Enrica Pessione

Lactic acid bacteria contribution to gut microbiota complexity: lights and shadows

Lactic Acid Bacteria (LAB) are ancient organisms that cannot biosynthesize functional cytochromes, and cannot get ATP from respiration. Besides sugar fermentation, they evolved electrogenic decarboxylations and ATP-forming deiminations. The right balance between sugar fermentation and decarboxylation/deimination ensures buffered environments thus enabling LAB to survive in human gastric trait and colonize gut. A complex molecular cross-talk between LAB and host exists. LAB moonlight proteins are made in response to gut stimuli and promote bacterial adhesion to mucosa and stimulate immune cells. Similarly, when LAB are present, human enterocytes activate specific gene expression of specific genes only. Furthermore, LAB antagonistic relationships with other microorganisms constitute the basis for their anti-infective role. Histamine and tyramine are LAB bioactive catabolites that act on the CNS, causing hypertension and allergies. Nevertheless, some LAB biosynthesize both gamma-amino-butyrate (GABA), that has relaxing effect on gut smooth muscles, and beta-phenylethylamine, that controls satiety and mood. Since LAB have reduced amino acid biosynthetic abilities, they developed a sophisticated proteolytic system, that is also involved in antihypertensive and opiod peptide generation from milk proteins. Short-chain fatty acids are glycolytic and phosphoketolase end-products, regulating epithelial cell proliferation and differentiation. Nevertheless, they constitute a supplementary energy source for the host, causing weight gain. Human metabolism can also be affected by anabolic LAB products such as conjugated linoleic acids (CLA). Some CLA isomers reduce cancer cell viability and ameliorate insulin resistance, while others lower the HDL/LDL ratio and modify eicosanoid production, with detrimental health effects. A further appreciated LAB feature is the ability to fix selenium into seleno-cysteine. Thus, opening interesting perspectives for their utilization as antioxidant nutraceutical vectors.

Towards lactic acid bacteria-based biorefineries.

Lactic acid bacteria (LAB) have long been used in industrial applications mainly as starters for food fermentation or as biocontrol agents or as probiotics. However, LAB possess several characteristics that render them among the most promising candidates for use in future biorefineries in converting plant-derived biomass-either from dedicated crops or from municipal/industrial solid wastes-into biofuels and high value-added products. Lactic acid, their main fermentation product, is an attractive building block extensively used by the chemical industry, owing to the potential for production of polylactides as biodegradable and biocompatible plastic alternative to polymers derived from petrochemicals. LA is but one of many high-value compounds which can be produced by LAB fermentation, which also include biofuels such as ethanol and butanol, biodegradable plastic polymers, exopolysaccharides, antimicrobial agents, health-promoting substances and nutraceuticals. Furthermore, several LAB strains have ascertained probiotic properties, and their biomass can be considered a high-value product. The present contribution aims to provide an extensive overview of the main industrial applications of LAB and future perspectives concerning their utilization in biorefineries. Strategies will be described in detail for developing LAB strains with broader substrate metabolic capacity for fermentation of cheaper biomass.

Purification, biochemical properties and substrate specificity of a catechol 1,2-dioxygenase from a phenol degrading Acinetobacter radioresistens

A catechol 1,2‐dioxygenase (C1,2O) has been purified to homogeneity from Acinetobacter radioresistens grown on phenol as the sole carbon and energy source. The C1,2O appears to be a homodimer, with a molecular mass of 78 000 Da. At relatively high ionic strengths (0.5 M Na2SO4) subunit dissociation occurs and the monomeric unit (38 700 Da) is shown to be active. This phenomenon has never been observed before in dioxygenases. The purified C1,2O contains 0.96 iron(III) ions per unit and spectroscopic measurements suggest the presence of one high‐spin iron(III) ion in an environment characteristic of intradiol cleaving enzymes. The NH2‐terminal amino acid sequence has been determined and compared to the primary structures of intradiol rings cleaving dioxygenases from other Acinetobacter strains revealing 45% homology with the benzoate‐grown A. calcoaceticus ADP‐1 and an identity of only one of the 20 amino acids sequenced for the phenol‐grown A. calcoaceticus NCIB 8250.

