Mucosal TLR5 activation controls healthspan and longevity.

Addressing age-related immunological defects through therapeutic interventions is essential for healthy aging, as the immune system plays a crucial role in controlling infections, malignancies, and in supporting tissue homeostasis and repair. In our study, we show that stimulating toll-like receptor 5 (TLR5) via mucosal delivery of a flagellin-containing fusion protein effectively extends the lifespan and enhances the healthspan of mice of both sexes. This enhancement in healthspan is evidenced by diminished hair loss and ocular lens opacity, increased bone mineral density, improved stem cell activity, delayed thymic involution, heightened cognitive capacity, and the prevention of pulmonary lung fibrosis. Additionally, this fusion protein boosts intestinal mucosal integrity by augmenting the surface expression of TLR5 in a certain subset of dendritic cells and increasing interleukin-22 (IL-22) secretion. In this work, we present observations that underscore the benefits of TLR5-dependent stimulation in the mucosal compartment, suggesting a viable strategy for enhancing longevity and healthspan.

Bacterial cancer therapy using the attenuated fowl-adapted Salmonella enterica serovar Gallinarum.

We report here a novel anti-cancer therapy based on an avian-host-specific serotype Salmonella enterica serovar Gallinarum (S. Gallinarum) deficient in ppGpp synthesis. To monitor the tumor targeting, a bioluminescent ΔppGpp S. Gallinarum was constructed and injected intravenously into mice bearing syngeneic and human xenograft tumors. Strong bioluminescent signals were detected specifically in all grafted tumors at 2 days post-injection (dpi). The bacterial counts in normal and tumor tissue at 1 dpi revealed that ΔppGpp S. Gallinarum reached >108 CFU/g in tumor tissue and 106-107 CFU/g in endothelial organs; counts were much lower in other organs. At 16 dpi, ΔppGpp S. Gallinarum counts in tumor tissue decreased to ∼106 CFU/g, while those in the other organs became undetectable. A strong anti-cancer effect was observed after the injection of ΔppGpp S. Gallinarum into BALB/c mice grafted with CT26 colon cancer cells. This could be attributed to reduced virulence, which allowed the administration of at least a 10-fold greater dose (108 CFU) of ΔppGpp S. Gallinarum than other attenuated strains of S. enterica serovar Typhimurium (≤107 CFU). An advantage of the avian-specific S. Gallinarum as a cancer therapeutic should be a reduced capacity to cause infections or harm in humans.

Analysis of random mutations in Salmonella Gallinarum dihydropteroate synthase conferring sulfonamide resistance.

In bacteria and primitive eukaryotes, sulfonamide antibiotics block the folate pathway by inhibiting dihydropteroate synthase (FolP) that combines para-aminobenzoic acid (pABA) and dihydropterin pyrophosphate (DHPP) to form dihydropteroic acid (DHP), a precursor for tetrahydrofolate synthesis. However, the emergence of resistant strains has severely compromised the use of pABA mimetics as sulfonamide drugs. Salmonella enterica serovar Gallinarum (S. Gallinarum) is a significant source of antibiotic-resistant infections in poultry. Here, a sulfonamide-resistant FolP mutant library of S. Gallinarum was generated through random mutagenesis. Among resistant strains, substitution of amino acid Arginine 171 with Proline (R171P) in the FolP protein conferred the highest resistance against sulfonamide. Substitution of Phe28 with Leu or Ile (F28L/I) led to modest sulfonamide resistance. Structural modeling indicates that R171P and Phenylalanine 28 with leucine or isoleucine (F28L/I) substitution mutations are located far from the substrate-binding site and cause insignificant conformational changes in the FolP protein. Rather, in silico studies suggest that the mutations altered the stability of the protein, potentially resulting in sulfonamide resistance. Identification of specific mutations in FolP that confer resistance to sulfonamide would contribute to our understanding of the molecular mechanisms of antibiotic resistance.

