T cell proliferation is reduced in MOG_35-55 treated animals.

<p><b>(A)</b> T cell proliferation in response to rechallenge with MOG_35-55 at the indicated concentrations. Spleen cell proliferation at day 15 of EAE of animals of the indicated groups was measured by [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155082#pone.0155082.ref003" target="_blank">3</a>]H-thymidine incorporation in the presence of increasing concentrations of MOG_35-55 (0–50 μg/ml) in quadruplicates of four independent experiments. Depicted is the mean ± SEM of six mice per group. <b>(B)</b> Number of FoxP3 positive cells gated on CD4 T cells of lymphoid organs. Cells from the indicated organs were prepared at day 15 of EAE from the four groups. Intranuclear FoxP3 staining of was used to determine frequencies of regulatory T cell within the CD4 population in three independent experiments with 4 mice per group. dLN: immunization site draining lymph node; mLN: mesenteric (gut-draining) lymph node; PP: Peyer’s patches. EAE = immunized mice, untreated; CU ctrl = <i>C</i>. <i>utilis</i> wild-type; CU MOG = CBCu17. Asterisks indicate significance (* p < 0.05; *** p < 0.01 *** p < 0.001) student’s t test.</p

Clinical manifestation of EAE in mice after oral gavage of <i>C</i>. <i>utilis</i> expressing MOG_35-55.

<p><b>(A)</b> Mean clinical score of four independent experiments testing the preventive potential of <i>C</i>. <i>utilis</i> expressing MOG_35-55. C57BL/6 mice (n = 6–8) were immunized and 1.5 x 10⁸ <i>C</i>. <i>utilis</i> were applied daily by oral gavage for 7 days prior immunization for 10 days. <i>C</i>. <i>utilis</i> expressing MOG_35-55 (CBCu17) was tested either alive or heat killed. Control groups received 1.5 x 10⁸ wild-type <i>Candida utilis</i> (ctrl.) or no cells (EAE). <b>(B)</b> Incidence of EAE and <b>(C)</b> maximal score at the peak of the disease after oral gavage of <i>C</i>. <i>utilis</i> to C57BL/6 mice in the indicated groups. Incidence was calculated as percentage of mice that displayed clinical symptoms of a score of one for more than two days deviated to number of immunized mice in the given groups for each experiment. Clinical scoring was assessed daily using the following system: 0 = no clinical signs; 1 = tail paralysis; 2 = hind limb paresis; 3 = hind limb paralysis; 4 = fore limp paresis; 5 = death. Depicted is the mean ± SEM of four independent experiments. Asterisks indicate significance (* p < 0.05; *** p < 0.01 *** p < 0.001) student’s t test.</p

MOG production and localization in <i>C</i>. <i>utilis</i>.

<p><b>(A)</b> Transformants of CBCu7 and CBCu8 were incubated for 72 h in YPD medium supplemented with 10 μg/ml Nourseothricin. Cells were harvested and washed two times with PBS. 20 μl of cells were loaded on a SDS gel (4–20% acrylamide) and the MOG peptide was detected in an immunoblot with a mouse anti-MOG_35-55 antibody and a secondary horseradish peroxidase coupled anti-mouse antibody. Protein masses are indicated in kDa. Strain MKCu1 (vc) was used as control. Lane 1–3: three independent transformants. <b>(B)</b> Strain CBCu8 was incubated for 72 h in YPD medium and MOG was detected on the surface of non permeabilized cells with a mouse anti-MOG_35-55 antibody and a secondary FITC-coupled anti-mouse antibody. Nuclear DNA was stained with 4′,6-Diamidin-2-phenylindol (DAPI). Strain MKCu1 (<i>TDH3</i>p) was used as negative control. <b>(C)</b> Feeding protocol. On day 0 (d0) fecal pellets were collected and one group (n = 6) was administered with 1x10⁷ CBCu17 cells, another group with 1x10⁸ cells. Fecal pellets were collected every 24 hours and on day 2 (d2) mice were fed with CBCu17 cells again. On day 11 the experiment was stopped. The collected fecal pellets were plated out on YPD agar plates containing ampicillin (100 μg/ml) and Nourseothricin (10 μg/ml) to prevent bacterial growth and colony forming units were counted. Mean values of three measurements and standard deviations are shown.</p

MOG expression vector.

