Food-grade titanium dioxide can affect microbiota physiology, adhesion capability, and interbacterial interactions: A study on L. rhamnosus and E. faecium.

Food-grade titanium dioxide (TiO2-FG) is a widespread metal oxide used in the food industries. Recently, the European Food Safety Authority concluded that TiO2-FG cannot be considered safe for consumption due to its genotoxicity; however, its effect on the gut microbiota has not yet been completely unraveled. We studied the effects of TiO2-FG (0.125 mg/mL) on Lactobacillus rhamnosus GG (LGG) and Enterococcus faecium NCIMB10415 (Ent), in particular some physiological and phenotypic traits (growth kinetics, bile salts, and ampicillin resistance) and their interactions with the host (auto-aggregation, biofilm formation, and adhesion on Caco-2/TC7 monolayers) and other gut microorganisms (antimicrobial activity towards pathogens). The results obtained revealed that TiO2-FG alters both LGG and Ent growth and lowers bile resistance (62 and 34.5%, respectively) and adhesion on Caco-2/TC7 monolayers (34.8 and 14.16%, respectively). The other outcomes were strictly species-specific: Ent showed a lower ampicillin sensitivity (14.48%) and auto-aggregation (38.1%), while LGG showed a reduced biofilm formation (37%) and antimicrobial activity towards Staphylococcus aureus (35.73%). Overall, these results suggest an adverse effect of TiO2-FG on both the endogenous and exogenously administered probiotics, contributing to the argument against using TiO2-FG as a food additive.

Putative probiotics decrease cell viability and enhance chemotherapy effectiveness in human cancer cells: role of butyrate and secreted proteins.

Recent advances have highlighted probiotic role in preventing colorectal cancer, by promoting differentiation, inhibiting proliferation, and inducing apoptosis in colonocytes. Here, three ascertained probiotics (L. rhamnosus GG ATCC 53103, L. reuterii DSM 17938 and L. johnsonii LC1) and four food-isolated putative probiotics (L. plantarum S2, L. plantarum O2, L. pentosus S3, L. rhamnosus 14E4) were investigated for their ability to adhere to HT29 cancer cells and to inhibit their and the chemoresistant counterpart (HT29-dx cells) proliferation. Three putative probiotics (S2, S3 and 14E4) were able to decrease viability of both sensitive and chemo-resistant HT-29 cells. Supposing this effect related to secreted metabolites (namely short chain fatty acids (SCFA), exopolysaccharides (EPS) and extracellular proteins) we tested the efficacy of extracellular extracts and butyrate with or without the chemotherapeutic agent doxorubicin (DOXO) (10 µM, 4 h). Increased production of mitochondrial reactive oxygen species (ROS) in HT29 and HT29-dx cells was observed. Moreover, cell exposure to DOXO (10 µM, 24 h) and extracellular extracts (48 h) reduced cell viability. Comparative phenotypic and secretome analyses on the effective/non effective strains, revealed quantitative/qualitative differences in EPS content and protein profiles, suggesting that P40, phage-tail-like and capsid-like proteins may be also involved. These results suggest that food-isolated bacteria releasing bioactive compounds (butyrate, EPS and peculiar proteins) may control cancer cell proliferation and improve their response to chemotherapy.

Enterococcus faecium NCIMB10415 responds to norepinephrine by altering protein profiles and phenotypic characters.

The long-term established symbiosis between gut microbiota and humans is based upon a dynamic equilibrium that, if unbalanced, could lead to the development of diseases. Despite the huge amount of data concerning the microbiota-gut-brain-axis, little information is available on what happens at the molecular level in bacteria, when exposed to human signals. In the present study, the physiological effects exerted by norepinephrine (NE), a human hormone present in significant amounts in the host gut, were analyzed using the commensal/probiotic strain Enterococcus faecium NCIMB10415 as a target. The aim was to compare the protein profiles of treated and untreated bacteria and relating these proteome patterns to some phenotypic modifications important for bacteria-host interaction. Actually, to date, only pathogens have been considered. Combining a gel-free/label-free proteomic analysis with the evaluation of bile salts resistance, biofilm formation and autoaggregation ability (as well as with the bacterial growth kinetics), allowed to detect changes induced by NE treatment on all the tested probiotic properties. Furthermore, exposure to the bioactive molecule increased the abundance of proteins related to stress response and to host-microbe interaction, such as moonlight proteins involved in adhesion and immune stimulation. The results of this investigation demonstrated that, not only pathogens, but also commensal gut bacteria are affected by host-derived hormones, underlining the importance of a correct cross-signalling in the maintenance of gut homeostasis. SIGNIFICANCE: The crucial role played by the human gut microbiota in ensuring host homeostasis and health is definitively ascertained as suggested by the holobiome concept. The present research was intended to shed light on the endocrinological perturbations possibly affecting microbiota. The microbial model used in this study belongs to Enterococcus faecium species, whose controversial role as gut commensal and opportunistic pathogen in the gut ecosystem is well recognized. The results obtained in the present investigation clearly demonstrate that E. faecium NCIMB10415 can sense and respond to norepinephrine, a human hormone abundant at the gut level, by changing protein profiles and physiology, inducing changes that could favor survival and colonization of the host tissues. To our knowledge, this is the first proteomic report concerning the impact of a human hormone on a commensal/probiotic bacterium, since previous research has focused on exploring the effects of neuroendocrine molecules on growth and virulence of pathogenic species.