Analysis of antibiotic resistance gene cassettes in a newly identified Salmonella enterica serovar Gallinarum strain in Korea.

Antimicrobial resistant pathogens are a global health threat driven by the indiscriminate use of antimicrobials. Antimicrobial resistance can be acquired by resistance genes encoded by mobile genetic elements. In this study, we identified a strain of Salmonella enterica serovar Gallinarum (SG4021) from an infected chicken in Korea and characterized the presence of resistance genes in its plasmid by whole genome sequencing. The sequence was then compared with that of a plasmid (P2) from strain SG_07Q015, the only other strain of S. Gallinarum isolated in Korea for which a genome sequence is available. The results revealed that both strains harbored nearly identical DNA carrying antibiotic resistance gene cassettes inserted into integron In2 of the transposable element Tn21, namely an aadA1 resistance gene conferring resistance to aminoglycosides and a sul1 resistance gene conferring resistance to sulfonamide. Interestingly, despite the presence of sul1 in SG4021, an antibiotic sensitivity test revealed that it was sensitive to sulfonamides. Further analysis revealed that this disparity was due to the insertion of a ~ 5 kb ISCR16 sequence downstream of the promoter driving sul1 expression in SG4021. Using various mutants, we showed that the insertion of ISCR16 blocked the expression of the sul1 gene from the upstream promoter. Therefore, the functionality of antimicrobial resistance genes determines phenotypic antimicrobial resistance.

Constitutive Expression of a Cytotoxic Anticancer Protein in Tumor-Colonizing Bacteria.

Bacterial cancer therapy is a promising next-generation modality to treat cancer that often uses tumor-colonizing bacteria to deliver cytotoxic anticancer proteins. However, the expression of cytotoxic anticancer proteins in bacteria that accumulate in the nontumoral reticuloendothelial system (RES), mainly the liver and spleen, is considered detrimental. This study examined the fate of the Escherichia coli strain MG1655 and an attenuated strain of Salmonella enterica serovar Gallinarum (S. Gallinarum) with defective ppGpp synthesis after intravenous injection into tumor-bearing mice (~108 colony forming units/animal). Approximately 10% of the injected bacteria were detected initially in the RES, whereas approximately 0.01% were in tumor tissues. The bacteria in the tumor tissue proliferated vigorously to up to 109 colony forming units/g tissue, whereas those in the RES died off. RNA analysis revealed that tumor-associated E. coli activated rrnB operon genes encoding the rRNA building block of ribosome needed most during the exponential stage of growth, whereas those in the RES expressed substantially decreased levels of this gene and were cleared soon presumably by innate immune systems. Based on this finding, we engineered ΔppGpp S. Gallinarum to express constitutively a recombinant immunotoxin comprising TGFα and the Pseudomonas exotoxin A (PE38) using a constitutive exponential phase promoter, the ribosomal RNA promoter rrnB P1. The construct exerted anticancer effects on mice grafted with mouse colon (CT26) or breast (4T1) tumor cells without any notable adverse effects, suggesting that constitutive expression of cytotoxic anticancer protein from rrnB P1 occurred only in tumor tissue.

Therapeutic strategy targeting host lipolysis limits infection by SARS-CoV-2 and influenza A virus.

