scholarly journals Flexible cobamide metabolism in Clostridioides (Clostridium) difficile 630 Δerm

2019 ◽  
Author(s):  
Amanda N. Shelton ◽  
Xun Lyu ◽  
Michiko E. Taga
Keyword(s):  
Clostridium Difficile ◽  
De Novo ◽  
Genomic Analysis ◽  
Opportunistic Pathogen ◽  
Vitamin B ◽  
Uptake System ◽  
Human Gut ◽  
Clostridioides Difficile ◽  
Lower Ligand

AbstractClostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent metabolisms encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro. Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis, and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinc acid or the late intermediate cobinamide, and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in in the btuFCD locus of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile.ImportanceThe ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, though it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo, but produces them when given cobamide precursors. Better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

scholarly journals Flexible Cobamide Metabolism in Clostridioides (Clostridium) difficile 630 Δerm

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Amanda N. Shelton ◽  
Xun Lyu ◽  
Michiko E. Taga
Keyword(s):  
De Novo ◽  
Aminolevulinic Acid ◽  
Genomic Analysis ◽  
Opportunistic Pathogen ◽  
Vitamin B ◽  
Uptake System ◽  
Human Gut ◽  
Content Type ◽  
Clostridioides Difficile

ABSTRACT Clostridioides (Clostridium) difficile is an opportunistic pathogen known for its ability to colonize the human gut under conditions of dysbiosis. Several aspects of its carbon and amino acid metabolism have been investigated, but its cobamide (vitamin B12 and related cofactors) metabolism remains largely unexplored. C. difficile has seven predicted cobamide-dependent pathways encoded in its genome in addition to a nearly complete cobamide biosynthesis pathway and a cobamide uptake system. To address the importance of cobamides to C. difficile, we studied C. difficile 630 Δerm and mutant derivatives under cobamide-dependent conditions in vitro. Our results show that C. difficile can use a surprisingly diverse array of cobamides for methionine and deoxyribonucleotide synthesis and can use alternative metabolites or enzymes, respectively, to bypass these cobamide-dependent processes. C. difficile 630 Δerm produces the cobamide pseudocobalamin when provided the early precursor 5-aminolevulinic acid or the late intermediate cobinamide (Cbi) and produces other cobamides if provided an alternative lower ligand. The ability of C. difficile 630 Δerm to take up cobamides and Cbi at micromolar or lower concentrations requires the transporter BtuFCD. Genomic analysis revealed genetic variations in the btuFCD loci of different C. difficile strains, which may result in differences in the ability to take up cobamides and Cbi. These results together demonstrate that, like other aspects of its physiology, cobamide metabolism in C. difficile is versatile. IMPORTANCE The ability of the opportunistic pathogen Clostridioides difficile to cause disease is closely linked to its propensity to adapt to conditions created by dysbiosis of the human gut microbiota. The cobamide (vitamin B12) metabolism of C. difficile has been underexplored, although it has seven metabolic pathways that are predicted to require cobamide-dependent enzymes. Here, we show that C. difficile cobamide metabolism is versatile, as it can use a surprisingly wide variety of cobamides and has alternative functions that can bypass some of its cobamide requirements. Furthermore, C. difficile does not synthesize cobamides de novo but produces them when given cobamide precursors. A better understanding of C. difficile cobamide metabolism may lead to new strategies to treat and prevent C. difficile-associated disease.

scholarly journals Identification of a Novel Cobamide Remodeling Enzyme in the Beneficial Human Gut Bacterium Akkermansia muciniphila

mBio ◽  
2020 ◽  
Vol 11 (6) ◽  
Author(s):  
Kenny C. Mok ◽  
Olga M. Sokolovskaya ◽  
Alexa M. Nicolas ◽  
Zachary F. Hallberg ◽  
Adam Deutschbauer ◽  
...  
Keyword(s):  
De Novo ◽  
Structural Diversity ◽  
Vitamin B ◽  
Human Gut ◽  
Diverse Range ◽  
Content Type ◽  
Akkermansia Muciniphila ◽  
Binding Domains ◽  
Domains Of Life ◽  
Lower Ligand

