close to as understood by one of ordinary skill in the art. In one non-limiting embodiment, the term “about” is defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0092] The phrase “pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.

[0093] The term “preventing is art-recognized, and when used in relation to a condition, such as a local recurrence, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. A therapeutic that prevents a disorder or condition refers to a bacterium or composition that, inAttorney Docket No. : UCH-42725

[0094] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0095] a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample.

[0096] The term “prophylactic” or “therapeutic” treatment is art-recognized and includes administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic (i.e., it protects the host against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0097] A “therapeutically effective amount” of a compound with respect to the subject method of treatment refers to an amount of the compound(s) in a preparation which, when administered as part of a desired dosage regimen (to a mammal, preferably a human) alleviates a symptom, ameliorates a condition, or slows the onset of disease conditions according to clinically acceptable standards for the disorder or condition to be treated or the cosmetic purpose, e.g., at a reasonable benefit / risk ratio applicable to any medical treatment.

[0098] As used herein, the term “treating or “treatment” includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner to improve or stabilize a subject's condition. The term “treating” includes prophylactic and / or therapeutic treatments. The term “prophylactic or therapeutic” treatment is art-recognized and includes administration to the subject of one or more of the disclosed compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the subject) then the treatment is prophylactic (i.e., it protects the subject against developing the unwanted condition), whereas if it is administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate, or stabilize the existing unwanted condition or side effects thereof).

[0099] As used herein, the terms “modulate” or “modulation,” or “regulate” or “regulation” and “differentially regulated" can refer to both up regulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and down regulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting), unless otherwise specified or clear from the context of a specific usage.

[0100] “Impaired neural development” and / or “brain damage" as used herein, refers to abnormalities in brain function and behavior. Examples of impaired neural developmentAttorney Docket No. : UCH-42725

[0101] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0102] include, but are not limited to, increased anxiety, impaired learning, and impaired memory (e.g., poor performance on cognitive or spatial tests). Examples of “healthy neural development,” as used herein, include, but are not limited to, healthy development in fetal brain gene expression, fetal axonogenesis, fetal axon development, and / or adult tactile sensory behavior.

[0103] As used herein, “cognitive impairment” includes a lessening in the overall capacity or the speed of mental processes. Cognitive impairment can be caused by, e.g., higher than recommended exposure to or accumulation of mercury and / or methylmercury in one or more tissues of a subject.

[0104] As used herein, “mercury and / or methylmercury poisoning” (e.g., mercury and / or methylmercury exposure or accumulation) refers to a type of metal poisoning due to exposure from mercury and / or methylmercury that causes symptoms. Mercury and / or methylmercury poisoning can cause damage to the brain and nervous system and / or such symptoms as tremors, memory loss, coordination issues, vision / hearing problems, kidney malfunction, developmental issues, muscle weakness, numbness in the hands and feet, skin rashes, anxiety, memory problems, trouble speaking, trouble hearing, trouble seeing, peripheral neuropathy (presenting, for example, as paresthesia or itching, burning, pain, or even a sensation that resembles small insects crawling on or under the skin (formication)), skin discoloration (pink cheeks, fingertips and toes); swelling; desquamation (shedding or peeling of skin) and a range of neurological symptoms associated with erethism. Symptoms depend upon the type, dose, method, and duration of exposure, and can be diagnosed by a clinician. Exposure to mercury and / or methylmercury can be, among other sources, from water / soil, bioaccumulates in fish, industrial exposure, and / or broken thermometers / fluorescent bulbs. Provided herein are compositions and methods for treating or preventing mercury and / or methylmercury poisoning.

[0105] As used herein, “methylmercury-degrading enzyme” refers to a protein that binds to mercury and / or methylmercury. The protein can have enzymatic functions. The protein can “degrade” or “detoxify” methylmercury, i.e., convert (e.g., demethylate and / or reduce) methylmercury (MeHg) into other mercury compounds (e.g., Hg2+and / or Hg°). The protein may degrade methylmercury into other mercury species (e.g., Hg2+or Hg°) that are less bioaccessible, less likely to bioaccumultate in a subject, and / or are more likely to be excreted from the subject. In some embodiments, the methylmercury-degrading enzyme is part of and / or derived from a mercury resistance (mer) system. In some embodiments, theAttorney Docket No. : UCH-42725

[0106] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0107] methylmercury-degrading enzyme is found in mercury-resistant strains of bacteria (e.g., Pseudomonas aeruginosa) that may be isolated from mercury-polluted environments.

[0108] “Microbiome,” as used herein, refers to the microorganisms in a given environment, such as the body or a part of the body. The “maternal microbiome,” as used herein, refers to the microorganisms in a subject (i.e., a pregnant or gestating subject), particularly in the gut of the subject. The gut microbiome modulates the bioavailability of hundreds of biochemicals in the circulating blood. During pregnancy, the maternal gut environment supplies nutrients and growth factors, from the maternal diet and other nutritional intake, to nurture offspring growth.

[0109] As used herein, the term “identity” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Methods for the calculation of a percent identity as between two provided sequences are known in the art. The relative sequence identity of genes can be calculated based on their DNA and / or RNA sequences. The relative sequence identity of proteins can be calculated based on their polypeptide sequences, or based on the sequences of the DNA and / or RNA molecules that encode them.

[0110] The term “subject” to which administration is contemplated includes, but is not limited to, humans (i.e., a male or female of any age group, e.g., a pediatric subject (e.g., infant, child, adolescent) or adult subject (e.g., young adult, middle-aged adult or senior adult)) and / or other primates (e.g., cynomolgus monkeys, rhesus monkeys); and / or mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, goats, cats, and / or dogs. In some preferred embodiments, the subject is a human.

[0111] “Antibiotic-treated” subjects, as used herein, are subjects treated with one or more antibiotic compounds, many representative examples of which are known in the art.

[0112] As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different therapeutic compounds such that the second compound is administered while the previously administered therapeutic compound is still effective in the body (e.g., the two compounds are simultaneously effective in the subject, which may include synergistic effects of the two compounds). For example, the different therapeutic compounds can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially. In certain embodiments, the different therapeutic compounds can be administered within one hour, 12 hours, 24 hours, 36 hours, 48Attorney Docket No. : UCH-42725

[0113] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0114] hours, 72 hours, or a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic compounds.

[0115] Therapeutic Methods

[0116] The disclosure herein, relates, in part, to the discovery that engineered bacteria, such as Bacteroides, containing different methylmercury-degrading enzymes, such as MerA and MerB, can be used in compositions. Such compositions are suitable for use in methods of preventing, treating, or reducing methylmercury and / or mercury accumulation in a subject.

[0117] In some aspects, the methods comprise depleting the gut microbiota of the subject prior to administration with a composition described herein (e.g., by administering antibiotics to the subject).

[0118] In some embodiments, the bacteria comprise one or more genes each encoded by an exogenous nucleic acid, e.g., a plasmid or other vector in which a gene is operably coupled to a promoter that promotes expression (e.g., constitutively or inducibly) of the gene in the bacterium. In some embodiments, the one or more genes encode one or more methylmercury -degrading enzymes. In some embodiments, the promoter is pBfPlE6.

[0119] Provided herein are methods of making a bacterium described herein, comprising transforming a bacterium with a gene expression construct encoding a methylmercurydegrading enzyme operably coupled to a promoter that promotes expression (e.g., constitutively or inducibly) of the gene in the bacterium. In some embodiments, the method comprises formulating the bacterium for administration to a subject, e.g., in a pharmaceutical composition or in a food or beverage product. In some embodiments, the method further comprises culturing the bacterium to allow expression of the genes. In some embodiments, the bacterium (e.g., Bacteroides thetaiotaomicrori) regulates the gene expression.