The biosynthesis of host lipids and/or lipid droplets (LDs) has been studied extensively as a putative therapeutic target in diverse viral infections. However, directly targeting the LD lipolytic catabolism in virus-infected cells has not been widely investigated. Here, we show the linkage of the LD-associated lipase activation to the breakdown of LDs for the generation of free fatty acids (FFAs) at the late stage of diverse RNA viral infections, which represents a broad-spectrum antiviral target. Dysfunction of membrane transporter systems due to virus-induced cell injury results in intracellular malnutrition at the late stage of infection, thereby making the virus more dependent on the FFAs generated from LD storage for viral morphogenesis and as a source of energy. The replication of SARS-CoV-2 and influenza A virus (IAV), which is suppressed by the treatment with LD-associated lipases inhibitors, is rescued by supplementation with FFAs. The administration of lipase inhibitors, either individually or in a combination with virus-targeting drugs, protects mice from lethal IAV infection and mitigates severe lung lesions in SARS-CoV-2-infected hamsters. Moreover, the lipase inhibitors significantly reduce proinflammatory cytokine levels in the lungs of SARS-CoV-2- and IAV-challenged animals, a cause of a cytokine storm important for the critical infection or mortality of COVID-19 and IAV patients. In conclusion, the results reveal that lipase-mediated intracellular LD lipolysis is commonly exploited to facilitate RNA virus replication and furthermore suggest that pharmacological inhibitors of LD-associated lipases could be used to curb current COVID-19- and future pandemic outbreaks of potentially troublesome RNA virus infection in humans.

Structural features of a minimal intact methyltransferase of a type I restriction-modification system.

Type I restriction-modification enzymes are oligomeric proteins composed of methylation (M), DNA sequence-recognition (S), and restriction (R) subunits. The different bipartite DNA sequences of 2-4 consecutive bases are recognized by two discerned target recognition domains (TRDs) located at the two-helix bundle of the two conserved regions (CRs). Two M-subunits and a single S-subunit form an oligomeric protein that functions as a methyltransferase (M2S1 MTase). Here, we present the crystal structure of the intact MTase from Vibrio vulnificus YJ016 in complex with the DNA-mimicking Ocr protein and the S-adenosyl-L-homocysteine (SAH). This MTase includes the M-domain with a helix tail (M-tail helix) and the S1/2-domain of a TRD and a CR α-helix. The Ocr binds to the cleft of the TRD surface and SAH is located in the pocket within the M-domain. The solution- and negative-staining electron microscopy-based reconstructed (M1S1/2)2 structure reveals a symmetric (S1/2)2 assembly using two CR-helices and two M-tail helices as a pivot, which is plausible for recognizing two DNA regions of same sequence. The conformational flexibility of the minimal M1S1/2 MTase dimer indicates a particular state resembling the structure of M2S1 MTases.

Optimized Doxycycline-Inducible Gene Expression System for Genetic Programming of Tumor-Targeting Bacteria.

PURPOSE: In the programming of tumor-targeting bacteria, various therapeutic or reporter genes are expressed by different gene-triggering strategies. Previously, we engineered pJL87 plasmid with an inducible bacterial drug delivery system that simultaneously co-expressed two genes for therapy and imaging by a bidirectional tet promoter system only in response to the administration of exogenous doxycycline (Doxy). In this multi-cassette expression approach, tetA promoter (PtetA) was 100-fold higher in expression strength than tetR promoter (PtetR). In the present study, we developed pJH18 plasmid with novel Doxy-inducible gene expression system based on a tet promoter. PROCEDURES: In this system, Tet repressor (TetR) expressed by a weak constitutive promoter binds to tetO operator, resulting in the tight repression of gene expressions by PtetA and PtetR, and Doxy releases TetR from tetO to de-repress PtetA and PtetR. RESULTS: In Salmonella transformed with pJH18, the expression balance of bidirectional tet promoters in pJH18 was remarkably improved (PtetA:PtetR = 4~6:1) compared with that of pJL87 (PtetA:PtetR = 100:1) in the presence of Doxy. Also, the expression level by novel tet system was much higher in Salmonella transformed with pJH18 than in those with pJL87 (80-fold in rluc8 and 5-fold in clyA). Interestingly, pJH18 of the transformed Salmonella was much more stably maintained than pJL87 in antibiotic-free tumor-bearing mice (about 41-fold), because only pJH18 carries bom sequence with an essential role in preventing the plasmid-free population of programmed Salmonella from undergoing cell division. CONCLUSIONS: Overall, doxycycline-induced co-expression of two proteins at similar expression levels, we exploited bioluminescence reporter proteins with preclinical but no clinical utility. Future validation with clinically compatible reporter systems, for example, suitable for radionuclide imaging, is necessary to develop this system further towards potential clinical application.

