EPS production by Propionibacterium freudenreichii facilitates its immobilization for propionic acid production.

AIMS: Immobilization of microbial cells is a useful strategy for developing high cell density bioreactors with improved stability and productivity for production of different chemicals. Functionalization of the immobilization matrix or biofilm forming property of some strains has been utilized for achieving cell attachment. The aim of the present study was to investigate the production of exopolysaccharide (EPS) by Propionibacterium freudenreichii C.I.P 59.32 and utilize this feature for immobilization of the cells on porous glass beads for production of propionic acid. METHODS AND RESULTS: Propionibacterium freudenreichii was shown to produce both capsular and excreted EPS during batch cultivations using glucose as carbon source. Different electron microscopy techniques confirmed the secretion of EPS and formation of cellular aggregates. The excreted EPS was mainly composed of mannose and glucose in a 5·3 : 1 g g-1 ratio. Immobilization of the cells on untreated and polyethyleneimine (PEI)-treated Poraver beads in a bioreactor was evaluated. Higher productivity and yield of propionic acid (0·566 g l-1  h-1 and 0·314 g g-1 , respectively) was achieved using cells immobilized to untreated beads and EPS production reached 617·5 mg l-1 after 48 h. CONCLUSION: These results suggest an important role of EPS-producing strains for improving cell immobilization and propionic acid production. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the EPS-producing microbe to be easily immobilized on a solid matrix and to be used in a bioprocess. Such a system could be optimized for achieving high cell density in fermentations without the need for functionalization of the matrix.

Potent In Vitro Antiproliferative Synergism of Combinations of Ergosterol Biosynthesis Inhibitors against Leishmania amazonensis.

Leishmaniases comprise a spectrum of diseases caused by protozoan parasites of the Leishmania genus. Treatments available have limited safety and efficacy, high costs, and difficult administration. Thus, there is an urgent need for safer and more-effective therapies. Most trypanosomatids have an essential requirement for ergosterol and other 24-alkyl sterols, which are absent in mammalian cells. In previous studies, we showed that Leishmania amazonensis is highly susceptible to aryl-quinuclidines, such as E5700, which inhibit squalene synthase, and to the azoles itraconazole (ITZ) and posaconazole (POSA), which inhibit C-14α-demethylase. Herein, we investigated the antiproliferative, ultrastructural, and biochemical effects of combinations of E5700 with ITZ and POSA against L. amazonensis. Potent synergistic antiproliferative effects were observed against promastigotes, with fractional inhibitory concentration (FIC) ratios of 0.0525 and 0.0162 for combinations of E5700 plus ITZ and of E5700 plus POSA, respectively. Against intracellular amastigotes, FIC values were 0.175 and 0.1125 for combinations of E5700 plus ITZ and E5700 plus POSA, respectively. Marked alterations of the ultrastructure of promastigotes treated with the combinations were observed, in particular mitochondrial swelling, which was consistent with a reduction of the mitochondrial transmembrane potential, and an increase in the production of reactive oxygen species. We also observed the presence of vacuoles similar to autophagosomes in close association with mitochondria and an increase in the number of lipid bodies. Both growth arrest and ultrastructural/biochemical alterations were strictly associated with the depletion of the 14-desmethyl endogenous sterol pool. These results suggest the possibility of a novel combination therapy for the treatment of leishmaniasis.

Tomatidine promotes the inhibition of 24-alkylated sterol biosynthesis and mitochondrial dysfunction in Leishmania amazonensis promastigotes.

Leishmaniasis is a set of clinically distinct infectious diseases caused by Leishmania, a genus of flagellated protozoan parasites, that affects ~12 million people worldwide, with ~2 million new infections annually. Plants are known to produce substances to defend themselves against pathogens and predators. In the genus Lycopersicon, which includes the tomato, L. esculentum, the main antimicrobial compound is the steroidal glycoalkaloid α-tomatine. The loss of the saccharide side-chain of tomatine yields the aglycone tomatidine. In the present study, we investigated the effects of tomatidine on the growth, mitochondrial membrane potential, sterol metabolism, and ultrastructure of Leishmania amazonensis promastigotes. Tomatidine (0·1 to 5 µM) inhibited parasite growth in a dose-dependent manner (IC(50)=124±59 nM). Transmission electron microscopy revealed lesions in the mitochondrial ultrastructure and the presence of large vacuoles and lipid storage bodies in the cytoplasm. These structural changes in the mitochondria were accompanied by an effective loss of mitochondrial membrane potential and a decrease in ATP levels. An analysis of the neutral lipid content revealed a large depletion of endogenous 24-alkylated sterols such as 24-methylene-cholesta-5, 7-dien-3ß-ol (5-dehydroepisterol), with a concomitant accumulation of cholesta-8, 24-dien-3ß-ol (zymosterol), which implied a perturbation in the cellular lipid content. These results are consistent with an inhibition of 24-sterol methyltransferase, an important enzyme responsible for the methylation of sterols at the 24 position, which is an essential step in the production of ergosterol and other 24-methyl sterols.