Representative granulome histopathology of lungs from infected mice after 40 and 90 days of infection.

<p>BALB/c mice were euthanized 40 and 90 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with <i>P. brasiliensis</i>. Infected/T, same as Infected, but treated with fluconazole. rPb27/T, group infected and posteriorly immunized with rPb27 and treated with fluconazole. In each photo, the scale bar represents 25.9 µm.</p

IgG isotypes production against rPb27 by infected and immunized mice.

<p>Antibody response against rPb27 in mice infected and immunized with this recombinant protein associated or not with fluconazole chemotherapy was determined by ELISA assay after 40 (A) and 90 (B) days of treatment. Control, mice without any intervention. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. Bars represent the means and standard deviations of optical density (O.D.) at 1∶400 serum dilution in each experimental group (n = 3). * significant (p<0,05) difference in relation to the control group. # significant (p<0,05) difference in relation to the rPb27 group.</p

Representative histopathology of lungs, livers and spleens from infected mice, after 40 days of treatment.

<p>BALB/c mice were euthanized 40 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with <i>P. brasiliensis</i>. Infected/T, same as Infected, but treated with fluconazole. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. In each lung photos, the scale bar represents 427.3 µm, while in each liver and spleen photos, the scale bar represents 56.9 µm.</p

Representative histopathology of lungs, livers and spleens from infected mice, after 90 days of treatment.

<p>BALB/c mice were euthanized 90 days after treatment. The lungs, spleens, and livers were excised, fixed in 10% buffered formalin, and embedded in paraffin for sectioning. The sections were stained with hematoxylin–eosin and examined microscopically. Infected, group only infected with <i>P. brasiliensis</i>. Infected/T, same as Infected, but treated with fluconazole. rPb27, group infected and posteriorly immunized with rPb27. rPb27/T, same as rPb27, but treated with fluconazole. In each lung photos, the scale bar represents 416.6 µm, while in each liver and spleen photos, the scale bar represents 55.6 µm.</p

Fungal recovery in lung, spleen and liver of infected mice.

<p>The CFUs were estimated 40 (A) and 90 days (B) post treatment in organs from mice infected intratracheally with 3×10⁵<i>P. brasiliensis</i> yeast cells and subjected to fluconazole treatment combined or not with rPb27 immunization. Control mice were only infected with <i>P. brasiliensis</i> (Infected), adjuvant mice were inoculated with <i>C. parvum</i>-Al(OH)₃ with fluconazole treatment (Adjuvant/T) or not (Adjuvant), and rPb27 mice were immunized with recombinant protein combined to fluconazole treatment (rPb27/T) or not (rPb27). All groups of mice were infected with the same number of yeast cells. Bars represent the Log₁₀(UFC/g) means and standard deviations from organs of 3 to 5 animals in each group. * significant (p<0,05) difference in relation to the group of mice only infected.</p

Crystal Structures of Apo and Liganded 4‑Oxalocrotonate Decarboxylase Uncover a Structural Basis for the Metal-Assisted Decarboxylation of a Vinylogous β‑Keto Acid

The enzymes in the catechol <i>meta</i>-fission pathway have been studied for more than 50 years in several species of bacteria capable of degrading a number of aromatic compounds. In a related pathway, naphthalene, a toxic polycyclic aromatic hydrocarbon, is fully degraded to intermediates of the tricarboxylic acid cycle by the soil bacteria <i>Pseudomonas putida</i> G7. In this organism, the 83 kb NAH7 plasmid carries several genes involved in this biotransformation process. One enzyme in this route, NahK, a 4-oxalocrotonate decarboxylase (4-OD), converts 2-oxo-3-hexenedioate to 2-hydroxy-2,4-pentadienoate using Mg²⁺ as a cofactor. Efforts to study how 4-OD catalyzes this decarboxylation have been hampered because 4-OD is present in a complex with vinylpyruvate hydratase (VPH), which is the next enzyme in the same pathway. For the first time, a monomeric, stable, and active 4-OD has been expressed and purified in the absence of VPH. Crystal structures for NahK in the apo form and bonded with five substrate analogues were obtained using two distinct crystallization conditions. Analysis of the crystal structures implicates a lid domain in substrate binding and suggests roles for specific residues in a proposed reaction mechanism. In addition, we assign a possible function for the NahK N-terminal domain, which differs from most of the other members of the fumarylacetoacetate hydrolase superfamily. Although the structural basis for metal-dependent β-keto acid decarboxylases has been reported, this is the first structural report for that of a vinylogous β-keto acid decarboxylase and the first crystal structure of a 4-OD