Reporter gene-based optoacoustic imaging of E. coli targeted colon cancer in vivo.

Bacteria-mediated cancer-targeted therapy is a novel experimental strategy for the treatment of cancers. Bacteria can be engineered to overcome a major challenge of existing therapeutics by differentiating between malignant and healthy tissue. A prerequisite for further development and study of engineered bacteria is a suitable imaging concept which allows bacterial visualization in tissue and monitoring bacterial targeting and proliferation. Optoacoustics (OA) is an evolving technology allowing whole-tumor imaging and thereby direct observation of bacterial colonization in tumor regions. However, bacterial detection using OA is currently hampered by the lack of endogenous contrast or suitable transgene fluorescent labels. Here, we demonstrate improved visualization of cancer-targeting bacteria using OA imaging and E. coli engineered to express tyrosinase, which uses L-tyrosine as the substrate to produce the strong optoacoustic probe melanin in the tumor microenvironment. Tumors of animals injected with tyrosinase-expressing E. coli showed strong melanin signals, allowing to resolve bacterial growth in the tumor over time using multispectral OA tomography (MSOT). MSOT imaging of melanin accumulation in tumors was confirmed by melanin and E. coli staining. Our results demonstrate that using tyrosinase-expressing E. coli enables non-invasive, longitudinal monitoring of bacterial targeting and proliferation in cancer using MSOT.

Genomic characterization of nine Clostridioides difficile strains isolated from Korean patients with Clostridioides difficile infection.

BACKGROUND: Clostridioides difficile infection (CDI) is an infectious nosocomial disease caused by Clostridioides difficile, an opportunistic pathogen that occurs in the intestine after extensive antibiotic regimens. RESULTS: Nine C. difficile strains (CBA7201-CBA7209) were isolated from nine patients diagnosed with CDI at the national university hospital in Korea, and the whole genomes of these strains were sequenced to identify their genomic characteristics. Comparative genomic analysis was performed using 51 reference strains and the nine isolated herein. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that all 60 C. difficile strains belong to the genus Clostridioides, while core-genome tree indicated that they were divided into five groups, which was consistent with the results of MLST clade analysis. All strains were confirmed to have a clindamycin antibiotic resistance gene, but the other antibiotic resistance genes differ depending on the MLST clade. Interestingly, the six strains belonging to the sequence type 17 among the nine C. difficile strains isolated here exhibited unique genomic characteristics for PaLoc and CdtLoc, the two toxin gene loci identified in this study, and harbored similar antibiotic resistance genes. CONCLUSION: In this study, we identified the specific genomic characteristics of Korean C. difficile strains, which could serve as basic information for CDI prevention and treatment in Korea.

Orphan nuclear receptor ERR-γ regulates hepatic FGF23 production in acute kidney injury.

Fibroblast growth factor 23 (FGF23), a hormone generally derived from bone, is important in phosphate and vitamin D homeostasis. In acute kidney injury (AKI) patients, high-circulating FGF23 levels are associated with disease progression and mortality. However, the organ and cell type of FGF23 production in AKI and the molecular mechanism of its excessive production are still unidentified. For insight, we investigated folic acid (FA)-induced AKI in mice. Interestingly, simultaneous with FGF23, orphan nuclear receptor ERR-γ expression is increased in the liver of FA-treated mice, and ectopic overexpression of ERR-γ was sufficient to induce hepatic FGF23 production. In patients and in mice, AKI is accompanied by up-regulated systemic IL-6, which was previously identified as an upstream regulator of ERR-γ expression in the liver. Administration of IL-6 neutralizing antibody to FA-treated mice or of recombinant IL-6 to healthy mice confirms IL-6 as an upstream regulator of hepatic ERR-γ-mediated FGF23 production. A significant (P < 0.001) interconnection between high IL-6 and FGF23 levels as a predictor of AKI in patients that underwent cardiac surgery was also found, suggesting the clinical relevance of the finding. Finally, liver-specific depletion of ERR-γ or treatment with an inverse ERR-γ agonist decreased hepatic FGF23 expression and plasma FGF23 levels in mice with FA-induced AKI. Thus, inverse agonist of ERR-γ may represent a therapeutic strategy to reduce adverse plasma FGF23 levels in AKI.