[0120] The composition may be formulated for oral delivery. In some embodiments, the composition may comprise probiotics. In some embodiments, the compositions disclosed herein are food products. The composition may be in the form of a pill, tablet, or capsule. In some embodiments, the subject may be a mammal (e.g., a human). In some embodiments, the composition is self-administered. While it is preferred for a single composition to comprise all the bacteria to be administered, it will be recognized that for any of the various embodiments described herein, the combination of bacteria can similarly be administered in multiple compositions that together comprise the combination of bacteria.

[0121] In some embodiments, the composition is formulated for rectal delivery (e.g., a fecal sample). In some embodiments, the subject undergoes fecal microbiota transplant, whereinAttorney Docket No. : UCH-42725

[0122] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0123] the transplant comprises a composition disclosed herein. Fecal microbiota transplantation (FMT), also commonly known as 'fecal bacteriotherapy' represents a therapeutic protocol that allows the reconstitution of colon microbial communities. The process involves the transplantation of fecal bacteria from a healthy individual into a recipient. FMT restores colonic microflora by introducing healthy bacterial flora through infusion of a fecal sample, e.g., by enema, orogastric tube or by mouth in the form of a capsule containing freeze-dried material, obtained from a healthy donor. In some embodiments, the fecal sample is from a fecal bank.

[0124] In some embodiments, the bacterial DNA in subject’s gut microbiota is sequenced. The subject’s gut bacterial DNA may be sequenced prior to administration of the composition. For example, a sample comprising bacterial DNA may be obtained from the subject, and the bacterial DNA is then sequenced for Bacteroides thetaiotaomicron, therefore measuring the presence or level of such bacteria in the subject’s gut microbiota. The composition disclosed herein may then be administered to the subject if the level of the bacteria is low. In some embodiments, the subject is deemed to have low levels of B. thetaiotaomicron if less than 0.0001%, less than 0.001%, less than 0.01%, less than 0.02%, less than 0.03%, less than 0.04%, less than 0.05%, less than 0.06%, less than 0.07%, less than 0.08%, less than 0.09%, less than 0.1%, less than 0.2%, less than 0.3%, less than 0.4%, less than 0.5%, less than 0.6%, less than 0.7%, less than 0.8%, less than 0.9%, less than 1%, less than 2%, less than 3%, less than 5%, less than 7%, less than 10%, less than 20%, less than 30%, less than 40%, or less than 50% of the bacteria in the sample is the bacteria of interest. Bacterial DNA to be sequenced may be obtained through any means known in the art, including, but not limited to, obtaining a fecal sample from the subject and isolating the bacterial DNA. Bacterial DNA sequencing by any known technique in the art, including, but not limited to, Maxam Gilbert sequencing, Sanger sequencing, shotgun sequencing, bridge PCR, or next generation sequencing methods, such as massively parallel signature sequencing (MPSS), polony sequencing, 454 pyrosequencing, Illumina (Solexa) sequencing, SOLiD sequencing, Ion torrent semiconductor sequencing, DNA nanoball sequencing, heliscope single molecule sequencing, single molecule real time (SMRT) sequencing, or nanopore DNA sequencing.

[0125] Compositions

[0126] In some aspects, the invention relates to a composition comprising the bacteria described herein. Compositions may comprise at least one, at least two, at least three, at leastAttorney Docket No. : UCH-42725

[0127] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0128] four, at least five, at least six at least seven, at least eight, at least nine, at least ten, at least fifteen, or at least twenty types of bacteria. Any combination of the bacteria may be included in the composition.

[0129] The composition may comprise a pharmaceutically acceptable carrier. The composition may comprise probiotics. The pharmaceutical compositions disclosed herein may be delivered by any suitable route of administration, including orally, bucally, sublingually, parenterally, and rectally, as by powders, ointments, drops, liquids, gels, tablets, capsules, pills, or creams. In certain embodiments, the pharmaceutical compositions are delivered generally (e.g., via oral administration). In certain other embodiments, the compositions disclosed herein are delivered rectally.

[0130] In certain embodiments, the invention provides kits comprising the compositions described herein.

[0131] Compositions described herein may be used for oral administration to the gastrointestinal tract, directed at the objective of introducing the bacteria (e.g., the bacteria disclosed herein) to tissues of the gastrointestinal tract. The formulation for a composition (e.g., a probiotic composition) of the present invention may also include other probiotic agents or nutrients which promote spore germination and / or bacterial growth. An exemplary material is a bifidogenic oligosaccharide, which promotes the growth of beneficial probiotic bacteria. In some embodiments, the probiotic bacterial composition is administered with a therapeutically-effective dose of an (preferably, broad spectrum) antibiotic, or an anti-fungal agent. In some embodiments, the compositions described herein are encapsulated into an enterically-coated, time-released capsule or tablet. The enteric coating allows the capsule / tablet to remain intact (i.e., undissolved) as it passes through the gastrointestinal tract, until after a certain time and / or until it reaches a certain part of the GI tract (e.g., the small intestine). The time-released component prevents the “release” of the probiotic bacterium in the compositions described herein for a pre-determined time period.

[0132] The composition may be a food product, such as, but not limited to, a dairy product. The dairy product may be cultured or a non-cultured (e.g., milk) dairy product. Non-limiting examples of cultured dairy products include yogurt, cottage cheese, sour cream, kefir, buttermilk, etc. Dairy products also often contain various specialty dairy ingredients, e.g. whey, non-fat dry milk, whey protein concentrate solids, etc. The dairy product may be processed in any way known in the art to achieve desirable qualities such as flavor, thickening power, nutrition, specific microorganisms and other properties such as moldAttorney Docket No. : UCH-42725

[0133] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0134] growth control. The compositions of the present invention may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins, or minerals.

[0135] In some embodiments, the compositions of the present invention are combined with a carrier (e.g., a pharmaceutically acceptable carrier) which is physiologically compatible with the gastrointestinal tissue of the subject(s) to which it is administered. Carriers can be comprised of solid-based, dry materials for formulation into tablet, capsule or powdered form; or the carrier can be comprised of liquid or gel -based materials for formulations into liquid or gel forms. The specific type of carrier, as well as the final formulation depends, in part, upon the selected route(s) of administration. The therapeutic composition of the present invention may also include a variety of carriers and / or binders. Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gelbased materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration. Typical carriers for dry formulations include, but are not limited to: trehalose, malto-dextrin, rice flour, microcrystalline cellulose (MCC) magnesium sterate, inositol, FOS, GOS, dextrose, sucrose, and like carriers. Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e.g., methanol, ethanol, propanol, butanol); glycols (e.g., ethylene glycol, propylene glycol, and the like). Preferably, water-based carriers possess a neutral pH value (i.e., pH 7.0). Other carriers or agents for administering the compositions described herein are known in the art, e.g., in U.S. Patent No. 6,461,607.

[0136] In some embodiments, the composition further comprises other bacteria or microorganisms known to colonize the gastrointestinal tract. For example, the composition may comprise species belonging to the Firmicutes phylum, the Proteobacteria phylum, the Tenericutes phylum, the Actinobacteria phylum, or a combination thereof. Examples of additional bacteria and microorganisms that may be included in the subject compositions include, but are not limited to, Saccharomyces, Bacteroides, Eubacterium, Clostridium, Lactobacillus, Fusobacterium, Propionibacterium, Streptococcus, Enteroccus, Lactococcus and Staphylococcus, Peptostreptococcus. In certain embodiments, the composition is substantially free of bacteria that increase the risk of metabolic disorder. Such bacteria include Bifidobacterium bacteria. Thus, in some embodiments, the composition is substantially free of Bacteroides bacteria. A composition is substantially free of a bacterial type if that type makes up less than 10% of the bacteria in a composition, preferably less thanAttorney Docket No. : UCH-42725

[0137] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0138] 5%, even more preferably less than 1%, most preferably less than 0.5%, or even 0% of the bacteria in the composition.