ABSTRACT The beneficial human gut bacterium Akkermansia muciniphila provides metabolites to other members of the gut microbiota by breaking down host mucin, but most of its other metabolic functions have not been investigated. A. muciniphila strain MucT is known to use cobamides, the vitamin B12 family of cofactors with structural diversity in the lower ligand. However, A. muciniphila MucT is unable to synthesize cobamides de novo, and the specific forms that can be used by A. muciniphila have not been examined. We found that the levels of growth of A. muciniphila MucT were nearly identical with each of seven cobamides tested, in contrast to nearly all bacteria that had been studied previously. Unexpectedly, this promiscuity is due to cobamide remodeling—the removal and replacement of the lower ligand—despite the absence of the canonical remodeling enzyme CbiZ in A. muciniphila. We identified a novel enzyme, CbiR, that is capable of initiating the remodeling process by hydrolyzing the phosphoribosyl bond in the nucleotide loop of cobamides. CbiR does not share similarity with other cobamide remodeling enzymes or B12-binding domains and is instead a member of the apurinic/apyrimidinic (AP) endonuclease 2 enzyme superfamily. We speculate that CbiR enables bacteria to repurpose cobamides that they cannot otherwise use in order to grow under cobamide-requiring conditions; this function was confirmed by heterologous expression of cbiR in Escherichia coli. Homologs of CbiR are found in over 200 microbial taxa across 22 phyla, suggesting that many bacteria may use CbiR to gain access to the diverse cobamides present in their environment. IMPORTANCE Cobamides, comprising the vitamin B12 family of cobalt-containing cofactors, are required for metabolism in all domains of life, including most bacteria. Cobamides have structural variability in the lower ligand, and selectivity for particular cobamides has been observed in most organisms studied to date. Here, we discovered that the beneficial human gut bacterium Akkermansia muciniphila can use a diverse range of cobamides due to its ability to change the cobamide structure via a process termed cobamide remodeling. We identify and characterize the novel enzyme CbiR that is necessary for initiating the cobamide remodeling process. The discovery of this enzyme has implications for understanding the ecological role of A. muciniphila in the gut and the functions of other bacteria that produce this enzyme.

scholarly journals Bifidobacterium adolescentis shows potential to strengthen host defence against gastrointestinal infection via inhibition of the opportunistic pathogen Candida albicans and stimulation of human-isolated macrophages killing capacity in vitro

2020 ◽  
Vol 2 (7A) ◽  
Author(s):  
Liviana Ricci ◽  
Joanna Mackie ◽  
Megan D. Lenardon ◽  
Caitlin Jukes ◽  
Ahmed N. Hegazy ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Bacterial Species ◽  
Opportunistic Pathogen ◽  
Limited Information ◽  
Human Gut ◽  
Bifidobacterium Adolescentis ◽  
Host Immune Responses ◽  
Opportunistic Fungal Pathogen ◽  
Antimicrobial Metabolites

The human gut microbiota enhances the host’s resistance to enteric pathogens via colonisation resistance, a phenomenon that is driven by multiple mechanisms, such as production of antimicrobial metabolites and activation of host immune responses. However, there is limited information on how individual gut bacterial species, particularly many of the dominant anaerobes, might impact the host’s defence. This study investigated the potential of specific human gut isolates to bolster the host’s resistance to infection. First, by antagonising the opportunistic fungal pathogen Candida albicans, and secondly, by modulating the killing capacity of human-isolated macrophages in vitro. Co-culturing C. albicans with faecal microbiota from different healthy individuals revealed varying levels of fungal inhibition. In vitro assays with a panel of representative human gut anaerobes confirmed that culture supernatants from certain bacterial isolates, in particular of Bifidobacterium adolescentis, significantly inhibited C. albicans growth. Mechanistic studies revealed that microbial fermentation acids including acetate and lactate, in combination with the associated decrease in pH, were strong drivers of this inhibitory activity. In the second in vitro assay, human-isolated macrophages were exposed to bacterial supernatants, and subsequently tested for their capacity to eliminate adherent-invasive Escherichia coli. Among the gut anaerobes tested, B. adolescentis was revealed to exert the strongest immunostimulatory and killing effect when compared to the unstimulated macrophages control. B. adolescentis is known to be stimulated by dietary consumption of resistant starch andmay therefore represent an attractive target for the development of probiotic and prebiotic interventions tailored to enhancethe host’s natural defences against infection.

