Ruminococcus flavefaciens is a vital cellulosome-producing fibrolytic rumen bacterium. The arrangement of the cellulosomal scaffoldin gene cluster (scaC-scaA-scaB-cttA-scaE) is conserved in two R. flavefaciens strains (17 and FD-1). Sequence analysis revealed a high mosaic conservation of the intergenic regions in the two strains that contrasted sharply with the divergence of the structural sca gene sequences. Based on the conserved intergenic regions, we designed PCR primers in order to examine the sca gene cluster in additional R. flavefaciens strains (C94, B34b, C1a and JM1). Using these conserved and/or degenerate primers, the scaC, scaA and scaB genes were amplified in all six strains, while the entire sca gene cluster and the proximal genes cttA and scaE were successfully amplified in four of the strains (17, FD-1, C94 and JM1). The sequencing of scaA and scaC genes in all the strains yielded additional insight into the variability of the structural genes with regard to the number and type of cohesin modules contained in a conserved molecular skeleton. Moreover, the scaC gene, being short and variable, appears to be a promising functional phylotyping target for metagenomic population studies of R. flavefaciens in the rumen as a function of the individual host animal.

Graft-versus-host-disease (GVHD) is a major obstacle to successful allogeneic hematopoietic cell transplantation (alloHCT). Cannabidiol (CBD), a nonpsychotropic ingredient of Cannabis sativa, possesses potent anti-inflammatory and immunosuppressive properties. We hypothesized that CBD may decrease GVHD incidence and severity after alloHCT. We conducted a phase II study. GVHD prophylaxis consisted of cyclosporine and a short course of methotrexate. Patients transplanted from an unrelated donor were given low-dose anti-T cell globulin. CBD 300 mg/day was given orally starting 7 days before transplantation until day 30. Forty-eight consecutive adult patients undergoing alloHCT were enrolled. Thirty-eight patients (79%) had acute leukemia or myelodysplastic syndrome and 35 patients (73%) were given myeloablative conditioning. The donor was either an HLA-identical sibling (n = 28), a 10/10 matched unrelated donor (n = 16), or a 1-antigen-mismatched unrelated donor (n = 4). The median follow-up was 16 months (range, 7 to 23). No grades 3 to 4 toxicities were attributed to CBD. None of the patients developed acute GVHD while consuming CBD. In an intention-to-treat analysis, we found that the cumulative incidence rates of grades II to IV and grades III to IV acute GVHD by day 100 were 12.1% and 5%, respectively. Compared with 101 historical control subjects given standard GVHD prophylaxis, the hazard ratio of developing grades II to IV acute GVHD among subjects treated with CBD plus standard GVHD prophylaxis was .3 (P = .0002). Rates of nonrelapse mortality at 100 days and at 1 year after transplantation were 8.6% and 13.4%, respectively. Among patients surviving more than 100 days, the cumulative incidences of moderate-to-severe chronic GVHD at 12 and 18 months were 20% and 33%, respectively. The combination of CBD with standard GVHD prophylaxis is a safe and promising strategy to reduce the incidence of acute GVHD. A randomized double-blind controlled study is warranted. (clinicaltrials.gov: NCT01385124).

Background Ruminococcus flavefaciens is one of the predominant fiber-degrading bacteria found in the rumen of herbivores. Bioinformatic analysis of the recently sequenced genome indicated that this bacterium produces one of the most intricate cellulosome systems known to date. A distinct ORF, encoding for a multi-modular protein, RflaF_05439, was discovered during mining of the genome sequence. It is composed of two tandem modules of currently undefined function that share 45% identity and a C-terminal X-dockerin modular dyad. Gaining insight into the diversity, architecture and organization of different types of proteins in the cellulosome system is essential for broadening our understanding of a multi-enzyme complex, considered to be one of the most efficient systems for plant cell wall polysaccharide degradation in nature.Methodology/Principal FindingsFollowing bioinformatic analysis, the second tandem module of RflaF_05439 was cloned and its selenium-labeled derivative was expressed and crystallized. The crystals belong to space group P21 with unit-cell parameters of a = 65.81, b = 60.61, c = 66.13 Å, β = 107.66° and contain two protein molecules in the asymmetric unit. The crystal structure was determined at 1.38-Å resolution by X-ray diffraction using the single-wavelength anomalous dispersion (SAD) method and was refined to Rfactor and Rfree of 0.127 and 0.152 respectively. The protein molecule mainly comprises a β-sheet flanked by short α-helixes, and a globular α-helical domain. The structure was found to be structurally similar to members of the NlpC/P60 superfamily of cysteine peptidases.Conclusions/SignificanceThe 3D structure of the second repeat of the RflaF_05439 enabled us to propose a role for the currently undefined function of this protein. Its putative function as a cysteine peptidase is inferred from in silico structural homology studies. It is therefore apparent that cellulosomes integrate proteins with other functions in addition to the classic well-defined carbohydrate active enzymes.

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Edited by Wilhelm JustKeywords: Cohesin Type III Dockerin Cellulose degradation Cohesin-dockerin complex Rumen bacteria Ruminococcus flavefaciens a b s t r a c tThe cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. Dockerin-bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin-borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface.

PDB reference: cohesin from R. flavefaciens, 4n2oRuminococcus flavefaciens is a cellulolytic bacterium found in the rumen of herbivores and produces one of the most elaborate and variable cellulosome systems. The structure of an R. flavefaciens protein (RfCohG, ZP_06142108), representing a freestanding (non-cellulosomal) type III cohesin module, has been determined. A selenomethionine derivative with a C-terminal histidine tag was crystallized and diffraction data were measured to 2.44 Å resolution. Its structure was determined by single-wavelength anomalous dispersion, revealing eight molecules in the asymmetric unit. RfCohG exhibits the most complex among all known cohesin structures, possessing four -helical elements and a topographical protuberance on the putative dockerin-binding surface.

The second type II cohesin module of the cellulosomal scaffoldin polypeptide ScaB from Acetivibrio cellulolyticus (CohB2) was cloned into two constructs: one containing a short (five-residue) C-terminal linker (CohB2_S) and the second incorporating the full native 45-residue linker (CohB2_L). Both constructs encode proteins that also include the full native six-residue N-terminal linker. The CohB2_S and CohB2_L proteins were expressed, purified and crystallized in the orthorhombic crystal system, but with different unit cells and symmetries: space group P2 1 2 1 2 1 with unit-cell parameters a = 90.36, b = 68.65, c = 111.29 Å for CohB2_S and space group P2 1 2 1 2 with unitcell parameters a = 68.76, b = 159.22, c = 44.21 Å for CohB2_L. The crystals diffracted to 2.0 and 2.9 Å resolution, respectively. The asymmetric unit of CohB2_S contains three cohesin molecules, while that of CohB2_L contains two molecules.

Graft-versus-host-disease (GVHD) affects more than 50% of transplanted patients and is a major obstacle to successful allogeneic stem cell transplantation (alloSCT). Cannabidiol (CBD), a non-psychotropic ingredient of Cannabis has been shown to exhibit potent anti-inflammatory properties in animal models of various autoimmune diseases like multiple sclerosis, rheumatoid arthritis, inflammatory bowel disease and diabetes mellitus. We hypothesized that the addition of CBD to standard GVHD prophylaxis may decrease GVHD incidence and severity.