[0139] In some embodiments, the composition comprises a fecal sample comprising a bacterium described herein. In some embodiments, the fecal sample is from a fecal bank. In some embodiments, the compositions may be added to a fecal sample prior to administration to the subject.

[0140] The composition may further comprise a nutrient. In some embodiments, the nutrient promotes protein intake or absorption. In some embodiments, the nutrient aids in the growth of bacteria (e.g., bacteria disclosed herein). In some embodiments the nutrient is a fiber (e.g., fructan, levan, inulin). In some embodiments, the nutrient is a lipid (e.g., lineoleic acid, stearic acid, or palmitic acid). In some embodiments, the nutrient may be conjointly administered with a composition disclosed herein. As used herein, the phrase “conjoint administration” refers to any form of administration of two or more different agents (e.g., a composition disclosed herein and a nutrient disclosed herein) such that the second agent is administered while the previously administered agent is still effective in the body. For example, the compositions disclosed herein and the nutrients disclosed herein can be administered either in the same formulation or in a separate formulation, either concomitantly or sequentially.

[0141] Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0142] The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0143] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could prescribe and / or administer doses of the compounds employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desiredAttorney Docket No. : UCH-42725

[0144] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0145] effect is achieved.

[0146] EXAMPLES

[0147] Example 1: An engineered gut bacterium protects against dietary methylmercury exposure in pregnant mice

[0148] MeHg is a global problem that continues to rise, due to its long residence time and possible worsening with climate change, and warrants new solutions that effectively address risk from chronic exposures. Engineering an efficient and safe commensal strain for MeHg demethylation could be more efficacious and targeted than other approaches. This study demonstrates that an engineered bacterium for reductive demethylation of MeHg limits MeHg accumulation and protects against adverse outcomes of dietary MeHg exposure in pregnant gnotobiotic mice.

[0149] Expression of key enzymes from an environmental bacterium in Bacteroides

[0150]

[0151] confers MeHg demethylation activity

[0152] Mercury resistance (mer) systems detoxify MeHg via reductive demethylation in select soil and marine bacteria but are largely absent from the human and mouse gut microbiota. To achieve targeted MeHg detoxification by a gut bacterium, key mer enzymes for the demethylation of MeHg and reduction of divalent mercury were expressed in the commensal bacterium Bacteroides thetaiotaomicron. based on its high prevalence in the human gut microbiota, functional importance as a keystone species, demonstrated safety as a probiotic candidate, and high genetic tractability. Gene sequences for organomercury lyase (merB) and mercuric reductase (merA) were obtained from a mercury-resistant strain of Pseudomonas aeruginosa previously isolated from polluted mines and demonstrated to exhibit high MeHg demethylation activity. merA and merB sequences were stably integrated into the type strain of B. thetaiotaomicron (Bt) at two different intergenic sites under control of the strong constitutive promoter pBfPlE6 (FIG. 1A). There were no noticeable growth defects in the engineered strain (BtmerA / B) when compared with the wild-type parental Bt (FIG. IB) When grown in minimal media supplemented with MeHg-Cl (3.83 ± 0.13 ng Hg / mL), BtmerA / Breduced MeHg with a half-life (t%) of 5.07 ± 0.14 hours, whereas Bt displayed minimal native ability to reduce MeHg (FIG. 1C). As MeHg readily forms metallo-organic conjugates in complex matrices (e.g. biological tissues), the efficacy of i"'e'A 11in demethylating MeHg bioaccumulated in fish was tested. Meat from a whole Pacific bluefin tuna (Thunnus orientalist was aseptically powdered and digested in vitro to simulateAttorney Docket No. : UCH-42725

[0153] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0154] breakdown within the gut, and supplemented to minimal media at a MeHg concentration of 2.21 ± 0.17 ng Hg / mL. tme'' 'Beliminated fish-derived MeHg compared to Bt controls, with a t% of 2.43 ± 0.21 hours (FIG. ID). Both MeHg-Cl and fish-derived MeHg were fully demethylated within 12 hours (FIGs. 1C and ID).

[0155] j^^nerA / B decreasesMeHg in adult monocolonized mice acutely exposed to oral MeHg or fed MeHg-containing diets

[0156] Oral ingestion is the main route of human exposure to MeHg. To determine whether BfnerA / Beffectively decreases MeHg from oral exposure, germ-free mice were first monocolonized with BfnerA / Bo Bt and then gavaged with single high dose of MeHg-Cl (250 pg / kg) (FIG. IE). Mice monocolonized with Bt"'"'A Bhad lower levels of MeHg in feces within 3 hours and over 24 hours after gavage when compared with / ^ / -colonized controls (FIG. IF), with no statistically significant differences in fecal loads of Blme,A 11or Bt (FIG. 1G). These results suggest rapid reduction decrease of MeHg by BerA / Bwithin the mouse intestine. Consistent with this, fecal levels of MeHg remained decreased in B ”er ZS-colonized mice at 4 days after MeHg gavage (FIG. 2B), suggesting that BfnerA / Bcontinues to eliminate MeHg over long time scales. To further assess bacterial activity at the site of densest colonization, colon contents at 4 days after MeHg exposure were examined (FIG. 2C). Mice colonized with BtmerA / Bexhibited significantly decreased levels of MeHg in colon contents at 4 days post exposure with concurrent increases in levels of total mercury (THg: the sum of all mercury forms) (FIG. 2C), suggesting that the lower fecal levels of MeHg reflect active bacterial demethylation and reduction of MeHg into Hg2+or Hg° within the intestine.

[0157] However, high levels of MeHg were observed in livers and brains of mice at 4 days after oral gavage of MeHg-Cl, with no significant decrease by BerA / B(FIG. 2D). Taken together, these results suggest that BfnerA / Bsuccessfully demethylates MeHg locally within the intestine, but may not prevent MeHg absorption and tissue bioaccumulation when challenged with high dose MeHg-Cl by oral gavage. Based on these findings, it was hypothesized that BerA / Bmay be more effective at decreasing tissue MeHg levels when exposure occurs gradually through chronic dietary intake, rather than as a single high-dose bolus.

[0158] Billions of people rely on fish as a source of animal protein and essential micronutrients, but fish for consumption are a major source of MeHg exposure. To determine whether BfnerA / Balleviates MeHg bioaccumulation in response to chronic dietary exposure to MeHg, Bt or BerA / B-colonized mice were fed with custom diets formulated with 20% bluefin tuna over 10 days (FIG. 2D). The diets contained 107.14 ng Hg per gram hydrated diet,Attorney Docket No. : UCH-42725

[0159] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0160] which amounted to an exposure of approximately 18.75 pg / kg per day and a total dosage that was -75% of the single gavage of MeHg-Cl but spread across 10 days (FIGs. 1E-G and 2A-D). Consistent with results from the acute exposure paradigm, mice colonized with Btme- 'Bhad lower levels of fecal MeHg by 3 days and through 10 days of consuming MeHg-rich diets, as compared to Bt-colonized controls (FIG. 2F). There were no significant differences in mouse weight and the amount of diet consumed by Bt- or BfnerA / B-colonized mice (FIG.

[0161] 5A and 5B), indicating that they were exposed to equivalent levels of MeHg. Similarly, mice colonized with BtmerA / Bexhibited significantly lower levels of MeHg and increased total mercury in colon contents, suggesting active bacterial demethylation of intestinal MeHg (FIG. 2G). Despite having high tissue loads of MeHg due to the chronic dietary exposure, mice colonized with BerA / Bexhibited modest, but statistically significant, decreases in brain levels of MeHg compared to Bt-colonized controls, with no differences in levels of MeHg or pathology in liver tissue (FIGs. 2H and 5C). These results indicate that BerA / Beffectively demethylates MeHg in the mouse intestine and further suggest that BerA / Bis more effective at limiting MeHg bioaccumulation in the context of chronic, not acute, exposure to MeHg.