ATM mediated-p53 signaling pathway forms a novel axis for senescence control.

Previously, we uncovered a novel mechanism in which senescence is controlled by mitochondrial functional recovery upon Ataxia-telangiectasia mutated (ATM) inhibition. However, it remains elusive how ATM controls signaling pathways to achieve restorative effect. In this study, we performed microarray and found that p53 pathway was differentially expressed upon ATM inhibition. We found that ATM inhibition yields senescence amelioration through p53-dependent manner. The restorative effect was also afforded by direct p53 inhibition. Furthermore, mitochondrial metabolic reprogramming via p53 inhibition was a prerequisite for senescence amelioration. Taken together, our data indicated that p53 pathway functions as potential target for ATM-mediated senescence amelioration.

Lipocalin2 Induced by Bacterial Flagellin Protects Mice against Cyclophosphamide Mediated Neutropenic Sepsis.

Neutropenic sepsis is a fatal consequence of chemotherapy, and septic complications are the principal cause of mortality. Chemotherapy-induced neutropenia leads to the formation of microscopic ulcers in the gastrointestinal epithelium that function as a portal of entry for intraluminal bacteria, which translocate across the intestinal mucosal barrier and gain access to systemic sites, causing septicemia. A cyclophosphamide-induced mouse model was developed to mimic the pathophysiologic sequence of events that occurs in patients with neutropenic sepsis. The TLR5 agonist bacterial flagellin derived from Vibrio vulnificus extended the survival of cyclophosphamide-treated mice by reducing the bacterial load in internal organs. The protective effect of flagellin was mediated by the antimicrobial protein lipocalin 2 (Lcn2), which is induced by TLR5-NF-κB activation in hepatocytes. Lcn2 sequestered iron from infecting bacteria, particularly siderophore enterobactin-dependent members of the Enterobacteriaceae family, thereby limiting their proliferation. Lcn2 should be considered for the treatment of neutropenic sepsis and gastrointestinal damage during chemotherapy to prevent or minimize the adverse effects of cancer chemotherapy.

Development of Oxytolerant Salmonella typhimurium Using Radiation Mutation Technology (RMT) for Cancer Therapy.

A critical limitation of Salmonella typhimurium (S. typhimurium) as an anti-cancer agent is the loss of their invasive or replicative activities, which results in no or less delivery of anti-cancer agents inside cancer cells in cancer therapy. Here we developed an oxytolerant attenuated Salmonella strain (KST0650) from the parental KST0649 (ΔptsIΔcrr) strain using radiation mutation technology (RMT). The oxytolerant KST0650 strain possessed 20-times higher replication activity in CT26 cancer cells and was less virulent than KST0649. Furthermore, KST0650 migrated effectively into tumor tissues in mice. KST0650 was further equipped with a plasmid harboring a spliced form of the intracellular pro-apoptotic protein sATF6, and the expression of sATF6 was controlled by the radiation-inducible recN promoter. The new strain was named as KST0652, in which sATF6 protein expression was induced in response to radiation in a dose-dependent manner. This strain was effectively delivered inside cancer cells and tumor tissues via the Salmonella type III secretion system (T3SS). In addition, combination treatment with KST0652 and radiation showed a synergistic anti-tumor effect in murine tumor model with complete inhibition of tumor growth and protection against death. In conclusion, we showed that RMT can be used to effectively develop an anti-tumor Salmonella strain for delivering anti-cancer agents inside tumors.

