Dataset Information

Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production.

ABSTRACT: We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for the import of aromatic amino acids. In contrast, the transporter gene loss did not measurably affect bacterial growth or production of enterotoxins. Phylogenomic analysis of publically available genome sequences indicated that this transporter gene deletion had occurred multiple times in diverse clonal lineages of C. difficile, with a particularly high prevalence in ribotype 027 isolates, where 48 of 195 genomes (25%) were affected. The transporter gene deletion likely was facilitated by the repetitive structure of its genomic location. While at least some of the observed transporter gene deletions are likely to have occurred during the natural life cycle of C. difficile, we also provide evidence for the emergence of this mutation during long-term laboratory cultivation of reference strain R20291.

SUBMITTER: Steglich M

PROVIDER: S-EPMC5951980 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production.

Steglich Matthias M Hofmann Julia D JD Helmecke Julia J Sikorski Johannes J Spröer Cathrin C Riedel Thomas T Bunk Boyke B Overmann Jörg J Neumann-Schaal Meina M Nübel Ulrich U

Frontiers in microbiology 20180508

We report the frequent, convergent loss of two genes encoding the substrate-binding protein and the ATP-binding protein of an ATP-binding cassette (ABC) transporter from the genomes of unrelated Clostridioides difficile strains. This specific genomic deletion was strongly associated with the reduced uptake of tyrosine and phenylalanine and production of derived Stickland fermentation products, including p-cresol, suggesting that the affected ABC transporter had been responsible for ...[more]

PMID: 29867812

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Project description:Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and Pσ54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCEClostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ≤0.25 mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage.

Project description:Infections due to Clostridioides difficile (previously known as Clostridium difficile) are a major problem in hospitals, where cases can be caused by community-acquired strains as well as by nosocomial spread. Whole genome sequences from clinical samples contain a lot of information but that needs to be analyzed and compared in such a way that the outcome is useful for clinicians or epidemiologists. Here, we compare 663 public available complete genome sequences of C. difficile using average amino acid identity (AAI) scores. This analysis revealed that most of these genomes (640, 96.5%) clearly belong to the same species, while the remaining 23 genomes produce four distinct clusters within the Clostridioides genus. The main C. difficile cluster can be further divided into sub-clusters, depending on the chosen cutoff. We demonstrate that MLST, either based on partial or full gene-length, results in biased estimates of genetic differences and does not capture the true degree of similarity or differences of complete genomes. Presence of genes coding for C. difficile toxins A and B (ToxA/B), as well as the binary C. difficile toxin (CDT), was deduced from their unique PfamA domain architectures. Out of the 663 C. difficile genomes, 535 (80.7%) contained at least one copy of ToxA or ToxB, while these genes were missing from 128 genomes. Although some clusters were enriched for toxin presence, these genes are variably present in a given genetic background. The CDT genes were found in 191 genomes, which were restricted to a few clusters only, and only one cluster lacked the toxin A/B genes consistently. A total of 310 genomes contained ToxA/B without CDT (47%). Further, published metagenomic data from stools were used to assess the presence of C. difficile sequences in blinded cases of C. difficile infection (CDI) and controls, to test if metagenomic analysis is sensitive enough to detect the pathogen, and to establish strain relationships between cases from the same hospital. We conclude that metagenomics can contribute to the identification of CDI and can assist in characterization of the most probable causative strain in CDI patients.

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.

Dataset Information

Convergent Loss of ABC Transporter Genes From Clostridioides difficile Genomes Is Associated With Impaired Tyrosine Uptake and p-Cresol Production.

Publications

Convergent Loss of ABC Transporter Genes From <i>Clostridioides difficile</i> Genomes Is Associated With Impaired Tyrosine Uptake and <i>p</i>-Cresol Production.

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