[0162] Bfn‘’r [ BdecreasesMeHg and attenuates adverse fetal responses to dietary MeHg exposure during pregnancy

[0163] MeHg from exposures during pregnancy can cross the placenta and bioaccumulate in the fetus to impair neurocognitive development of the offspring. To test effects of BerA / Bon MeHg burden in pregnant dams and their developing offspring, germ-free female mice were monocolonized with BerA / Bor Bt, time-mated with germ-free male mice, and the resulting pregnant dams were fed with MeHg-rich diets containing 20% bluefin tuna throughout gestation (FIG. 3A). There were no differences between experimental groups in gross fetal outcome measures, including litter size, viability, and deformities, on E18.5 (FIG. 6A and 6B). However, dams colonized with BtmerA / Bexhibited significantly lower MeHg in maternal colon content, but not maternal liver (FIG. 3B), aligning with previous results from adult non-pregnant mice fed MeHg-rich diets (FIGs. 2G and 2H). There were also no differences between groups in levels of MeHg in the decidua, which forms the maternal compartment of the placenta. However, dams colonized with BerA / Bexhibited significant reductions in MeHg in the placental labyrinth (FIG. 3B), the fetal compartment of the placenta responsible for nutrient exchange between the mother and fetus. Levels of MeHg in the placental labyrinth correlated with MeHg levels in the fetal brain (FIG. 3C), which were modestly reduced in fetuses from dams colonized with BfnerA / Bcompared to Bt controls (FIG. 3B). Additionally,Attorney Docket No. : UCH-42725

[0164] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0165] levels of MeHg in maternal liver correlate with those in fetal brain, suggesting that decreasing MeHg in the maternal circulation limits fetal MeHg burden (Figure 6B). These decreases in MeHg in fetal but not maternal tissues could be explained by the preferential distribution of MeHg to fetuses and placentas. Taken together, these findings demonstrate that BerA / Bsimilarly lowers intestinal MeHg in both pregnant dams and non-pregnant mice, but only modestly limits the tissue bioaccumulation of MeHg when chronically exposed to high levels of MeHg.

[0166] Human consumption of dietary MeHg typically occurs in low to moderate doses, where regular consumption of smaller fish can lead to cumulative health effects over time. To model scenarios closer to real-world chronic dietary exposure to MeHg, the effects of BerA / Bin pregnant dams fed custom diets formulated with 20% salmon (2.39 ng Hg / g hydrated diet) throughout gestation, amounting to a dose of approximately 0.37 pg / kg per day (~50x less than in the MeHg-rich bluefin tuna-based diets) were examined (FIG. 3D). There were no differences in gross fetal outcomes from BfnerA / B- and Bt-colonized dams (FIG. 6C-6F). Consistent with results from MeHg-rich bluefin-tuna-based diets, pregnant dams colonized with Blme,A Bexhibited reduced MeHg in colon content compared to Bt controls (FIG. 3E).

[0167] Notably, significant decreases in MeHg were also seen in maternal liver, placental decidua, placental labyrinth, and fetal brains from dams colonized with BfnerA / B(FIGs. 6D, 3E and 3F). These results suggest that BerA / Beffectively limits MeHg bioaccumulation in response toxicologically relevant exposure levels.

[0168] Statistically significant reductions in MeHg levels do not necessarily equate to biologically meaningful reductions in MeHg-induced physiological abnormalities. To gain insight into whether the lower levels of MeHg seen with maternal Bf"erA / Bcolonization led to improved functional outcomes, the transcriptomes of fetal brains from Bt- or BfnerA / B-colonized dams fed diets containing 20% bluefin tuna (“MeHg-rich”), salmon (“MeHg-modesf ’), or casein as a protein source containing no MeHg (“standard”), were profiled. Compared to controls fed standard diets, maternal consumption of the MeHg-rich diet during pregnancy yielded fetal brains with differential expression of 1593 genes, whereas maternal consumption of the MeHg-modest diet induced fetal brain alterations in the expression of 724 genes (FIG. 7A-F), with no other differences across groups in gross fetal outcomes (FIG. 6).

[0169] Gene set enrichment analysis revealed alterations in multiple pathways that were commonly enriched by both MeHg diets, including nonsense-mediated decay, translation initiation, and ribosomal protein and RNA pathways (FIG. 4A, 1stand 3rdcolumns), which might reflectAttorney Docket No. : UCH-42725

[0170] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0171] compensatory transcriptional responses to MeHg-induced defects in protein translation. Maternal consumption of MeHg, and particularly the MeHg-modest diet, also led to upregulated fetal brain expression of genes related to cellular stress, dysregulated protein translation, and cell cycle (i.e., G2 / M checkpoint, Hedgehog “off’ state, GTSE1, SCF(Skp2)-p21 related pathways that govern G2 / M and Gl / S progression) (FIG. 4A, 1stand 3rdcolumns). This aligns with reported MeHg-induced increases in reactive oxygen species and cell cycle dysfunction due to delays in the G2 / M transition through p21. Notably, the aforementioned transcriptional signatures of MeHg exposure in the fetal brain were reversed by maternal colonization with BfnerA / Bcompared with Bt (FIG. 4A, 2ndand 4thcolumns, FIG.

[0172] 7C and 7G), suggesting that even modest decreases in fetal brain MeHg help to alleviate MeHg-induced abnormalities in neurodevelopmental cell states. Some differential pathways were rescued only in the MeHg-modest diet condition, including iron uptake and transport, and GLP1 and adrenaline / noradrenaline regulation of insulin levels, which parallel existing studies reporting that modulating the GLP1 pathway protects against MeHg-induced neurotoxicity and that MeHg exposure promotes ferroptosis (iron-dependent cell death) and dysregulated energy metabolism. To gain further insight into the biological states represented by the transcriptomic data, they were analyzed according to key features of aging in brain tissue from the single-cell transcriptomic atlas Tabula Muris Senis. Both MeHg diet conditions yielded fetal brains with increased expression of gene sets related to aging of microglia, oligodendrocytes, pericytes, and endothelial cells, which were attenuated by maternal colonization with BlmerA Bcompared with Bt (FIG. 4B). Consistent with the former, fetuses from dams colonized with BfnerA / Bexhibited elevated density of IbaU microglia in the hippocampus (FIG. 4D), a primary site affected by MeHg exposure, when compared to Bt controls. Gene-level results from fetal brain RNAseq also showed several microglial genes (Gains, Oplm!, Npm 7, and RpH8a) that were modulated by dietary MeHg. Gains encodes galactosamine-6-sulfatase, which is involved in lysosomal degradation of glycosaminoglycans, which contains sulfur groups that mercury could bind strongly; Ophnl encodes Rho-GTPase-activating protein, which is implicated in neurogenesis and dendritic spine maturation; Npml encodes a phosphoprotein that has broad functionality, including suppressing apoptosis and mitigating cellular oxidative stress; Rpll8a forms the 60S ribosomal subunit and is downregulated in neural stem cells in response to acute DNA damage. There were no significant differences between groups in fetal hippocampal levels of markers for apoptosis (TUNEL),cell proliferation (Ki67) or neuronal progenitor cells / neuralAttorney Docket No. : UCH-42725

[0173] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0174] stem cells (Nestin) (FIG. 4C, 7K). IbaU cell counts were inversely associated with levels of fetal brain MeHg in littermates (FIG. 4E), suggesting that Bf'erA / B-Am&n reductions in MeHg bioaccumulation directly in the fetal brain promote levels of neuroprotective microglia and / or protect against microglial cell dealth. Overall, these data reveal that maternal colonization with BerA / Breduces tissue burden of MeHg with effect sizes that protect against adverse transcriptomic and cellular outcomes in the fetal brain.