<p><b>(A)</b> Scheme of the expression unit of the single MOG plasmid pCB13 and the corresponding encoded amino acid sequence. <b>(B)</b> Scheme of the tandem MOG expression plasmid pCB10 and amino acid sequence. The coding sequence for the MOG epitope was inserted between the secretion signal sequence (red) and GPI anchor sequence (blue) of the <i>C</i>. <i>utilis GAS1</i> sequence. The immunogenic MOG epitope (green) is linked to the <i>GAS1</i> GPI sequence with a non immunogenic MOG spacer sequence (black). The potential GPI attachment site (ω) is highlighted in orange. <i>Nhe</i>I restriction sites are in <i>italics</i>.</p

Oral Tolerance Induction in Experimental Autoimmune Encephalomyelitis with <i>Candida utilis</i> Expressing the Immunogenic MOG35-55 Peptide

<div><p>Multiple sclerosis (MS) is an autoimmune disease that attacks myelinated axons in the central nervous system. Induction of oral tolerance is a potent mechanism to prevent autoimmunity. The food yeast <i>Candida utilis</i> was used to test the therapeutic potential of oral tolerance induction in an animal model of human multiple sclerosis (MS). We constructed a <i>C</i>. <i>utilis</i> strain, which displays a fusion peptide composed of the encephalitogenic MOG_35-55 peptide and the <i>C</i>. <i>utilis</i> Gas1 cell wall protein on its surface.By immunizing mice with MOG_35-55 peptide experimental autoimmune encephalomyelitis (EAE) was induced in a mouse model. Feeding of mice with <i>C</i>. <i>utilis</i> that expresses MOG_35-55 peptide on its surface was started seven days prior to immunization and was continued for ten days. Control animals were treated with wild-type fungus or left untreated. Untreated mice developed first clinical symptoms ten days post immunization (p. i.) with an ascending paralysis reaching maximal clinical disability at day 18 to 20 p. i.. Treatment with the wild-type strain demonstrated comparable clinical symptoms. In contrast, oral gavage of MOG_35-55-presenting fungus ameliorated the development of EAE. In addition, incidence as well as maximal clinical disease severity were significantly reduced. Interestingly, reduction of disease severity also occurred in animals treated with heat-inactivated <i>C</i>. <i>utilis</i> cells indicating that tolerance induction was independent of fungal viability. Better disease outcome correlated with reduced demyelination and cellular inflammation in the spinal cord, lower T cell proliferation against rechallenge with MOG_35-55 and more regulatory T cells in the lymph nodes. Our data demonstrate successful that using the food approved fungus <i>C</i>. <i>utilis</i> presenting the immunogenic MOG_35-55 peptide on its surface induced an oral tolerance against this epitope in EAE. Further studies will reveal the nature and extent of an anti-inflammatory environment established by the treatment that prevents the development of an autoimmune disorder affecting the CNS.</p></div

The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nucleotide polymorphisms

Rupp O, Brinkrolf K, Buerth C, et al. The structure of the Cyberlindnera jadinii genome and its relation to Candida utilis analyzed by the occurrence of single nucleotide polymorphisms. Journal of Biotechnology. 2015;211:20-30.The yeast Cyberlindnera jadinii is a close relative of Candida utilis that is being used in the food and feed industries. Here, we present the 12.7Mb genome sequence of C. jadinii strain CBS 1600 generated by next generation sequencing. The deduced draft genome sequence consists of seven large scaffolds analogous to the seven largest chromosomes of C. utilis. An automated annotation of the C jadinii genome identified 6147 protein-coding sequences. The level of ploidy for both genomes was analyzed by calling single nucleotide polymorphisms (SNPs) and was verified measuring nuclear DNA contents by florescence activated cell sorting (FACS). Both analyses determined the level of ploidy to diploid for C. jadinii and to triploid for C utilis. However, SNP calling for C. jadinii also identified scaffold regions that seem to be haploid, triploid or tetraploid. (C) 2015 Elsevier B.V. All rights reserved