Correction: Selenium effects on the metabolism of a Se-metabolizing Lactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes.

Correction for 'Selenium effects on the metabolism of a Se-metabolizing Lactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes' by E. Mangiapane et al., Mol. BioSyst., 2014, 10, 1272-1280.

Ten years of subproteome investigations in lactic acid bacteria: A key for food starter and probiotic typing.

The definition of safety and efficacy of food-employed bacteria as well as probiotic strains is a continuous, often unattended, challenge. Proteomic techniques such as 2DE, DIGE and LC/LC-MS/MS are suitable and powerful tools to reveal new aspects (positive and negative) of "known" and "unknown" strains that can be employed in food making and as nutraceutical supplements for human health. Unfortunately, these techniques are not used as extensively as it should be wise. The present report describes the most significant results obtained by our research group in 10years of study on subproteomes in bacteria, chiefly lactic acid bacteria. Production of desired and undesired metabolites, differences between strains belonging to same species but isolated from different ecological niches, the effect of cryoprotectants on survival to lyophilization as well as the adhesive capability of strains, were elucidated by analysis of cytosolic, membrane-enriched, surface and extracellular proteomes. The present review opens a window on a yet largely underexplored field and highlights the huge potential of subproteome investigations for more rational choice of microbial strains as food starters, probiotics and for production of nutraceuticals. These analyses will hopefully contribute to manufacturing safer and healthier food and food supplements in the near future. This article is part of a Special Issue entitled: HUPO 2014.

Selenium effects on the metabolism of a Se-metabolizing Lactobacillus reuteri: analysis of envelope-enriched and extracellular proteomes.

Selenium (Se) has received great attention in the last few years, as it is considered to be essential for human health (prevention of viral infections, heart diseases and ageing-related diseases). Se deficiency can be counteracted by the administration of selenium-enriched probiotics that are able to convert inorganic selenium into less toxic and more bio-available organic forms. This study was performed on Lactobacillus reuteri Lb2 BM DSM 16143, a probiotic LAB previously demonstrated to be able to fix Se into selenocysteines. The aim was to assess Se influence on its metabolism, by a 2-DE proteomic approach, on two different cellular districts: envelope-enriched and extracellular proteomes. While in the envelope-enriched fraction 15 differentially expressed proteins were identified, in the extracellular proteome no quantitative difference was detected. However, at a molecular level, we observed the insertion of Se into selenocysteine, exclusively under the stimulated conditions. The obtained results confirmed the possibility to use L. reuteri Lb2 BM DSM 16143 as a carrier of organic Se that can be easily released in the gut becoming available for the human host.

Mare's colostrum globules stimulate fibroblast growth in vitro: a biochemical study.

The wound repair function of mare's milk and colostrum was investigated. Mare's colostrum improved wound healing in vivo; thus fibroblast growth activation by mare's milk and colostrum was examined. As expected, colostrum was more effective than milk. To establish the biochemical nature of the bioactive molecules involved, colostrum was fractionated into whey, casein, and fat globules, and the efficacy of these fractions on fibroblast proliferation was studied. The fat globule fraction provided the strongest stimulation; its composition was studied and compared with the less-active milk fat globule fraction. The lipid pattern highlighted several differences between mare's colostrum and milk; in particular, total lipid, linoleic acid, linolenic acid, ganglioside, and glycolipid contents were higher in colostrum. A proteomic investigation revealed some differences between the protein composition of colostrum and milk fat globules. Adipophylin and lactadherin were significantly overexpressed in colostrum fat globules. The role of specific lipids on skin wound repair and that of the epidermal growth factor-like domain, embedded within the lactadherin molecule and probably released in conditions stimulating proteolysis, are discussed.