[0175] Bfn rA / B decreases MeHg in adult mice with intact gut microbiome and can colonize stably when co-colonized with wild-type Bt

[0176] To determine the effectiveness of Bf"erA / Bin the context of a native gut microbiome, BfnerA / Bor Bt were administered by daily oral gavage to specific-pathogen-free (SPF) mice concurrently with MeHg-rich diets containing 20% bluefin tuna (Figure 8A). Blmer Bdecreased MeHg in liver tissue, but not in fecal or colon content (Figure 8B and 8D), suggesting that in the presence of a native microbiome, probiotic treatment with BerA / Bmore effectively limits extraintestinal accumulation of MeHg. Additionally, the levels of BerA / Bremained high throughout the daily administration (Figure 8C), suggesting that they maintain presence at least within 16 hours between oral gavage and fecal sampling, above the typical mouse gut transit time of 6-8 hours. The apparent lack of effectiveness of BerA / Bin colonic content and feces could be due to the low abundance of Bt"'e'A Bin the colon of SPF mice compared to monocolonized (Figure 8G). In contrast, the increased effectiveness of BerA / Bfor reducing liver MeHg might be due to interactions between BtmejA Band native microbes in the upper gastrointestinal tract which is the main site of MeHg absorption.

[0177] The ability of BerA / Bto colonize and compete with wild-type Bt or SPF mouse fecal microbes was assessed by colonizing germ-free mice with Blme,ABlme,A B+ Bt mixture (1:1, v / v), or Bti,le,A B+ fecal slurry mixture (1:20, w / w) (Figure 8D). When co-administered with wild-type Bt or fecal microbiome, BerA / Bcolonized and stably persisted over 10 days, albeit at a lower abundance than seen with monocolonization (Figure 8E). Additionally, BerA / Bmaintained presence along the gastrointestinal tract (Figure 8G), suggesting it could prevent early absorption of MeHg in the small intestine. These showed that Btine'A Bcould stably persist upon engraftment in the gut microbiome. Overall, these results support the potential value of the engineered bacterial approach for reducing MeHg in subjects with a normal microbiome.Attorney Docket No. : UCH-42725

[0178] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0179] Summary

[0180] This disclosure demonstrates that an engineered gut bacterium can demethylate MeHg, lower MeHg accumulation in host tissues, and mitigate adverse physiological outcomes in monocolonized mice exposed to dietary MeHg during pregnancy. BerA / Bexhibited persistent reductive MeHg demethylation activity, which was highly effective in decreasing MeHg locally within the mouse intestine, and showed promise for decreasing tissue burden of MeHg, particularly in distant extraintestinal sites (i.e., the adult brain, placenta, and fetal brain). tme'-'11showed efficacy when tested in two translationally relevant contexts - chronic ingestion of MeHg derived from fish sold for human consumption, as the most common route of MeHg exposure, and in pregnant dams and their developing fetuses, as groups that are particularly vulnerable to adverse effects of MeHg. Targeted MeHg demethylation and reduction by tme'-'11mitigates exposures to MeHg already in seafood, by converting MeHg into less bioaccessible species, Hg2+or Hg° for excretion.

[0181] BfnerA / Bconsjstently and effectively lowered MeHg in intestinal contents of MeHg-exposed mice. Data from the study suggest that BerA / Bwas best in mitigating MeHg bioaccumulation from exposures that were prolonged and of lower dosages, particularly for tissues that were more protected or distal from the exposure site (i.e., brain, placenta, and fetal brain, as opposed to colon and liver). The results raise the possibility that any muted effects of BerA / Bare due to high exposure levels of MeHg that overload the processing capacity of the bacteria. Indeed, the effective dosages of MeHg used in the disclosed animal studies (250 pg / kg for MeHg-Cl, 18.75 pg / kg / day for MeHg-rich diet, 0.37 pg / kg / day for MeHg-modest diet) exceed those reported for human exposure to dietary MeHg, which range from 0.25 ng / kg / day in inland countries with limited seafood consumption to 0.25 pg / kg / day in countries with high seafood consumption. They also surpass the United States Environmental Protection Agency’s safe limit of 0.1 ug / kg / day, which aims to protect sensitive populations, such as pregnant women and young children, from the neurotoxic effects of MeHg. These comparisons, together with the empirical data, demonstrate that BfnerA / Beffectively mitigated the effects of chronic exposure to relevant levels of MeHg.

[0182] BfnerA / B significantly attenuated MeHg-induced transcriptomic and cellular alterations in the fetal brain, toward signatures seen in non-exposed mice. Generally, the fetus exhibits high susceptibility towards MeHg exposure, as animal and human epidemiological studies of MeHg exposure commonly report higher levels of MeHg in fetal cord blood than in maternal blood, suggesting preferential MeHg bioaccumulation in the fetus. The effects of chronicAttorney Docket No. : UCH-42725

[0183] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0184] dietary exposure to MeHg during gestation vary across study populations; however, a growing body of research suggests that domains of attention / hyperactivity, memory, visuospatial and motor outcomes are affected in the developing child. Even at non-toxic levels, exposure to MeHg has been associated with various psychomotor and cognitive defects. The present disclosure indicates that Btme'-'11-driven reductions in MeHg bioaccumulation attenuate molecular hallmarks of MeHg exposure in the fetal brain, including features of oxidative damage, impaired cell cycle regulation, and altered microglial development. These results provide foundational evidence supporting a functional role for BfnerA / Binlimiting adverse physiological outcomes of MeHg exposure.

[0185] Example 2: Methods associated with Example 1

[0186] Bacterial strains and culturing

[0187] Wild-type B. thetaiotaomicron (VPI-5482, ATCC) was used as the chassis to express demethylation genes. Escherichia coli S17 X-pir (BAA-2428, ATCC) was used to as conjugation partner for transferring plasmid into B. thetaiotaomicron. B. thetaiotaomicron was cultured in BHIS — Brain Heart Infusion (BD) supplemented with 5 pg / ml hemin and vitamin KI (Sigma-Aldrich) — in an anaerobic atmosphere of 85% nitrogen, 10% carbon dioxide, and 5% hydrogen, while E. coli was cultured in LB (Sigma- Aldrich). Growth curves were conducted in Minimal media (MM), which contains 100 mM potassium phosphate buffer (pH 7.2), 7.5 mM (NH^SCU, 9.4 mM Na2CO3, 4mM L-cysteine, 1.4 pM FeSC>4.7(H2O), 1 pg / ml vitamin K3, 5 ng / ml vitamin Bn, 1.9 pM hematin / 200 pM L-histidine, 15 mMNaCl, 180 pM CaCl2.2H2O, 98 pM MgCl2.6H2O, 50 pM MnCl2.4H2O, and 42 pM CoCl2.6H2O. For all growth curves, single colonies of bacteria were grown overnight in liquid BHIS, sub-cultured 1 : 1000 to synchronize their growth for 2 hours, washed with MM, and sub-cultured 1:25 into MM for 12 hours. Growth curves were obtained by sampling 200 pl from each tube and reading OD600 with a Synergy Hl plate reader at each timepoint. Additionally, growth curves by counting colony forming units (CFUs) were obtained by sampling 10 pl from each tube, performing serial dilutions, and plating 10 pl of each serial dilution on BHIS plates at each timepoint.

[0188] Mice

[0189] Mice used for data collection were male and female germ-free (GF) wild-type Swiss Webster mice, at least 7-8 weeks of age. GF Swiss Webster mice were purchased fromAttorney Docket No. : UCH-42725

[0190] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0191] Laconic Farms and bred in flexible film isolators at the UCLA Goodman-Luskin Microbiome Center Gnotobiotics Core Facility. Specific-pathogen free (SPF) mice for daily gavage of engineered microbe paradigm were purchased at 4-5 weeks old from Laconic Farms and were acclimatize to UCLA animal facility for at least 1 week. Mice were housed on a 12-h lightdark schedule in a temperature-controlled (22-25 °C) and humidity-controlled environment with ad libitum access to water and sterile “breeder” chow (5K52, Lab Diets) or experimental diets as described below.