First evidence of a membrane-bound, tyramine and β-phenylethylamine producing, tyrosine decarboxylase in Enterococcus faecalis: A two-dimensional electrophoresis proteomic study

The soluble and membrane proteome of a tyramine producing Enterococcus faecalis, isolated from an Italian goat cheese, was investigated. A detailed analysis revealed that this strain also produces small amounts of β‐phenylethylamine. Kinetics of tyramine and β‐phenylethylamine accumulation, evaluated in tyrosine plus phenylalanine‐enriched cultures (stimulated condition), suggest that the same enzyme, the tyrosine decarboxylase (TDC), catalyzes both tyrosine and phenylalanine decarboxylation: tyrosine was recognized as the first substrate and completely converted into tyramine (100% yield) while phenylalanine was decarboxylated to β‐phenylethylamine (10% yield) only when tyrosine was completely depleted. The presence of an aspecific aromatic amino acid decarboxylase is a common feature in eukaryotes, but in bacteria only indirect evidences of a phenylalanine decarboxylating TDC have been presented so far. Comparative proteomic investigations, performed by 2‐DE and MALDI‐TOF/TOF MS, on bacteria grown in conditions stimulating tyramine and β‐phenylethylamine biosynthesis and in control conditions revealed 49 differentially expressed proteins. Except for aromatic amino acid biosynthetic enzymes, no significant down‐regulation of the central metabolic pathways was observed in stimulated conditions, suggesting that tyrosine decarboxylation does not compete with the other energy‐supplying routes. The most interesting finding is a membrane‐bound TDC highly over‐expressed during amine production. This is the first evidence of a true membrane‐bound TDC, longly suspected in bacteria on the basis of the gene sequence.

Media containing aromatic compounds induce peculiar proteins in Acinetobacter radioresistens, as revealed by proteome analysis

An Acinetobacter radioresistens strain able to grow on phenol or benzoate as sole carbon and energy source through the β‐ketoadipate pathway was isolated in our laboratories. In previous research, we found a different expression of catechol‐1,2‐dioxygenase isoenzymes (C‐1,2‐O) depending on the growth substrate (phenol or benzoate). In the present study, we used proteome techniques to extend our investigation to other enzymes involved in the aromatic degradation pathway. Since the first nontoxic metabolite in this route is cis,cis‐muconic acid, we focused our attention on the enzymes leading to this compound, chiefly phenol hydroxylase (PH), benzoate dioxygenase (BD), cis‐1,2‐dihydroxycyclohexa‐3,5‐diene‐1‐carboxylate dehydrogenase (D) and C‐1,2‐O. In particular, the A. radioresistens proteome was monitored under different growth substrate conditions, using acetate, benzoate, or phenol as sole carbon source. We compared the protein maps by software image analysis and detected marked differences, suggesting the inducibility of most enzymes. This research also sought to evaluate the conditions allowing the best expression of enzymes to be used in immobilized systems suitable for bioremediation. The experimental data indicate that benzoate is the best carbon source to gain the highest amount of C‐1,2‐O and D, while phenol is the best growth substrate to obtain PH.

Bioactive Molecules Released in Food by Lactic Acid Bacteria: Encrypted Peptides and Biogenic Amines

Lactic acid bacteria (LAB) can produce a huge amount of bioactive compounds. Since their elective habitat is food, especially dairy but also vegetal food, it is frequent to find bioactive molecules in fermented products. Sometimes these compounds can have adverse effects on human health such as biogenic amines (tyramine and histamine), causing allergies, hypertensive crises, and headache. However, some LAB products also display benefits for the consumers. In the present review article, the main nitrogen compounds produced by LAB are considered. Besides biogenic amines derived from the amino acids tyrosine, histidine, phenylalanine, lysine, ornithine, and glutamate by decarboxylation, interesting peptides can be decrypted by the proteolytic activity of LAB. LAB proteolytic system is very efficient in releasing encrypted molecules from several proteins present in different food matrices. Alpha and beta-caseins, albumin and globulin from milk and dairy products, rubisco from spinach, beta-conglycinin from soy and gluten from cereals constitute a good source of important bioactive compounds. These encrypted peptides are able to control nutrition (mineral absorption and oxidative stress protection), metabolism (blood glucose and cholesterol lowering) cardiovascular function (antithrombotic and hypotensive action), infection (microbial inhibition and immunomodulation) and gut-brain axis (opioids and anti-opioids controlling mood and food intake). Very recent results underline the role of food-encrypted peptides in protein folding (chaperone-like molecules) as well as in cell cycle and apoptosis control, suggesting new and positive aspects of fermented food, still unexplored. In this context, the detailed (transcriptomic, proteomic, and metabolomic) characterization of LAB of food interest (as starters, biocontrol agents, nutraceuticals, and probiotics) can supply a solid evidence-based science to support beneficial effects and it is a promising approach as well to obtain functional food. The detailed knowledge of the modulation of human physiology, exploiting the health-promoting properties of fermented food, is an open field of investigation that will constitute the next challenge.

Purification and catalytic properties of two catechol 1,2-dioxygenase isozymes from benzoate-grown cells of Acinetobacter radioresistens.

