Dataset Information

Regulation and Anaerobic Function of the Clostridioides difficile ?-Lactamase.

ABSTRACT: Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to ?-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to ?-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of ?-lactam resistance in C. difficile are not fully understood. Our data reinforce prior evidence that C. difficile produces a ?-lactamase, which is a common ?-lactam resistance mechanism found in other bacterial species. Here, we characterize the C. difficile bla operon that encodes a lipoprotein of unknown function and a ?-lactamase that was greatly induced in response to several classes of ?-lactam antibiotics. An in-frame deletion of the operon abolished ?-lactamase activity in C. difficile strain 630?erm and resulted in decreased resistance to the ?-lactam ampicillin. We found that the activity of this ?-lactamase, BlaCDD, is dependent upon the redox state of the enzyme. In addition, we observed that transport of BlaCDD out of the cytosol and to the cell surface is facilitated by an N-terminal signal sequence. Our data demonstrate that a cotranscribed lipoprotein, BlaX, aids in BlaCDD activity. Further, we identified a conserved BlaRI regulatory system and demonstrated via insertional disruption that BlaRI controls transcription of the blaXCDD genes in response to ?-lactams. These results provide support for the function of a ?-lactamase in C. difficile antibiotic resistance and reveal the unique roles of a coregulated lipoprotein and reducing environment in C. difficile ?-lactamase activity.

SUBMITTER: Sandhu BK

PROVIDER: S-EPMC7187622 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Publications

Regulation and Anaerobic Function of the Clostridioides difficile β-Lactamase.

Sandhu Brindar K BK Edwards Adrianne N AN Anderson Sarah E SE Woods Emily C EC McBride Shonna M SM

Antimicrobial agents and chemotherapy 20191220 1

Clostridioides difficile causes severe antibiotic-associated diarrhea and colitis. C. difficile is an anaerobic, Gram-positive sporeformer that is highly resistant to β-lactams, the most commonly prescribed antibiotics. The resistance of C. difficile to β-lactam antibiotics allows the pathogen to replicate and cause disease in antibiotic-treated patients. However, the mechanisms of β-lactam resistance in C. difficile are not fully understood. Our data reinforce prior ...[more]

PMID: 31611350

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Project description:The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (760 g/l) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron transporters were found induced by Fur regulation during low iron (11 g/l) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were found significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some initial intermediates (phenylpyruvate, oxo-isocaproate, 3-hydroxy-butanoyl-CoA, crotonyl-CoA) were found accumulated, while some end product like isocaproate and butanoate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were found enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB,) of the F0F1-type was found induced while the formation of a V-type, mostly proton-pumping, ATP-consuming ATPase (atpDBAFCEKI, was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. An intensive remodeling of the cell wall was observed most likely to increase antibiotic resistance. Polyamine biosynthesis (spe) was found induced leading to an accumulation of spermine, spermidine and putrescine. The fur mutant lost most of its flagella. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, significant decrease in metabolic iron utilization and protection during the complex transition.

Project description:The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (15 ?M) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron and siderophore transporter genes were induced by Fur during low iron (0.2 ?M) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some intermediates (phenylpyruvate, 2-oxo-isocaproate, 3-hydroxy-butyryl-CoA, crotonyl-CoA) accumulated, while end products like isocaproate and butyrate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB) of the F0F1-type was induced while the formation of a ATP-consuming, proton-pumping V-type ATPase (atpDBAFCEKI) was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. The fur mutant showed an increased sensitivity to vancomycin and polymyxin B. An intensive remodeling of the cell wall was observed, Polyamine biosynthesis (spe) was induced leading to an accumulation of spermine, spermidine, and putrescine. The fur mutant lost most of its flagella and motility. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of around 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, a significant replacement of iron requiring metabolic pathways and the restructuring of the cell surface for protection during the complex adaptation phase and was only partly directly regulated by Fur.