[0192] B. thetaiotaomicron engineering and in vitro activity testing

[0193] BtmerA / Bmutant was generated using genomic integration with pNBU2-based plasmid pWW3837 as described previously and counterselectable allelic exchange with pSIEl (Addgene plasmid #136355) as described previously. MerA and merB sequences were derived from Hg-resistant . aeruginosa found in polluted gold mines. Briefly, MerA and MerB sequences were synthesized de novo (Lwist Biosciences) and was ligated, together with upstream and downstream genomic overlap sequences (1000 bp) and constitutive promoter pBfPlE6, into pWW3837 or pSIEl respectively via Gibson assembly (GeneArt Hifi Assembly, Lhermo Fisher Scientific). Constructed plasmid was then transformed via heat shock and amplified inE. coli S17 -pir, before sequentially conjugated into Pt. B. thetaiotaomicron colonies that had received the vector were selected with gentamicin (200 pg / ml) and erythromycin (12.5 pg / ml), and counterselection was performed with anhydrotetracycline (100 ng / ml). After two rounds of countersei ection, the desired mutants with genomic integration of MerA and MerB were identified by PCR screening.

[0194] Lo test the heterologous expression and activity of MerA and MerB, BfierA / B

[0195]

[0196] cultured in MM as described above. Notably, MM was supplemented with MeHg-Cl until a final concentration of 5 ng / mL. Culture was sampled every 1-2 hours for MeHg measurements by pipetting 200 pl into 50 ml 0.14% HC1. Additionally, the efficacy of BtmerA / B indemethylating MeHg in complex, human-relevant, matrix -digested bluefin tuna tissue- was tested. Luna tissue was prepared and powdered as described below and was digested following INFOGESL in vitro digestion protocol. BfnerA / Bwas cultured in MM supplemented with digested tuna tissue until final MeHg concentration of 2.5 ng / mL. Culture was sampled every 1-2 hours for MeHg measurements.

[0197] Table 2. Oligonucleotides

[0198]

[0199] Attorney Docket No. : UCH-42725

[0200] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0201]

[0202] In vitro samples MeHg analysis

[0203] In vitro samples were first diluted with ultrapure H20-HC1 solution (0.14% v / v) at 1 :500 ratio and stored at 4°C until analysis. MeHg was measured with isotope dilution (ID) using a synthesized Me201Hg tracer as previously described. Me201Hg working solutions (ca.

[0204] 70 ng / mL, 15 ng / mL, 1 ng / mL) were quantified using reverse isotope dilution with standards prepared from a certified 1000 ppm MeHg (II) chloride standard (Alfa Aesar). Samples were further diluted 1 :225 with ultrapure H2O, and spiked with Me201Hg tracer and ethylated using ascorbic acid - sodium tetraethylborate (1% NaTEB in 2% potassium hydroxide, Strem Chemicals) protocol prior to analysis. Processed samples were purged with Hg-free ultra-high purity argon gas, concentrated on a Carbotrap (graphitized carbon black, Sigma-Aldrich), and analyzed using an automated Tekran 2700 coupled to an Agilent 8900 ICP-MS. Calculation of MeHg was conducted after signal deconvolution of201Hg and202Hg peak areas.Attorney Docket No. : UCH-42725

[0205] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0206] t%, defined as time needed for 50% reduction of MeHg, was calculated by first fitting a first-order decay regression model on natural log transformed and time 0 normalized MeHg concentrations over time.

[0207]

[0208] Reduction rate constant (k) derived from the model was then used to estimate the time needed for 50% reduction of MeHg (t%).

[0209]

[0210] Fish and diet preparation

[0211] A 23 kg freshly caught Pacific bluefin tuna was purchased at the Tuna Harbor Dockside Market in San Diego and dissected using a filleting knife. The fillets were skinned and ground using a KitchenAid® mixer with a meat grinder attachment. The ground fish meat was then dried in batches at 50 °C over three days, with regular stirring. Once dried, the meat was powdered using the mixer with a grain mill attachment, yielding approximately 2 kg of bluefin tuna powder containing 736 ng Hg per gram dry weight. The salmon meal was prepared using approximately 1kg of commercially obtained raw freeze-dried farmed salmon containing no additive, salt or any other preservatives (Military Surplus Meals). Salmon filets were powdered using the KitchenAid® mixer with a meat grinder attachment. Salmon powder contained 13.9 ng Hg per gram dry weight. Sterility of powdered fishes were assessed by plating with Schaedler Anaerobic Agar prior to usage (BD Biosciences). For dietary exposure experiments, diets were prepared every week by incorporating fish meals into a protein-free mice diet (TD.210869.PWD, Envigo) at 1:4 ratio. Prepared diets were analyzed for MeHg, yielding final concentrations of 107.14 ± 10.4 ng / g of hydrated tunabased diet and 2.39 ± 0.60 ng / g of hydrated salmon-based diet.

[0212] In vivo acute and dietary exposure of MeHg to mono-colonized mice

[0213] Prior to monocolonization of germ-free mice, Bt or BlmerAwere first streaked, and a single colony was grown overnight in BHIS until OD600 of 0.6-0.7. Germ-free female mice were then gavaged 200 pL of culture, weighted, and left 3 days for bacterial colonization prior to MeHg treatment. Mice were then kept in cages containing 100 pg / ml gentamicin-supplemented water to prevent contamination by other species.

[0214] Acute exposure of MeHg consists of a single dose of MeHg-Cl diluted in H2O-L-cysteine solution until final dosage of 250 pg MeHg / kg body weight (b.w.) per mice. ForAttorney Docket No. : UCH-42725

[0215] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0216] toxicokinetics study, mouse feces were sampled every 3 hours for 24 hours post-oral exposure. Feces were sent for MeHg analysis with methods described below. In a different cohort, mono-colonized mice were dosed, then euthanized and dissected them after 4 days. Colon content, colon tissue, liver (center lobe), and brain were harvested and snap-frozen with liquid nitrogen for MeHg and total mercury (THg) measurements as described below. Mono-colonized status of mice was checked by serially diluting mice fecal samples in BHIS on BHIS+100 pg / ml gentamicin plates and observing whether there was plurality of colony morphology when grown aerobically and anaerobically. Liver was fixed in formalin, embedded in paraffin, and stained with H&E staining prior to damage scoring based on Suzuki classification.

[0217] For experiments involving dietary exposures, germ-free female mice were first monocolonized with Bt or BfnerA / Bas described above and left to acclimatize to the fish diet odor for 3 days together with bacterial colonization. Fish diets were freshly prepared every week of the experiment. For 10 days, mice were fed solely on the fish diet and dietary intake was measured by weighing leftover diet at the end of the exposure course. At the end of the experiment, mice were euthanized and their organs (colon content, colon tissue, liver center lobe, and brain) were harvested for MeHg and THg measurements.