Two catechol 1,2-dioxygenase (C1,2O) isozymes (IsoA and IsoB) have been purified to homogeneity from a strain of Acinetobacter radioresistens grown on benzoate as the sole carbon and energy source. IsoA and IsoB are both homodimers composed of a single type of subunit with molecular mass of 38,600 and 37,700, Da respectively. In conditions of low ionic strength, IsoA can aggregate as a trimer, in contrast to IsoB, which maintains the dimeric structure, as also supported by the kinetic parameters (Hill numbers). IsoA is identical to the enzyme previously purified from the same bacterium grown on phenol, whereas the IsoB is selectively expressed using benzoate as carbon source. This is the first evidence of the presence of differently expressed C1,2O isozymes in A. radioresistens or more generally of multiple C1,2O isozymes in benzoate-grown Acinetobacter cells. Purified IsoA and IsoB contain approximately 1 iron(III) ion per subunit and both show electronic absorbance and EPR features typical of Fe(III) intradiol dioxygenases. The kinetic properties of the two enzymes such as the specificities toward substituted catechols, the main catalytic parameters, and their behavior in the presence of different kind of inhibitors are, unexpectedly, very similar, in contrast to most of the previously known dioxygenase isozymes.

Cloning and characterization of two catechol 1,2-dioxygenase genes from Acinetobacter radioresistens S13

Two novel catechol 1,2-dioxygenase (C 1,2-O) genes have been isolated from an Acinetobacter radioresistens strain that grows on phenol or benzoate as sole carbon and energy source. Designated as catA(A) and catA(B), they encode proteins composed of 314 and 306 amino acids, whose deduced sequences indicate that they have approximately 53% identity, whereas their NH2-terminal and COOH-terminal regions have no sequences in common. This may explain their different thermal and pH stability. Polyclonal antibodies raised against an amino-terminal CatA(A) peptide or the whole CatA(B) protein were used to establish their inducible and differential expression patterns upon bacterial growth in phenol or benzoate. The CatA(A) protein (IsoA) was induced by both phenol and benzoate though with different kinetics, whereas the catA(B) product (IsoB) was constitutively produced at low levels that increased only during growth in the presence of benzoate.

Proteomic characterization of a selenium-metabolizing probiotic Lactobacillus reuteri Lb2 BM for nutraceutical applications.

Selenium (Se), Se‐cysteines and selenoproteins have received growing interest in the nutritional field as redox‐balance modulating agents. The aim of this study was to establish the Se‐concentrating and Se‐metabolizing capabilities of the probiotic Lactobacillus reuteri Lb2 BM, for nutraceutical applications. A comparative proteomic approach was employed to study the bacteria grown in a control condition (MRS modified medium) and in a stimulated condition (4.38 mg/L of sodium selenite). The total protein extract was separated into two pI ranges: 4–7 and 6–11; the 25 identified proteins were divided into five functional classes: (i) Se metabolism; (ii) energy metabolism; (iii) stress/adhesion; (iv) cell shape and transport; (v) proteins involved in other functions. All the experimental results indicate that L. reuteri Lb2 BM is able to metabolize Se(IV), incorporating it into selenoproteins, through the action of a selenocysteine lyase, thus enhancing organic Se bioavailability. This involves endo‐ergonic reactions balanced by an increase of substrate‐level phosphorylation, chiefly through lactic fermentation. Nevertheless, when L. reuteri was grown on Se a certain degree of stress was observed, and this has to be taken into account for future applicative purposes. The proteomic approach has proven to be a powerful tool for the metabolic characterization of potential Se‐concentrating probiotics.

Detailed characterization of the lipid A fraction from the nonpathogen Acinetobacter radioresistens strain S13.

The genus Acinetobacter is composed of ubiquitous, generally nonpathogen environmental bacteria. Interest concerning these microorganisms has increased during the last 30 years, because some strains, belonging to the so-called A. baumannii-A. calcoaceticus complex, have been implicated in some severe pathological states in debilitated and hospitalized patients. The involvement of lipopolysaccharides (LPSs) as virulence factors in infections by Acinetobacter has been proven, and ongoing studies are aimed toward the complete serological characterization of the O-polysaccharides from LPSs isolated in clinical samples. Conversely, no characterization of the lipid A fraction from Acinetobacter strains has been performed. Here, the detailed structure of the lipid A fraction from A. radioresistens S13 is reported for the first time. A. radioresistens strains have never been isolated in cases of infectious disease. Nevertheless, it is known that the lipid A structure, with minor variations, is highly conserved across the genus; thus, structural details acquired from studies of this nonpathogen strain represent a useful basis for further studies of pathogen species.