Project description:Clostridioides difficile is a major cause of diarrhea associated with antibiotherapy. After germination of C. difficile spores in the small intestine, vegetative cells are exposed to low oxygen (O2) tensions. While considered strictly anaerobic, C. difficile is able to grow in nonstrict anaerobic conditions (1 to 3% O2) and tolerates brief air exposure indicating that this bacterium harbors an arsenal of proteins involved in O2 detoxification and/or protection. Tolerance of C. difficile to low O2 tensions requires the presence of the alternative sigma factor, σB, involved in the general stress response. Among the genes positively controlled by σB, four encode proteins likely involved in O2 detoxification: two flavodiiron proteins (FdpA and FdpF) and two reverse rubrerythrins (revRbr1 and revRbr2). As previously observed for FdpF, we showed that both purified revRbr1 and revRbr2 harbor NADH-linked O2- and H2O2-reductase activities in vitro, while purified FdpA mainly acts as an O2-reductase. The growth of a fdpA mutant is affected at 0.4% O2, while inactivation of both revRbrs leads to a growth defect above 0.1% O2 O2-reductase activities of these different proteins are additive since the quadruple mutant displays a stronger phenotype when exposed to low O2 tensions compared to the triple mutants. Our results demonstrate a key role for revRbrs, FdpF, and FdpA proteins in the ability of C. difficile to grow in the presence of physiological O2 tensions such as those encountered in the colon.IMPORTANCE Although the gastrointestinal tract is regarded as mainly anoxic, low O2 tension is present in the gut and tends to increase following antibiotic-induced disruption of the host microbiota. Two decreasing O2 gradients are observed, a longitudinal one from the small to the large intestine and a second one from the intestinal epithelium toward the colon lumen. Thus, O2 concentration fluctuations within the gastrointestinal tract are a challenge for anaerobic bacteria such as C. difficile This enteropathogen has developed efficient strategies to detoxify O2 In this work, we identified reverse rubrerythrins and flavodiiron proteins as key actors for O2 tolerance in C. difficile These enzymes are responsible for the reduction of O2 protecting C. difficile vegetative cells from associated damages. Original and complex detoxification pathways involving O2-reductases are crucial in the ability of C. difficile to tolerate O2 and survive to O2 concentrations encountered in the gastrointestinal tract.

Project description:The response to iron limitation of several bacteria is regulated by the ferric uptake regulator (Fur). The Fur-regulated transcriptional, translational and metabolic networks of the Gram-positive, pathogen Clostridioides difficile were investigated by a combined RNA sequencing, proteomic, metabolomic and electron microscopy approach. At high iron conditions (760 g/l) the C. difficile fur mutant displayed a growth deficiency compared to wild type C. difficile cells. Several iron transporters were found induced by Fur regulation during low iron (11 g/l) conditions. The major adaptation to low iron conditions was observed for the central energy metabolism. Most ferredoxin-dependent amino acid fermentations were found significantly down regulated (had, etf, acd, grd, trx, bdc, hbd). The substrates of these pathways phenylalanine, leucine, glycine and some initial intermediates (phenylpyruvate, oxo-isocaproate, 3-hydroxy-butanoyl-CoA, crotonyl-CoA) were found accumulated, while some end product like isocaproate and butanoate were found reduced. Flavodoxin (fldX) formation and riboflavin biosynthesis (rib) were found enhanced, most likely to replace the missing ferredoxins. Proline reductase (prd), the corresponding ion pumping RNF complex (rnf) and the reaction product 5-aminovalerate were significantly enhanced. An ATP forming ATPase (atpCDGAHFEB,) of the F0F1-type was found induced while the formation of a V-type, mostly proton-pumping, ATP-consuming ATPase (atpDBAFCEKI, was decreased. The [Fe-S] enzyme-dependent pyruvate formate lyase (pfl), formate dehydrogenase (fdh) and hydrogenase (hyd) branch of glucose utilization and glycogen biosynthesis (glg) were significantly reduced, leading to an accumulation of glucose and pyruvate. The formation of [Fe-S] enzyme carbon monoxide dehydrogenase (coo) was inhibited. An intensive remodeling of the cell wall was observed most likely to increase antibiotic resistance. Polyamine biosynthesis (spe) was found induced leading to an accumulation of spermine, spermidine and putrescine. The fur mutant lost most of its flagella. Finally, the CRISPR/Cas and a prophage encoding operon were downregulated. Fur binding sites were found upstream of 20 of the regulated genes. Overall, adaptation to low iron conditions in C. difficile focused on an increase of iron import, significant decrease in metabolic iron utilization and protection during the complex transition.

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Dataset Information

Regulation and Anaerobic Function of the Clostridioides difficile ?-Lactamase.

Publications

Regulation and Anaerobic Function of the <i>Clostridioides difficile</i> β-Lactamase.

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