[0218] Tissue measurements of total mercury (THg) and MeHg

[0219] For THg analysis, samples were thawed for 20 min, and subsamples weighing ca. 50-80 mg were analyzed using direct thermal decomposition-gold amalgamation-cold vapor atomic absorption spectroscopy on a Nippon Instruments Corporation MA-3000. Each batch included certified reference materials (CRMs) DORM-4 fish protein and TORT-3 lobster hepatopancreas, which had average measured concentrations of 386 ± 13 ng / g (n = 12) and 277 ± 8 ng / g (n = 12), respectively. Certified THg concentrations for CRMs DORM-4 and TORT-3 are 412 ± 36 ng / g and 292 ± 22 ng / g, respectively. Samples were prepared for MeHg analysis by microwave-assisted acid digestion with isotope dilution using a synthesized Me201Hg tracer as described above. Briefly, samples were digested with 5 M nitric acid (TraceMetal Grade, Fisher Chemical) spiked with an appropriate, known quantity of Me201Hg prior to digestion. An optimal202Hg / 201Hg mixing ratio of 0.23 was targeted for all samples to minimize uncertainty in signal deconvolution. Microwave digestion was carried out using an Anton Paar Multiwave 5000 programmed to heat samples to 70°C for 10 min following a 5 min ramp up. Sample digestates were diluted (2x-4x) with ultrapure water (Milli-Q, 18.2 MQ cm-1) based on expected MeHg concentration and stored at 4°C prior toAttorney Docket No. : UCH-42725

[0220] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0221] analysis with ICP-MS method described above. Each batch prepared for microwave digestion included either CRMs DORM-4 and TORT-3 or two replicates of standard reference material (SRM) 2974a mussel tissue, depending on which reference material(s) most closely matched expected sample MeHg concentration. Extraction efficiency when comparing measured MeHg content and reference MeHg amount was shown to be >90%. Results were analyzed with GraphPad Prism (v9.0.0).

[0222] In vivo dietary exposure of MeHg to pregnant dams

[0223] Four to five weeks-old germ-free female mice were mono-colonized with either Bt or BfnerA / Bvia oral gavage as described above. 2 mono-colonized females were then co-housed with 1 germ-free male each cage. Occurrence of cervical plugs were checked every day to determine E0.5 of potential pregnancy, and plugged females were immediately single housed and fed exclusively fish diet for 18 days. Fish diets were freshly prepared every week of the experiment, and dietary intake was measured longitudinally. Weight of plugged female mice were measured at days 0, 10, and 14 after observation of cervical plug to confirm pregnancy.

[0224] Maternal and fetal tissue collections

[0225] At El 8.5, dams were weighed and then euthanized by cervical dislocation. Fecal samples were extracted from the dam colon. Blood was collected by cardiac puncture into vacutainer SST tubes (Becton Dickinson), allowed to clot for 30 min at room temperature, before centrifuging at 1500xg, 4°C for 10 minutes. Serum supernatant was collected, snap-frozen, and stored at -80°C. The entire uterine horn, including all conceptuses, was removed and placed in ice cold lx PBS. Dams were weighed once again to record post-transection weight. Colon tissue, liver center lobe, and brain were collected from dissected dams for MeHg and THg measurements.

[0226] Each fetus and placenta were dissected from the amniotic sac and weighed. Placentas were separated into maternal (decidua) and fetal (labyrinth) compartments and snap-frozen in liquid nitrogen. Whole fetal brains were microdissected and either placed into RNAtoer (Invitrogen) for subsequent RNA sequencing, snap-frozen in liquid nitrogen for subsequent mercury quantification analysis or fixed in 4% PFA for subsequent immunostaining. All tissue samples were stored at -80°C. A total of 3 pairs of fetal brain, placental decidua, and placental labyrinth were collected per dam for MeHg quantification analysis.Attorney Docket No. : UCH-42725

[0227] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0228] Fetal brain RNA sequencing

[0229] Whole fetal brains were randomly chosen from each litter, dissected, and immediately placed into 400pL RNAtoer (ThermoFisher Scientific) and stored at -80°C. RNA was extracted using the PureLink RNA Mini Kit (Invitrogen). Equal amounts of RNA were pooled from 2 brains per litter, resulting in 3000ng RNA per sample. An RNA integrity number (RIN) of at least 9.7 was confirmed for each sample using the 4200 Tapestation system (Agilent). Paired end sequencing (150 bp) was performed using the Illumina NovaSeq X Plus platform by the UCLA Neuroscience Genomics Core.

[0230] Raw paired end sequences were quality filtered and trimmed using fastqc vO.12.1. Processed reads were mapped to mouse GENCODE primary assembly (GRCm39) using STAR v2.7.0a and quantified using Salmon vl .10.1. Quantified transcript was imported into R and summarized to gene counts using tximport vl.32.0. Differential expression analysis was conducted using DESeq2 vl.46.0 and gene set enrichment analysis was conducted using signed non-adjusted p-values derived from DESeq2 analysis with fgsea vl.32.0 against mouse MSigDB M2 CP (containing Reactome, Biocarta, and Wikipathway pathways) and curated M8 pathways (containing brain / neuron-specific and organogenesis cellular markers from Tabula Muris Senis and Descartes dataset). Significant pathways were then collapsed into independent pathways using collapsedPathways function within fgsea.

[0231] Fetal brain tissue staining

[0232] El 8.5 fetal brains were fixed in 4% PF A for 48 hours at 4°C, cryoprotected in 30% sucrose in PBS at 4°C until saturated, embedded in OCT (TissueTek), and serially sectioned at 15 pm, using a Leica CM1950 cryostat. Sagittal slices were placed directly onto coated slides and stored at -20°C until staining. Antigen retrieval was performed using 10% citrate buffer (Agilent) at 95°C for 20 minutes then at room temperature for 15 minutes. Sections were blocked with 10% goat serum for 1 hour at room temperature, then incubated with Ki67 (1:150, ThermoFisher Scientific) and Ibal (1:500, Fujifilm) for 16 hours at 4°C. Sections were then incubated for 1 hour at room temperature with anti-chicken, anti-rat, and antirabbit secondary antibodies (1:1000, Abeam) and DAPI (1:500, Invitrogen). TUNEL staining was performed according to manufacturer’s instructions (Click-iT™ Plus TUNEL Assay, ThermoFisher). Slides were cover-slipped with ProLong Gold (Invitrogen) and allowed to dry before imaging.Attorney Docket No. : UCH-42725

[0233] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0234] Image acquisition and analysis

[0235] All images were acquired using a Zeiss Axio Examiner LSM 780 confocal microscope with a 20x objective. 8 Z-stack images at 1.6 um intervals were acquired using Zen Black 2012 software (Zeiss). Images were stitched with a 10% overlap, and the complete range of z series was orthogonally projected using Zen Blue 2021 software (Zeiss). Prior to analysis and cell counting, images were blinded by a second researcher to decrease potential bias. All image analysis was done using ImageJ (NIH). ROIs for hippocampal subregions were drawn manually using the Allen Brain Atlas for developing 18.5 mouse brain (Sagittal, MPall, images #8-11) and confirmed by a second researcher. Proliferating cells were quantified by counting colocalization of Ki67 and DAPI. Apoptotic cells were quantified by counting colocalization of TUNEL and DAPI. Microglia were quantified by counting colocalization of Ibal and DAPI. All measures were normalized to DAPI tissue coverage area. Two technical replicates per fetal brain were quantified and averaged to obtain a biological n = 1. For Ki67 and Ibal quantification, n = 6-7 fetal brains per group, and for TUNEL quantification, n = 3-4 fetal brains per group.

[0236] In vivo dietary MeHg exposure to SPF mice administered with engineered bacteria SPF mice were administered Bt or BlmerA Bdaily for 10 days together with ad libitum feeding of MeHg-rich diet (20% bluefin tuna). Every day, a single colony of Bt or BfnerA / Bwas grown overnight in BHIS media until OD600 of 1.0 - 1.1 and 200 pL of culture was administered to mice via oral gavage in the evening to match their awake / feeding time. Fecal pellets were collected on day 3, 7, and 10 for MeHg measurement. On day 10, mice were euthanized, and colon content and liver were collected for MeHg and THg measurements.

