Strains used in this study.

a<p>–Plasmids listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone-0047033-t002" target="_blank">Table 2</a>.</p>b<p>–Phylogenetic species as defined by Matute et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone.0047033-Matute1" target="_blank">[17]</a> and Teixeira et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone.0047033-Teixeira1" target="_blank">[18]</a>.</p

The genome of <i>Paracoccidioides</i> encodes an α-pheromone.

<p>(A) Mating pheromone signaling pathway described in <i>S. cerevisiae </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047033#pone.0047033-Butler1" target="_blank">[21]</a>. (B) <i>S. cerevisiae</i> mating pheromone signaling pathway components and the homologous genes annotated in <i>Paracoccidioides</i> and <i>A. fumigatus</i> databases. Genes not annotated in databases are denoted N.A. (C) Alignment of α-pheromone precursor peptide sequences from <i>Paracoccidioides</i> strains and <i>H. capsulatum</i>. The Kex2 peptidase recognition site (KR) and the mature pheromone are indicated by blue and red boxes, respectively.</p

α-pheromone activates PreB receptor and induced <i>shmoo</i> formation in the <i>S. cerevisiae</i> model.

<p><i>Shmoo</i> formation in <i>S. cerevisiae</i> BY4741 induced with 20 µg/ml synthetic MFα pheromone after 0 and 5 hrs induction (A and B), and <i>S. cerevisiae</i> AGLPreB induced with 40 µg/ml synthetic Pbα pheromone after 0 and 5 hrs induction (C and D). Quantification of <i>shmoo</i> formation using <i>S. cerevisiae</i> BY4741 stimulated with MFα (2 µg/mL) or <i>S. cerevisiae</i> AGLPreB stimulated with Pbα (4 µg/mL) (E). Induction of <i>S. cerevisiae</i> AGLPreB using Pbα pheromone extracted from <i>P. lutzii</i> Pb01 monoculture or <i>Paracoccidioides</i> co-cultures (Pb01/Pb03 and Pb01/Pb60855), Pbα pheromone extracted from AGMPbα culture, as well stimulation with the synthetic Pbα pheromone (40 µg/mL) (F). Error bars are indicated, and asterisks show significant differences (<i>*</i>p<0.05, <i>***</i>p<0.001).</p

α-pheromone activates PreB receptor, inducing growth and cell cycle arrest in the <i>S. cerevisiae</i> model.

<p>Halo formation in <i>S. cerevisiae</i> AGLPreB stimulated with Pbα pheromone extracted from <i>P. lutzii</i> Pb01 monoculture (A); stimulated with 0.8, 4.0 and 40 µg synthetic Pbα pheromone (B–D respectively) or Pbα pheromone extracted from <i>S. cerevisiae</i> AGMPbα (E). <i>S. cerevisiae</i> BY4741 stimulated with 10 µg synthetic MFα (F) and with 20 µg synthetic Pbα (G). Quantification of growth inhibition zones (cm²) upon exposure to α-pheromones. Halos were recorded after 24 hrs of incubation and standard deviations indicated by error bars (H). Cell cycle arrest analysis for <i>S. cerevisiae</i> BY4741 upon addition of synthetic MFα (20 µg/ml) (I) and <i>S. cerevisiae</i> AGLPreB upon addition of synthetic Pbα pheromone (40 µg/ml) (J). (K–N) FCM profiles of α-pheromone-induced cell cycle arrest in haploid cells showing n and 2 n nuclear DNA content: BY4741 induced with 20 µg synthetic MFα (K–L) and <i>S. cerevisiae</i> AGLPreB induced with 40 µg synthetic Pbα pheromone (M–N). Growth rate evaluation of either <i>S. cerevisiae</i> BY4741 (O) or AGLPreB (P) upon addition of the cognate synthetic α-pheromone. Error bars are indicated, and asterisks show significant differences (<i>*</i>p<0.05, <i>**</i>p<0.01).</p

<i>Paracoccidioides</i> expresses the α-pheromone and the a- and α-receptor genes independently of the mating type.

<p>Mating gene expression of <i>Paracoccidioides MAT1-1</i> strains (Pb01 and T8B1) and <i>MAT1-2</i> strains (ATCC60855 and Pb03) in yeast (Y) and mycelial (M) form. Expression levels of <i>MAT1-1</i> (A) and <i>MAT1-2</i> (B), <i>PBα</i> (B, C), <i>PREB</i> (E, F) and <i>PREA</i> (G, H) genes were measured. Error bars are indicated, and asterisks show significant differences (<i>*</i>p<0.05 or <i>**</i>p<0.01).</p

α-pheromone does not induce mating gene expression in <i>Paracoccidioides</i>.

<p>Mating gene expression at different time points in <i>P. brasiliensis</i> ATCC60855 (A) and <i>P. lutzii</i> Pb01 (B) in mycelium form, upon induction with 50 µg/mL of synthetic Pbα pheromone. Basal expression levels of MAPK signaling pathway components in the mycelium form of <i>P. brasiliensis</i> Pb03 and ATCC60855 (<i>MAT1-2</i>) and <i>P. lutzii</i> Pb01 (<i>MAT1-1</i>) (C-K). Error bars are indicated, and asterisks show significant differences (<i>*</i>p<0.05, <i>**</i>p<0.01, <i>***</i>p<0.001).</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

α-pheromone and its cognate receptor functionally complement mating in the corresponding <i>S. cerevisiae</i> mutants.

<p> Mating efficiency of <i>S. cerevisiae</i> null mutants, expressing <i>Paracoccidioides</i> α–pheromone (AGLPbα and AGMPbα) and its cognate receptor (AGLPreB), crossed at different ratios after incubation for 5 hrs (A). (B) Mating efficiency after 5 hrs incubation of <i>S. cerevisiae</i> strains AGΔScα and AGLPreB, upon the addition of different concentrations of <i>Paracoccidioides</i> synthetic α-pheromone. Error bars are indicated, and asterisks show significant differences (<i>*</i>p<0.05).</p

<i>L. salivarius</i> UCC118 survives in, and adheres to, the pig ileum.

<p>Box-plots showing <i>L. salivarius</i> numbers on Day 29 in ileal digesta (A) and tissue (B) samples collected from pigs fed sterile milk (Control group) or milk incorporating 1×10¹⁰ CFU/day of <i>L. salivarius</i> UCC118 WT (Bac+ group) or <i>L. salivarius</i> UCC118 Δ<i>abpT</i> (Bac− group). Box-plots represent the median and the lower and upper quartiles. Whiskers extend to the last data point still within 1.5 inter-quartile range of the quartiles.</p

<i>L. salivarius</i> UCC118 administration effect on pig growth performance.

(a)<p>Values are mean ± SEM, <i>n</i> = 8.</p><p>*<i>p</i> = 0.006,</p>++<p>p = 0.06 and</p>+<p>p = 0.09, between the Bac+ and Bac− groups and the control group.</p