scholarly journals Cofactor Selectivity in Methylmalonyl Coenzyme A Mutase, a Model Cobamide-Dependent Enzyme

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Olga M. Sokolovskaya ◽  
Kenny C. Mok ◽  
Jong Duk Park ◽  
Jennifer L. A. Tran ◽  
Kathryn A. Quanstrom ◽  
...  
Keyword(s):  
Binding Affinity ◽  
Bacterial Growth ◽  
Microbial Interactions ◽  
Vitamin B ◽  
Content Type ◽  
Domains Of Life ◽  
Enzyme Cofactors ◽  
Lower Ligand ◽  
Enzyme Selectivity

ABSTRACT Cobamides, a uniquely diverse family of enzyme cofactors related to vitamin B12, are produced exclusively by bacteria and archaea but used in all domains of life. While it is widely accepted that cobamide-dependent organisms require specific cobamides for their metabolism, the biochemical mechanisms that make cobamides functionally distinct are largely unknown. Here, we examine the effects of cobamide structural variation on a model cobamide-dependent enzyme, methylmalonyl coenzyme A (CoA) mutase (MCM). The in vitro binding affinity of MCM for cobamides can be dramatically influenced by small changes in the structure of the lower ligand of the cobamide, and binding selectivity differs between bacterial orthologs of MCM. In contrast, variations in the lower ligand have minor effects on MCM catalysis. Bacterial growth assays demonstrate that cobamide requirements of MCM in vitro largely correlate with in vivo cobamide dependence. This result underscores the importance of enzyme selectivity in the cobamide-dependent physiology of bacteria. IMPORTANCE Cobamides, including vitamin B12, are enzyme cofactors used by organisms in all domains of life. Cobamides are structurally diverse, and microbial growth and metabolism vary based on cobamide structure. Understanding cobamide preference in microorganisms is important given that cobamides are widely used and appear to mediate microbial interactions in host-associated and aquatic environments. Until now, the biochemical basis for cobamide preferences was largely unknown. In this study, we analyzed the effects of the structural diversity of cobamides on a model cobamide-dependent enzyme, methylmalonyl-CoA mutase (MCM). We found that very small changes in cobamide structure could dramatically affect the binding affinity of cobamides to MCM. Strikingly, cobamide-dependent growth of a model bacterium, Sinorhizobium meliloti, largely correlated with the cofactor binding selectivity of S. meliloti MCM, emphasizing the importance of cobamide-dependent enzyme selectivity in bacterial growth and cobamide-mediated microbial interactions.

scholarly journals Investigation of the effect of the adsorbent DAV131A on the propensity of moxifloxacin to induce simulated Clostridioides (Clostridium) difficile infection (CDI) in an in vitro human gut model

2020 ◽  
Vol 75 (6) ◽  
pp. 1458-1465
Author(s):  
C H Chilton ◽  
G S Crowther ◽  
C Miossec ◽  
J de Gunzburg ◽  
A Andremont ◽  
...  
Keyword(s):  
Limit Of Detection ◽  
Toxin Production ◽  
Human Gut ◽  
Antibiotic Resistant ◽  
Delayed Onset ◽  
Clostridioides Difficile ◽  
Clostridioides Difficile Infection ◽  
Clinical Benefits ◽  
Bacteroides Fragilis Group

