On the Occurrence and Multimerization of Two-Polypeptide Phage Endolysins Encoded in Single Genes.

Bacteriophages (phages) and other viruses are extremely efficient in packing their genetic information, with several described cases of overlapping genes encoded in different open reading frames (ORFs). While less frequently reported, specific cases exist in which two overlapping ORFs are in frame and share the stop codon. Here, we studied the occurrence of this genetic arrangement in endolysins, the phage enzymes that cut the bacterial cell wall peptidoglycan to release the virion progeny. After screening over 3,000 endolysin sequences of phages infecting Gram-positive bacteria, we found evidence that this coding strategy is frequent in endolysin genes. Our bioinformatics predictions were experimentally validated by demonstrating that two polypeptides are indeed produced from these genes. Additionally, we show that in some cases the two polypeptides need to interact and multimerize to generate the active endolysin. By studying in detail one selected example, we uncovered a heteromeric endolysin with a 1:5 subunit stoichiometry that has never been described before. Hence, we conclude that the occurrence of endolysin genes encoding two polypeptide isoforms by in-frame overlapping ORFs, as well as their organization as enzymatic complexes, appears more common than previously thought, therefore challenging the established view of endolysins being mostly formed by single, monomeric polypeptide chains. IMPORTANCE Bacteriophages use endolysins to cleave the host bacteria cell wall, a crucial event underlying cell lysis for virion progeny release. These bacteriolytic enzymes are generally thought to work as single, monomeric polypeptides, but a few examples have been described in which a single gene produces two endolysin isoforms. These are encoded by two in-frame overlapping ORFs, with a shorter ORF being defined by an internal translation start site. This work shows evidence that this endolysin coding strategy is frequent in phages infecting Gram-positive bacteria, and not just an eccentricity of a few phages. In one example studied in detail, we show that the two isoforms are inactive until they assemble to generate a multimeric active endolysin, with a 1:5 subunit stoichiometry never described before. This study challenges the established view of endolysins, with possible implications in their current exploration and design as alternative antibacterials.

Chemico-Biological Characterization of Torpedino Di Fondi® Tomato Fruits: A Comparison with San Marzano Cultivar at Two Ripeness Stages.

Torpedino di Fondi (TF) is a hybrid tomato landrace developed in Sicily and recently introduced in the south Lazio area along with the classical San Marzano (SM) cultivar. The present study aimed at characterizing TF tomatoes at both pink and red ripening stages, and at comparing them with traditional SM tomatoes. A multidisciplinary approach consisting of morphological, chemical (FT-ICR MS, NMR, HPLC, and spectrophotometric methods), and biological (antioxidant and antifungal in vitro activity) analyses was applied. Morphological analysis confirmed the mini-San Marzano nature and the peculiar crunchy and solid consistency of TF fruits. Pink TF tomatoes displayed the highest content of hydrophilic antioxidants, like total polyphenols (0.192 mg/g), tannins (0.013 mg/g), flavonoids (0.204 mg/g), and chlorophylls a (0.344 mg/g) and b (0.161 mg/g), whereas red TF fruits were characterized by the highest levels of fructose (3000 mg/100 g), glucose (2000 mg/100 g), tryptophan (2.7 mg/100 g), phenylalanine (13 mg/100 g), alanine (25 mg/100 g), and total tri-unsaturated fatty acids (13% mol). Red SM fruits revealed the greatest content of lipophilic antioxidants, with 1234 mg/g of total carotenoids. In agreement with phenolics content, TF cultivar showed the greatest antioxidant activity. Lastly, red TF inhibited Candida species (albicans, glabrata and krusei) growth.

The proteome response to amyloid protein expression in vivo.

Protein misfolding disorders such as Alzheimer, Parkinson and transthyretin amyloidosis are characterized by the formation of protein amyloid deposits. Although the nature and location of the aggregated proteins varies between different diseases, they all share similar molecular pathways of protein unfolding, aggregation and amyloid deposition. Most effects of these proteins are likely to occur at the proteome level, a virtually unexplored reality. To investigate the effects of an amyloid protein expression on the cellular proteome, we created a yeast expression system using human transthyretin (TTR) as a model amyloidogenic protein. We used Saccharomyces cerevisiae, a living test tube, to express native TTR (non-amyloidogenic) and the amyloidogenic TTR variant L55P, the later forming aggregates when expressed in yeast. Differential proteome changes were quantitatively analyzed by 2D-differential in gel electrophoresis (2D-DIGE). We show that the expression of the amyloidogenic TTR-L55P causes a metabolic shift towards energy production, increased superoxide dismutase expression as well as of several molecular chaperones involved in protein refolding. Among these chaperones, members of the HSP70 family and the peptidyl-prolyl-cis-trans isomerase (PPIase) were identified. The latter is highly relevant considering that it was previously found to be a TTR interacting partner in the plasma of ATTR patients but not in healthy or asymptomatic subjects. The small ubiquitin-like modifier (SUMO) expression is also increased. Our findings suggest that refolding and degradation pathways are activated, causing an increased demand of energetic resources, thus the metabolic shift. Additionally, oxidative stress appears to be a consequence of the amyloidogenic process, posing an enhanced threat to cell survival.

Catalysis and structural properties of Leishmania infantum glyoxalase II: trypanothione specificity and phylogeny.

The glyoxalase pathway catalyzes the formation of d-lactate from methylglyoxal, a toxic byproduct of glycolysis. In trypanosomatids, trypanothione replaces glutathione in this pathway, making it a potential drug target, since its selective inhibition might increase methylglyoxal concentration in the parasites. Two glyoxalase II structures were solved. One with a bound spermidine molecule (1.8 A) and the other with d-lactate at the active site (1.9 A). The second structure was obtained by crystal soaking with the enzyme substrate (S)-d-lactoyltrypanothione. The overall structure of Leishmania infantum glyoxalase II is very similar to its human counterpart, with important differences at the substrate binding site. The crystal structure of L. infantum glyoxalase II is the first structure of this enzyme from trypanosomatids. The differential specificity of glyoxalase II toward glutathione and trypanothione moieties was revealed by differential substrate binding. Evolutionary analysis shows that trypanosomatid glyoxalases II diverged early from eukaryotic enzymes, being unrelated to prokaryotic proteins.