Fecal Clostridium symbiosum for Noninvasive Detection of Early and Advanced Colorectal Cancer: Test and Validation Studies.

OBJECTIVE: Current non-invasive early detection of colorectal cancer (CRC) requires improvement. We aimed to identified a fecal Clostridium symbiosum-based biomarker for early and advanced colorectal cancer detection. DESIGN: In the test stage, the relative abundance of Clostridium symbiosum (C. symbiosum) was measured by qPCR in 781 cases including 242 controls, 212 colorectal adenoma (CRA) patients, 109 early CRC (tumor restricted to the submucosa) patients, 218 advanced CRC patients. The prediction accuracy was compared to Fusobacterium nucleatum (F. nucleatum), fecal immunochemical test (FIT) and CEA (carcinoembryonic antigen) and validated in an independent cohort of 256 subjects. Current status of the trial:ongoing/still enrolling. Primary endpoint:June, 2017 (Clinicaltrials.gov Identifier NCT02845973). RESULTS: Significant stepwise increase of C. symbiosum abundance was found in CRA, early CRC and advanced CRC (P<0.01). C. symbiosum outperformed all the other markers in early CRC prediction performance. The combination of C. symbiosum and FIT achieved better performance (0.803 for test cohort and 0.707 for validation cohort). For overall discrimination of CRCs, the combination of all above markers achieved the performance of 0.876. CONCLUSIONS: Fecal C. symbiosum is a promising biomarker for early and noninvasive detection of colorectal cancer, being more effective than F. nucleatum, FIT and CEA. Combining C. symbiosum and FIT or CEA may improve the diagnosis power.

Re-engineering the discrimination between the oxidized coenzymes NAD+ and NADP+ in clostridial glutamate dehydrogenase and a thorough reappraisal of the coenzyme specificity of the wild-type enzyme.

Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2'-phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP(+) , although rates with NAD(+) were markedly diminished. This article reveals that the enzyme's discrimination in favour of NAD(+) and against NADP(+) had been greatly underestimated and has indeed been abated by a factor of > 16,000 by the mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed a burst increase of A(340) with NADP(+) but not NAD(+), with amplitude depending on the concentration of the coenzyme, rather than enzyme. Amplitude also varied with the commercial source of the NADP(+). FPLC, HPLC and mass spectrometry identified NAD(+) contamination ranging from 0.04 to 0.37% in different commercial samples. It is now clear that apparent rates of NADP(+) utilization mainly reflected the reduction of contaminating NAD(+), creating an entirely false view of the initial coenzyme specificity and also of the effects of mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.

Analysis of the mobilization functions of the vancomycin resistance transposon Tn1549, a member of a new family of conjugative elements.

Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::Delta nic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOB(p) superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOB(p) clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.

An asymmetric model for Na+-translocating glutaconyl-CoA decarboxylases.

Glutaconyl-CoA decarboxylase (Gcd) couples the biotin-dependent decarboxylation of glutaconyl-CoA with the generation of an electrochemical Na(+) gradient. Sequencing of the genes encoding all subunits of the Clostridium symbiosum decarboxylase membrane complex revealed that it comprises two distinct biotin carrier subunits, GcdC(1) and GcdC(2), which differ in the length of a central alanine- and proline-rich linker domain. Co-crystallization of the decarboxylase subunit GcdA with the substrate glutaconyl-CoA, the product crotonyl-CoA, and the substrate analogue glutaryl-CoA, respectively, resulted in a high resolution model for substrate binding and catalysis revealing remarkable structural changes upon substrate binding. Unlike the GcdA structure from Acidaminococcus fermentans, these data suggest that in intact Gcd complexes, GcdA is associated as a tetramer crisscrossed by a network of solvent-filled tunnels.

Swiveling domain mechanism in pyruvate phosphate dikinase.

Pyruvate phosphate dikinase (PPDK) catalyzes the reversible conversion of phosphoenolpyruvate (PEP), AMP, and Pi to pyruvate and ATP. The enzyme contains two remotely located reaction centers: the nucleotide partial reaction takes place at the N-terminal domain, and the PEP/pyruvate partial reaction takes place at the C-terminal domain. A central domain, tethered to the N- and C-terminal domains by two closely associated linkers, contains a phosphorylatable histidine residue (His455). The molecular architecture suggests a swiveling domain mechanism that shuttles a phosphoryl group between the two reaction centers. In an early structure of PPDK from Clostridium symbiosum, the His445-containing domain (His domain) was positioned close to the nucleotide binding domain and did not contact the PEP/pyruvate-binding domain. Here, we present the crystal structure of a second conformational state of C. symbiosum PPDK with the His domain adjacent to the PEP-binding domain. The structure was obtained by producing a three-residue mutant protein (R219E/E271R/S262D) that introduces repulsion between the His and nucleotide-binding domains but preserves viable interactions with the PEP/pyruvate-binding domain. Accordingly, the mutant enzyme is competent in catalyzing the PEP/pyruvate half-reaction but the overall activity is abolished. The new structure confirms the swivel motion of the His domain. In addition, upon detachment from the His domain, the two nucleotide-binding subdomains undergo a hinge motion that opens the active-site cleft. A similar hinge motion is expected to accompany nucleotide binding (cleft closure) and release (cleft opening). A model of the coupled swivel and cleft opening motions was generated by interpolation between two end conformations, each with His455 positioned for phosphoryl group transfer from/to one of the substrates. The trajectory of the His domain avoids major clashes with the partner domains while preserving the association of the two linker segments.

Clostridium symbiosum as a cause of bloodstream infection in an immunocompetent patient.

Bloodstream infection caused by Clostridium symbiosum was previously reported in only one case, from a highly immunocompromised patient with metastic colon cancer. We describe the second case of clinical bacteraemic sepsis caused by C. symbiosum from a previously healthy man, which underlines the pathogenicity of this species.

Transfer of vancomycin resistance transposon Tn1549 from Clostridium symbiosum to Enterococcus spp. in the gut of gnotobiotic mice.

The vancomycin resistance vanB2 gene cluster is disseminated worldwide and has been found in phylogenetically remote bacterial genera. The vanB2 operon is part of conjugative transposons Tn1549/Tn5382, but conjugative transposition of these elements has not been demonstrated. We have obtained transfer of a Tn1549-like element (referred to herein as "Tn1549-like") from Clostridium symbiosum MLG101 to Enterococcus faecium 64/3 and Enterococcus faecalis JH2-2 in the digestive tract of gnotobiotic mice and to E. faecium 64/3 in vitro. Retransfer of Tn1549-like from an E. faecium transconjugant also containing Tn916 to E. faecium BM77 was obtained in vitro, albeit at a very low frequency. Transfer efficiency was found to be both donor and recipient dependent. Pulsed-field gel electrophoresis analysis of total SmaI-digested DNA of 48 transconjugants indicated in 27 instances the acquisition of ca. 34 kb of DNA. Two transconjugants harbored two copies of the transposon. Sequencing of the flanking regions of Tn1549-like in 48 transconjugants revealed 29 integration events in 26 loci in the E. faecium genome, and two hot spots for insertion were identified. Integration of the transposon was associated with the acquisition of 5 (n = 18) or 6 (n = 7) bp of donor DNA or with 5-bp duplications of target DNA in the remaining transconjugants. These data demonstrate functionality of the Tn1549-like element and attest that the transfer of the vanB operon between enterococci and human commensal anaerobes occurs in the intestinal environment.