Influence of ethanol, malate and arginine on histamine production of Lactobacillus hilgardii isolated from an Italian red wine.

Wine, like other fermented foods, may contain biogenic amines produced by lactic acid bacteria via amino acids decarboxylation. The most relevant amines from the toxicological standpoint are histamine and tyramine. The complexity of fermented substrates makes it difficult to suggest a priori how variables can modulate amine production. Lactobacillus hilgardii ISE 5211 was isolated from an Italian red wine. Besides producing lactate from malate, this strain is also able to convert arginine to ornithine and histidine to histamine. In the present investigation we studied the influence of malate, arginine and ethanol on histamine accumulation by L. hilgardii ISE 5211. Ethanol concentrations above 13% inhibit both histamine accumulation and bacterial growth; concentrations below 9% affect neither growth nor histamine production. However, an ethanol concentration of 11% allows a low but continuous accumulation of histamine to occur. Arginine also delays histamine accumulation, while malate appears to have no effect on histidine-histamine conversion.

Catalytic and spectroscopic characterisation of a copper-substituted alcohol dehydrogenase from yeast.

Yeast alcohol dehydrogenase (Y-ADH) is widely studied for its biotechnological importance and various attempts to improve its catalytic properties have been made. In this paper, a catalytically active metal-substituted Y-ADH was prepared in vitro by substituting one zinc atom with copper. EPR and Raman spectroscopy suggest that copper maintains the same co-ordination geometry as zinc in native Y-ADH. The active Cu-ADH shows lower substrate affinity and lower specific activity (SA) than native ADH, but greater than a previously obtained Co-ADH. Furthermore, Cu-ADH maintains its catalytic efficiency in a wider pH range than native enzyme.

The catechol 1,2 dioxygenase system of Acinetobacter radioresistens: isoenzymes, inductors and gene localisation.

Two different isozymes (Iso A and Iso B) of catechol 1,2 dioxygenase (C1,2O) were isolated from cultures of A. radioresistens grown in two different media, containing phenol and benzoate respectively. In the phenol medium the bacteria expressed about 90% of Iso A, whereas in the benzoate medium the Iso A/Iso B ratio was 40:60. The two proteins have different molecular masses, isoelectric points and N-terminal sequences that are not consistent with simple post-translational modifications. Furthermore, their behaviour differs at high temperatures (42 degrees C-47 degrees C) and at moderately acidic pH (pH 6.0): Iso A proved to be the more stable under conditions of environmental stress. Hybridisation analysis with an A. calcoaceticus catA-derived probe revealed that A. radioresistens C1,2O proteins are encoded by two chromosomally located genes. Bidimensional electrophoresis (2DE) maps of crude extracts of cells grown in different carbon sources (phenol, benzoate and acetate) clearly demonstrated a differential induction pattern for the two proteins. The hypothesis of a double set of genes, one for benzoate catabolism and the other for phenol catabolism, is discussed, and analogies are drawn with other known C1,2Os.

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 beta-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.

Improved resistance to transition metals of a cobalt-substituted alcohol dehydrogenase 1 from Saccharomyces cerevisiae.

Cobalt-substituted alcohol dehydrogenase 1 was purified from a yeast culture of Saccharomyces cerevisiae. Its reactivity towards different transition metals was tested and compared with the native zinc enzyme. The cobalt enzyme displayed a catalytic efficiency 100-fold higher than that of the zinc enzyme. Copper, nickel and cadmium exerted a mixed-type inhibition, with a scale of inhibition efficiency: Cu(2+)>Ni(2+)>Cd(2+). In general, a higher resistance of the modified protein to the inhibitory action of transition metals was observed, with two orders of magnitude for copper I(50). The presence of nickel in the complexes enzyme-coenzyme-inhibitor-substrate resulted in a decrease of the ampholytic nature of the catalytic site. On the contrary, cadmium and copper exerted an enhancement of this parameter. Electrostatic or other types of interactions may be involved in conferring a good resistance in the basic pH range, making cobalt enzyme very suitable for biotechnological processes.

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.