Abstract Background Clostridioides difficile infection (CDI) remains a high burden worldwide. DAV131A, a novel adsorbent, reduces residual gut antimicrobial levels, reducing CDI risk in animal models. Objectives We used a validated human gut model to investigate the efficacy of DAV131A in preventing moxifloxacin-induced CDI. Methods C. difficile (CD) spores were inoculated into two models populated with pooled human faeces. Moxifloxacin was instilled (43 mg/L, once daily, 7 days) alongside DAV131A (5 g in 18 mL PBS, three times daily, 14 days, Model A), or PBS (18 mL, three times daily, 14 days, Model B). Selected gut microbiota populations, CD total counts, spore counts, cytotoxin titre and antimicrobial concentrations (HPLC) were monitored daily. We monitored for reduced susceptibility of CD to moxifloxacin. Growth of CD in faecal filtrate and medium in the presence/absence of DAV131A, or in medium pre-treated with DAV131A, was also investigated. Results DAV131A instillation reduced active moxifloxacin levels to below the limit of detection (50 ng/mL), and prevented microbiota disruption, excepting Bacteroides fragilis group populations, which declined by ∼3 log10 cfu/mL. DAV131A delayed onset of simulated CDI by ∼2 weeks, but did not prevent CD germination and toxin production. DAV131A prevented emergence of reduced susceptibility of CD to moxifloxacin. In batch culture, DAV131A had minor effects on CD vegetative growth, but significantly reduced toxin/spores (P < 0.005). Conclusions DAV131A reduced moxifloxacin-induced microbiota disruption and emergence of antibiotic-resistant CD. Delayed onset of CD germination and toxin production indicates further investigations are warranted to understand the clinical benefits of DAV131A in CDI prevention.

scholarly journals Evaluation of linezolid for the treatment of Clostridium difficile infection caused by epidemic strains using an in vitro human gut model

2011 ◽  
Vol 66 (7) ◽  
pp. 1537-1546 ◽  
Author(s):  
S. D. Baines ◽  
A. R. Noel ◽  
G. S. Huscroft ◽  
S. L. Todhunter ◽  
R. O'Connor ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Clostridium Difficile Infection ◽  
Human Gut

scholarly journals Evaluation of antimicrobial activity of ceftaroline against Clostridium difficile and propensity to induce C. difficile infection in an in vitro human gut model

2013 ◽  
Vol 68 (8) ◽  
pp. 1842-1849 ◽  
Author(s):  
S. D. Baines ◽  
C. H. Chilton ◽  
G. S. Crowther ◽  
S. L. Todhunter ◽  
J. Freeman ◽  
...  
Keyword(s):  
Antimicrobial Activity ◽  
Clostridium Difficile ◽  
Human Gut

scholarly journals C. difficile-associated antibiotics prime the host for infection by a microbiome-independent mechanism

2019 ◽  
Author(s):  
Jemila C. Kester ◽  
Douglas K. Brubaker ◽  
Jason Velazquez ◽  
Charles Wright ◽  
Douglas A. Lauffenburger ◽  
...  
Keyword(s):  
Innate Immune ◽  
Mucosal Barrier ◽  
Human Gut ◽  
Germ Free ◽  
Clostridioides Difficile ◽  
Increased Risk ◽  
Independent Mechanism ◽  
Relevant Risk Factor ◽  
Independent Effects

AbstractThe most clinically relevant risk factor for Clostridioides difficile-associated disease (CDAD) is recent antibiotic treatment. Though most broad-spectrum antibiotics significantly disrupt the structure of the gut microbiota, only particular ones increase CDAD risk, suggesting additional factors might increase the risk from certain antibiotics. Here we show that commensal-independent effects of antibiotics collectively prime an in vitro germ-free human gut for CDAD. We found a marked loss of mucosal barrier and immune function with CDAD-associated antibiotic pretreatment distinct from pretreatment with an antibiotic unassociated with CDAD, which did not reduce innate immune or mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibiotics sensitized mucosal barriers to C. difficile toxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent host changes in the increased risk of CDAD with specific antibiotics. Our findings are contrary to the previously held belief that antibiotics allow for CDAD solely through disruption of the microbiome. We anticipate this work to suggest potential avenues of research for host-directed treatment and preventive therapies for CDAD, and to impact human tissue culturing protocols.

scholarly journals Direct cobamide remodeling via additional function of cobamide biosynthesis protein CobS from Vibrio cholerae

2021 ◽  
Author(s):  
Amy T. Ma ◽  
Joris Beld
Keyword(s):  
Vibrio Cholerae ◽  
Intestinal Bacteria ◽  
Vitamin B ◽  
Bacterial Genetics ◽  
Additional Function ◽  
Metabolic Interactions ◽  
Polymicrobial Communities ◽  
Lower Ligand