Mitochondrial metabolic reprograming via BRAF inhibition ameliorates senescence.

Senescence is defined as irreversible cell cycle arrest and constitutes a major driving force in diseases related to aging or premature aging. Recent studies indicate that activation of the serine/threonine protein kinase B-raf (BRAF) plays important roles in oncogene-induced senescence. However, it remains elusive whether BRAF inhibition might be effective for abrogating senescence. In this study, we assessed several BRAF inhibitors to identify compounds that ameliorate senescence and revealed SB590885 as an effective agent. Senescence-ameliorating effect upon BRAF inhibition was evident from the observation that SB590885 treatment increased cellular proliferation but diminished senescent phenotypes. Moreover, BRAF inhibition induced the mitochondrial functional recovery along with the metabolic reprogramming, which comprises two salient features that are altered in senescent cells. Furthermore, mitochondrial metabolic reprogramming via BRAF inhibition was a prerequisite for senescence amelioration. Taken together, our data revealed a novel mechanism in which senescence amelioration is mediated by mitochondrial metabolic reprogramming upon BRAF inhibition.

GABAergic signaling linked to autophagy enhances host protection against intracellular bacterial infections.

Gamma-aminobutyric acid (GABA) is the principal inhibitory neurotransmitter in the brain; however, the roles of GABA in antimicrobial host defenses are largely unknown. Here we demonstrate that GABAergic activation enhances antimicrobial responses against intracellular bacterial infection. Intracellular bacterial infection decreases GABA levels in vitro in macrophages and in vivo in sera. Treatment of macrophages with GABA or GABAergic drugs promotes autophagy activation, enhances phagosomal maturation and antimicrobial responses against mycobacterial infection. In macrophages, the GABAergic defense is mediated via macrophage type A GABA receptor (GABAAR), intracellular calcium release, and the GABA type A receptor-associated protein-like 1 (GABARAPL1; an Atg8 homolog). Finally, GABAergic inhibition increases bacterial loads in mice and zebrafish in vivo, suggesting that the GABAergic defense plays an essential function in metazoan host defenses. Our study identified a previously unappreciated role for GABAergic signaling in linking antibacterial autophagy to enhance host innate defense against intracellular bacterial infection.

The hepcidin-ferroportin axis controls the iron content of Salmonella-containing vacuoles in macrophages.

Macrophages release iron into the bloodstream via a membrane-bound iron export protein, ferroportin (FPN). The hepatic iron-regulatory hormone hepcidin controls FPN internalization and degradation in response to bacterial infection. Salmonella typhimurium can invade macrophages and proliferate in the Salmonella-containing vacuole (SCV). Hepcidin is reported to increase the mortality of Salmonella-infected animals by increasing the bacterial load in macrophages. Here we assess the iron levels and find that hepcidin increases iron content in the cytosol but decreases it in the SCV through FPN on the SCV membrane. Loss-of-FPN from the SCV via the action of hepcidin impairs the generation of bactericidal reactive oxygen species (ROS) as the iron content decreases. We conclude that FPN is required to provide sufficient iron to the SCV, where iron serves as a cofactor for the generation of antimicrobial ROS rather than as a nutrient for Salmonella.

Cell mass-dependent expression of an anticancer protein drug by tumor-targeted Salmonella.