[0237] In vivo co-colonization of BtmerA / Bwith Bt or SPF fecal slurry

[0238] Prior to colonization, fresh fecal samples from SPF mice (age 5-6 weeks) were collected and weighted. Fecal samples were then diluted with pre-reduced PBS (final concentration 25 mg / mL) inside the anaerobic chamber and filtered through 70 pm strainer (Corning) to remove undigested materials. Fecal slurry was then mixed with an overnight BfierA / Bculture (OD600 1.0-1.1) at 1 :20 ratio (v / v). Additionally, a mixture of Bt and BtmerAABwas created by combining overnight culture (OD600 1.0-1.1) at 1:1 ratio (v / v)._Germ-free female mice were then gavaged 200 pL of either_71 / "'c'l / y, BtmerA'B+ Bt, or Bt"erA B+ fecalAttorney Docket No. : UCH-42725

[0239] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0240] slurry. Fecal samples were collected at day 1, 3, 5, 7, and 10 post-colonization for downstream qPCR of merB gene.

[0241] Quantification of

[0242]

[0243] Band Bt via quantitative PCR

[0244] Total DNA was extracted from fecal pellets following standard procedures for DNeasy PowerSoil Pro Kit (Qiagen). Amount of BfnerA / Bwere quantified using primer pair generated from Primer-BLAST93against merB gene sequence while excluding bacterial genomes (taxid:2). Primer pair targeting Bt (Pan-Bt) for fecal samples was developed elsewhere. DNA extracted from BfnerA / Bovernight culture was used to generate a standard curve. Copy number of standard DNA was calculated by multiplying amount of DNA (ng) with Avogadro’s constant and dividing by length of qPCR product and average mass of 1 base pair of DNA (6.6E11 ng / mol). Quantitative PCR was performed using recommended thermocycling conditions of PowerUp SYBR Green Master Mix (ThermoFisher Scientific) with final reaction volume of 10 pL and primer concentrations of 400 nM. Each sample was run in technical replicates. DNA copy number for each sample was calculated based on the standard curve and normalized to template DNA concentration. Limit of detection (LOD) was calculated based on the lowest dilution of standard curve where 90% of replicates were detected.

[0245] Statistical Analysis

[0246] All statistical analysis, except for RNASeq dataset, was performed using Prism software v9.0.0 (GraphPad). Data were plotted in the FIGs. as mean ± SD. For each FIG., n = number of independent biological replicates. Differences between two treatment groups were assessed using two-tailed, unpaired Student’s t test. Differences among >2 groups with two variables were assessed using two-way ANOVA followed by Dunnet’s post-hoc multiple comparisons test against control group. Significant differences emerging from the above tests are indicated in the FIGs. by *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Notable non-significant (and non-near significant) differences where p > 0.20 are indicated in the FIGs. by “n.s ”

[0247] Statistical analysis of RNAseq dataset was conducted with R. DESeq2 and fgsea packges were used to generate differential gene expression and differential pathways respectively. Signed (based on DESeq2 log2fold change results), unadjusted p-value were used as gene ranks for GSEA analysis and collapsing non-independent pathways. Figures were made with ggplot2 v3.5.1.Attorney Docket No. : UCH-42725

[0248] UCLA Ref. No.: [UCLA 2023-188-2] WO

[0249] INCORPORATION BY REFERENCE

[0250] All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0251] EQUIVALENTS

[0252] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

Claims

Attorney Docket No. : UCH-42725UCLA Ref. No.: [UCLA 2023-188-2] WOCLAIMSWe claim:

1. A bacterium comprising a gene encoding a methylmercury-degrading enzyme, wherein the gene is encoded by an exogenous nucleic acid.

2. The bacterium of claim 1, wherein the bacterium comprises an additional gene encoding a methylmercury-degrading enzyme, wherein the additional gene is encoded by an exogenous nucleic acid.

3. The bacterium of claim 1 or claim 2, wherein the gene or the additional gene encoding a methylmercury-degrading enzyme encodes a mercuric reductase (MerA).

4. The bacterium of claim 3, wherein the mercuric reductase has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to Pseudomonas aeruginosa MerA5. The bacterium of claim 4, wherein the mercuric reductase is Pseudomonas aeruginosa MerA6. The bacterium of any preceding claim, wherein the gene or the additional gene encoding a methylmercury-degrading enzyme encodes an organomercury lyase (MerB).

7. The bacterium of claim 6, wherein the organomercury lyase has at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to Pseudomonas aeruginosa MerB.

8. The bacterium of claim 7, wherein the organomercury lyase is Pseudomonas aeruginosa MerB.

9. The bacterium of any preceding claim, wherein the bacterium is of the Bacteroides genus.

10. The bacterium of claim 9, wherein the bacterium is of the species Bacteroides thetaiotaomicron.Attorney Docket No. : UCH-42725UCLA Ref. No.: [UCLA 2023-188-2] WO11. The bacterium of claim 10, wherein the bacterium is of the strain Bacteroides thetaiotaomicron VPI-5482.

12. A method of detoxifying or demethylating mercury and / or methylmercury in the gastrointestinal tract of a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

13. A method of preventing mercury and / or methylmercury accumulation in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

14. The method of claim 13, wherein the mercury and / or methylmercury accumulation is in the liver of the subject.

15. A method of preventing mercury and / or methylmercury accumulation in the liver of a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

16. A method of reducing mercury and / or methylmercury levels in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

17. The method of claim 16, wherein the mercury and / or methylmercury is in the liver of the subject.

18. A method of reducing mercury and / or methylmercury levels in the liver of a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

19. A method of treating a subject with mercury and / or methylmercury accumulation, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

20. A method of treating or preventing brain damage due to mercury and / or methylmercury exposure in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.Attorney Docket No. : UCH-42725UCLA Ref. No.: [UCLA 2023-188-2] WO21. A method of treating or preventing cognitive impairment after mercury and / or methylmercury exposure in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

22. A method of treating or preventing cognitive impairment due to mercury and / or methylmercury exposure in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

23. A method of treating mercury and / or methylmercury poisoning in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

24. A method of treating or preventing brain damage mercury and / or methylmercury poisoning in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

25. A method of treating or preventing cognitive impairment due to mercury and / or methylmercury poisoning in a subject, the method comprising administering to the subject a composition comprising the bacterium of any one of claims 1-11.

26. The method of any one of claims 12-25, wherein the subject is an adult.

27. The method of any one of claims 12-25, wherein the subject is a gestating a fetus.

28. The method of any one of claims 12-25, wherein the subject is a fetus.

29. The method of any one of claims 12-25, wherein the subject is a pediatric subject.

30. A method of promoting healthy neural development in a fetus, the method comprising administering to a subject gestating the fetus a composition comprising the bacterium of any one of claims 1-11.

31. A method of preventing impaired neural development in a fetus, the method comprising administering to a subject gestating the fetus a composition comprising the bacterium of any one of claims 1-11.

32. The method of claim 30 or 31, wherein the subject has been exposed to mercury and / or methylmercury.Attorney Docket No. : UCH-42725UCLA Ref. No.: [UCLA 2023-188-2] WO33 The method of any one of claims 12-32, wherein the subject is or has been exposed to mercury and / or methylmercury in their diet (e.g., high-mercury fish).

34. The method of any one of claims 12-33, wherein the subject is or has been exposed to mercury and / or methylmercury in their environment (e.g., water supply).

35. The method of any one of claims 12-34, wherein the composition is formulated for oral delivery.

36. The method of claim 35, wherein the composition is a food product.

37. The method of claim 36, wherein the food product is a dairy product.

38. The method of claim 37, wherein the food product is yogurt.

39. The method of any one of claims 12-38, wherein the composition is formulated for rectal delivery.

40. The method of any one of claims 12-39, wherein the composition is for selfadministration.

41. The method of any one of claims 12-40, wherein the subject is a human.

42. The method of claim 30, wherein healthy neural development comprises a reduction in anxiety-like behavioral deficits.

43. The method of claim 30, wherein healthy neural development comprises prevention of learning and memory deficits.

Images