Vitamin B12 belongs to a family of structurally-diverse cofactors with over a dozen natural analogs, collectively referred to as cobamides. Most bacteria encode cobamide-dependent enzymes, many of which can only utilize a subset of cobamide analogs. Some bacteria employ a mechanism called cobamide remodeling, a process in which cobamides are converted into other analogs, to ensure that compatible cobamides are available in the cell. Here we characterize an additional pathway for cobamide remodeling that is distinct from the previously-characterized ones. Cobamide synthase (CobS) is an enzyme required for cobamide biosynthesis that attaches the lower ligand moiety in which the base varies between analogs. In a heterologous model system, we previously showed Vibrio cholerae CobS (VcCobS) unexpectedly conferred remodeling activity, in addition to performing the known cobamide biosynthesis reaction. Here we show that additional Vibrio species perform the same remodeling reaction, and we further characterize VcCobS-mediated remodeling using bacterial genetics and in vitro assays. We demonstrate that VcCobS acts upon the cobamide pseudocobalamin directly to remodel it, a mechanism which differs from the known remodeling pathways in which cobamides are first cleaved into biosynthetic intermediates. This suggests that some CobS homologs have the additional function of cobamide remodeling, and we propose the term "direct remodeling" for this process. This characterization of yet another pathway for remodeling suggests that cobamide profiles are highly dynamic in polymicrobial environments, with remodeling pathways conferring a competitive advantage. Importance Cobamides are widespread cofactors that mediate metabolic interactions in complex microbial communities. Few studies directly examine cobamide profiles, but several have shown that mammalian gastrointestinal tracts are rich in cobamide analogs. Studies of intestinal bacteria, including beneficial commensals and pathogens, show variation in the ability to produce and utilize different cobamides. Some bacteria can convert imported cobamides into compatible analogs in a process called remodeling. Recent discoveries of additional cobamide remodeling pathways, including this work, suggest that remodeling is an important factor in cobamide dynamics. Characterization of such pathways is critical in understanding cobamide flux and nutrient cross-feeding in polymicrobial communities, and facilitates the establishment of microbiome manipulation strategies via modulation of cobamide profiles.

scholarly journals Multi-dimensional genomic analysis of myoepithelial carcinoma identifies prevalent oncogenic gene fusions

2017 ◽  
Author(s):  
Martin G. Dalin ◽  
Nora Katabi ◽  
Marta Persson ◽  
Ken-Wing Lee ◽  
Vladimir Makarov ◽  
...  
Keyword(s):  
Salivary Gland ◽  
Nuclear Dna ◽  
De Novo ◽  
Genomic Analysis ◽  
Salivary Gland Cancer ◽  
Gene Fusions ◽  
Myoepithelial Carcinoma ◽  
Molecular Alterations ◽  
Molecular Features

AbstractMyoepithelial carcinoma (MECA) is an aggressive type of salivary gland cancer with largely unknown molecular features. MECA may arise de novo or result from oncogenic transformation of a pre-existing pleomorphic adenoma (MECA ex-PA). We comprehensively analyzed the molecular alterations in MECA with integrated genomic analyses. We identified a low mutational load (0.5/MB), but a high prevalence of fusion oncogenes (28/40 tumors; 70%). We foundFGFR1-PLAG1in 7 (18%) cases, and the novelTGFBR3-PLAG1fusion in 6 (15%) cases.TGFBR3-PLAG1was specific for MECA de novo tumors or the malignant component of MECA ex-PA, was absent in 723 other salivary gland tumors, and promoted a tumorigenic phenotype in vitro. We discovered other novelPLAG1fusions, includingND4-PLAG1,which is an oncogenic fusion between mitochondrial and nuclear DNA. One tumor harbored anMSN-ALKfusion, which was tumorigenic in vitro, and targetable with ALK inhibitors. Certain gene fusions were predicted to result in neoantigens with high MHC binding affinity. A high number of copy number alterations was associated with poorer prognosis. Our findings indicate that MECA is a fusion-driven disease, nominateTGFBR3-PLAG1as a hallmark of MECA, and provide a framework for future steps of diagnostic and therapeutic research in this lethal cancer.

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