Metabolic adaption to extracellular pyruvate triggers biofilm formation in Clostridioides difficile
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ABSTRACT:
ORGANISM(S): Clostridioides difficile
PROVIDER: GSE165116 | GEO | 2021/05/04
REPOSITORIES: GEO
Similar Datasets
Project description:Metabolic adaption to extracellular pyruvate triggers biofilm formation in Clostridioides difficile
| PRJNA693288 | ENA
Project description:A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the human pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Here, we made use of the distinct MifR-dependent phenotypes to elucidate mechanisms associated with microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that cells located within microcolonies experience stressful, oxygen limited, and energy starving conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate partly restored microcolony formation in M-bM-^HM-^FmifR biofilms. Moreover, addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation. Consistent with the finding of denitrification genes not demonstrating distinct expression patterns in biofilms forming or lacking microcolonies, addition of nitrate did not alter microcolony formation. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation to the oxygen limitation and energy starvation of the P. aeruginosa biofilm environment. For biofilm growth experiments, three independent replicates of P. aeruginosa strains PAO1 and M-NM-^TmifR were grown as biofilms in a flow-through system using a once-through continuous flow tube reactor system for biofilm sample collection and in flow cells (BioSurface Technologies) for the analysis of biofilm architecture as previously described (Sauer et al., 2002, Sauer et al., 2004, Petrova & Sauer, 2009). Cells were treated with RNAprotect (Qiagen) and total RNA was extracted using an RNeasy mini purification kit (Qiagen) per the manufacturerM-bM-^@M-^Ys instructions. RNA quality and the presence of residual DNA were checked on an Agilent Bioanalyzer 2100 electrophoretic system pre- and post-DNase treatment. Ten micrograms of total RNA was used for cDNA synthesis, fragmentation, and labeling according to the Affymetrix GeneChip P. aeruginosa genome array expression analysis protocol. Sauer, K., A. K. Camper, G. D. Ehrlich, J. W. Costerton & D. G. Davies, (2002) Pseudomonas aeruginosa displays multiple phenotypes during development as a biofilm. J. Bacteriol. 184: 1140-1154. Sauer, K., M. C. Cullen, A. H. Rickard, L. A. H. Zeef, D. G. Davies & P. Gilbert, (2004) Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm. J. Bacteriol. 186: 7312-7326. Petrova, O. E. & K. Sauer, (2009) A novel signaling network essential for regulating Pseudomonas aeruginosa biofilm development. PLoS Pathogens 5: e1000668.
2012-04-30 | E-GEOD-35286 | biostudies-arrayexpress
Project description:A hallmark of the biofilm architecture is the presence of microcolonies. However, little is known about the underlying mechanisms governing microcolony formation. In the human pathogen Pseudomonas aeruginosa, microcolony formation is dependent on the two-component regulator MifR, with mifR mutant biofilms exhibiting an overall thin structure lacking microcolonies, and overexpression of mifR resulting in hyper-microcolony formation. Here, we made use of the distinct MifR-dependent phenotypes to elucidate mechanisms associated with microcolony formation. Using global transcriptomic and proteomic approaches, we demonstrate that cells located within microcolonies experience stressful, oxygen limited, and energy starving conditions, as indicated by the activation of stress response mechanisms and anaerobic and fermentative processes, in particular pyruvate fermentation. Inactivation of genes involved in pyruvate utilization including uspK, acnA and ldhA abrogated microcolony formation in a manner similar to mifR inactivation. Moreover, depletion of pyruvate from the growth medium impaired biofilm and microcolony formation, while addition of pyruvate significantly increased microcolony formation. Addition of pyruvate partly restored microcolony formation in ∆mifR biofilms. Moreover, addition of pyruvate to or expression of mifR in lactate dehydrogenase (ldhA) mutant biofilms did not restore microcolony formation. Consistent with the finding of denitrification genes not demonstrating distinct expression patterns in biofilms forming or lacking microcolonies, addition of nitrate did not alter microcolony formation. Our findings indicate the fermentative utilization of pyruvate to be a microcolony-specific adaptation to the oxygen limitation and energy starvation of the P. aeruginosa biofilm environment.
2012-05-01 | GSE35286 | GEO
Project description:Pyruvate is a central metabolite that connects many metabolic pathways in living organisms. To meet the cellular pyruvate requirements, Escherichia coli most likely has three pyruvate uptake systems, BtsT, YhjX and CstA. Transport studies in intact cells proved that CstA is a high specific pyruvate transporter with moderate affinity, which is energized by a proton gradient. When the cells of a reporter strain were cultured in complex medium, cstA expression was bimodal, with the maximum being reached in the stationary phase. A DNA affinity capture assay combined with mass spectrometry and an in vivo reporter assay identified Fis as repressor in addition to the known cAMP-CRP as activator for cstA expression.
2020-12-02 | PXD021798 | Pride
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.
2019-01-15 | GSE120189 | GEO
Project description:we report that succinate, a metabolite abundantly produced by the dysbiotic gut microbiota, induces in vitro biofilm formation of C. difficile strains. We characterized the morphology and spatial composition of succinate- induced biofilms, and compared to non-induced or deoxycholate-induced biofilms, biofilms induced by succinate are significantly thicker, structurally more complex, and poorer in proteins and exopolysaccharides (EPS).
2023-05-24 | GSE223108 | GEO
Project description:The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesised that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the lipoprotein CD1687 in the DCA-induced biofilm formation of C. difficile. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites induced for some of them in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and this localization is required for DCA-induced biofilm formation. Given this localization and that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA.
2023-04-19 | GSE218475 | GEO
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.
2019-01-11 | PXD011161 | Pride
Project description:The ability of bacterial pathogens to establish recurrent and persistent infections is frequently associated with their ability to form biofilms. Clostridioides difficile infections have a high rate of recurrence and relapses and it is hypothesised that biofilms are involved in its pathogenicity and persistence. Biofilm formation by C. difficile is still poorly understood. It has been shown that specific molecules such as deoxycholate (DCA) or metronidazole induce biofilm formation, but the mechanisms involved remain elusive. In this study, we describe the role of the lipoprotein CD1687 in the DCA-induced biofilm formation of C. difficile. We showed that the expression of CD1687, which is part of an operon within the CD1685-CD1689 gene cluster, is controlled by multiple transcription starting sites induced for some of them in response to DCA. Only CD1687 is required for biofilm formation and the overexpression of CD1687 is sufficient to induce biofilm formation. Using RNAseq analysis, we showed that CD1687 affects the expression of transporters and metabolic pathways and we identified several potential binding partners by pull-down assay, including transport-associated extracellular proteins. We then demonstrated that CD1687 is surface exposed in C. difficile, and this localization is required for DCA-induced biofilm formation. Given this localization and that C. difficile forms eDNA-rich biofilms, we confirmed that CD1687 binds DNA in a non-specific manner. We thus hypothesize that CD1687 is a component of the downstream response to DCA leading to biofilm formation by promoting interaction between the cells and the biofilm matrix by binding eDNA.
2023-05-16 | PXD038282 | Pride
Project description:Clostridium difficile Biofilm with and without deoxycholate at 48H
| PRJNA339928 | ENA