Bacterial cancer therapy relies on the properties of certain bacterial species capable of targeting and proliferating within solid malignancies. If these bacteria could be loaded with antitumor proteins, the efficacy of this approach could be greatly increased. However, because most antitumor proteins are also toxic to normal tissue, they must be expressed by bacteria that specifically target and exclusively localize to tumor tissue. As a strategy for treating solid malignancies, we recently evaluated L-asparaginase (L-ASNase) delivered by tumor-targeted Salmonella. In this system, L-ASNase was expressed under the control of the araBAD promoter (PBAD) of the E. coli arabinose operon, which is induced by injection of L-arabinose. Here, we further improved the performance of recombinant Salmonella in cancer therapy by exploiting the quorum-sensing (QS) system, which uses cell mass-dependent auto-induction logic. This approach obviates the necessity of monitoring intratumoral bacterial status and inducing cargo protein expression by administration of an exogenous compound. Recombinant Salmonella in tumors expressed and secreted active L-ASNase in a cell mass-dependent manner, yielding significant anticancer effects. These results suggest that expression of a therapeutic protein under the control of the QS system represents a promising engineering platform for the production of recombinant proteins in vivo.

Aggregation-prone GFAP mutation in Alexander disease validated using a zebrafish model.

BACKGROUND: Alexander disease (AxD) is an astrogliopathy that predominantly affects the white matter of the central nervous system (CNS), and is caused by a mutation in the gene encoding the glial fibrillary acidic protein (GFAP), an intermediate filament primarily expressed in astrocytes and ependymal cells. The main pathologic feature of AxD is the presence of Rosenthal fibers (RFs), homogeneous eosinophilic inclusions found in astrocytes. Because of difficulties in procuring patient' CNS tissues and the presence of RFs in other pathologic conditions, there is a need to develop an in vivo assay that can determine whether a mutation in the GFAP results in aggregation and is thus disease-causing. METHODS: We found a GFAP mutation (c.382G > A, p.Asp128Asn) in a 68-year-old man with slowly progressive gait disturbance with tendency to fall. The patient was tentatively diagnosed with AxD based on clinical and radiological findings. To develop a vertebrate model to assess the aggregation tendency of GFAP, we expressed several previously reported mutant GFAPs and p.Asp128Asn GFAP in zebrafish embryos. RESULTS: The most common GFAP mutations in AxD, p.Arg79Cys, p.Arg79His, p.Arg239Cys and p.Arg239His, and p.Asp128Asn induced a significantly higher number of GFAP aggregates in zebrafish embryos than wild-type GFAP. CONCLUSIONS: The p.Asp128Asn GFAP mutation is likely to be a disease-causing mutation. Although it needs to be tested more extensively in larger case series, the zebrafish assay system presented here would help clinicians determine whether GFAP mutations identified in putative AxD patients are disease-causing.

Functional validation of novel MKS3/TMEM67 mutations in COACH syndrome.

COACH syndrome is an autosomal recessive developmental disorder, a subtype of Joubert syndrome and related disorders, characterized by cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis. Although mutations in TMEM67 (transmembrane protein 67)/MKS3 (Meckel-Gruber syndrome, type 3) were reported to cause COACH syndrome, this causality has not verified by functional studies. In a 20-year-old Korean man, we found cerebellar ataxia, isolated elevation in serum γ-glutamyl transpeptidase (γ-GTP) activity, oligophrenia, the molar tooth sign (MTS) in the brain MR images and congenital hepatic fibrosis (CHF). Two novel compound heterozygous mutations were found in TMEM67 in the patient: i) missense mutation (c.395 G > C and p.Gly132Ala) in exon 3, and ii) deletion in exon 26 (c.2758delT and p.Tyr920ThrfsX40). Western blotting showed that the p.Tyr920ThrfsX40 mutation accelerates turnover of the TMEM67 protein. Although wild-type human TMEM67 RNA rescued phenotypes of zebrafish embryos injected with anti-sense oligonucleotide morpholinos against tmem67, the two human TMEM67 RNAs individually harboring the two mutations did not. Finally, Wnt signaling, but not Hedgehog signaling, was suppressed in tmem67 morphants. To the best of our knowledge, this is the first report verifying the causality between COACH syndrome and TMEM67, which will further our understanding of molecular pathogenesis of the syndrome.