Phenol hydroxylase from Acinetobacter radioresistens is a multicomponent enzyme. Purification and characterization of the reductase moiety.

This paper reports the isolation and characterization of phenol hydroxylase (PH) from a strain belonging to the Acinetobacter genus. An Acinetobacter radioresistens culture, grown on phenol as the only carbon and energy source, produced a multicomponent enzyme system, located in the cytoplasm and inducible by the substrate, that is responsible for phenol conversion into catechol. Because of the wide diffusion of phenol as a contaminant, the present work represents an initial step towards the biotechnological treatment of waste waters containing phenol. The reductase component of this PH system has been purified and isolated in large amounts as a single electrophoretic band. The protein contains a flavin cofactor (FAD) and an iron-sulfur cluster of the type [2Fe-2S]. The function of this reductase is to transfer reducing equivalents from NAD(P)H to the oxygenase component. In vitro, the electron acceptors can be cytochrome c as well as other molecules such as 2, 6-dichlorophenolindophenol, potassium ferricyanide, and Nitro Blue tetrazolium. The molecular mass of the reductase was determined to be 41 kDa by SDS/PAGE and 38.8 kDa by gel permeation; its isoelectric point is 5.8. The N-terminal sequence is similar to those of the reductases from A. calcoaceticus NCIB 8250 (10/12 identity) and Pseudomonas CF600 (8/12 identity) PHs, but much less similar (2/12 identity) to that of benzoate dioxygenase reductase from A. calcoaceticus BD413. Similarly, the internal peptide sequence of the A. radioresistens PH reductase displays a good level of identity (9/10) with both A. calcoaceticus NCIB 8250 and Pseudomonas CF600 PH reductase internal peptide sequences but a poorer similarity (3/10) to the internal peptide sequence of benzoate dioxygenase reductase from A. calcoaceticus BD413.

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.

Acinetobacter radioresistens metabolizing aromatic compounds. 2. Biochemical and microbiological characterization of the strain.

The metabolic potentialities of an Acinetobacter radioresistens strain, isolated from the soil adjacent to an activated sludge plant, were investigated. Among 26 aromatic substrates tested, only phenol, benzoate and catechol were metabolized. Since this strain possessed abundant plasmid DNA, the antibiotic and heavy metal resistance was examined, and the bacterial cells proved to be sensitive to all metals (Ni, Tl, Pb, Cd, Ag, Co, Zn) and antibiotics tested except for Fosfomycin and chloramphenicol. The degradation kinetics for phenol and benzoate as the sole carbon/energy source (pH 7, 30 degrees C) displayed different trends, confirmed by the bacterial growth curve. Crude extracts from phenol-grown cultures showed both phenol hydroxylating activity and catechol dioxygenating activity. Phenol hydroxylase possessed a reductase component able to reduce nitroblue tetrazolium (NBT) and cytochrome C, thus exhibiting differences from previously reported monocomponent phenol hydroxylases from the same genus. Catechol dioxygenase is an intradiol-cleaving enzyme recognizing also substituted catechols.

Acinetobacter radioresistens metabolizing aromatic compounds. 1. Optimization of the operative conditions for phenol degradation.

A strain of Acinetobacter radioresistens was able to utilize phenol as the only carbon and energy source, after an acclimatization period of 3 days in which increasing phenol concentrations from 50 to 200 mg/l were supplied. At 30 degrees C, the complete phenol utilization in batch degradation tests occurred in 2.5-3 h at pH 7 and 8, but it increased strongly at pH 6 (over 40 h). No microbial growth was detected at 40 degrees C, while at 20 degrees C (pH 7-8) the time necessary for complete phenol degradation was about twofold longer than that at 30 degrees C (pH 7-8) revealing a good capability of the strain as a seed-micro-organism for enhancing phenol degradation. The bacterial growth in acclimatized cultures, evaluated with the viable cell count, always displayed a trend consistent with the use of phenol as a substrate with an eventual lag phase and then an exponential phase, while in the non-acclimatized cultures an initial stage of cellular death was observed.

Factors affecting acetic acid production by yeasts in strongly clarified grape musts.

High acetate content in a wine, after strong clarification of the must, is due to depletion in the yeast cells of important metabolites (normally present in non-clarified musts) such as metals, amino acids, polyphenolic compounds and unsaturated fatty acids. These substances were added separately to a synthetic medium, comparable in composition to the clarified must, which was then inoculated with a high acetate producer strain. No effects were observed after the addition of various metals and amino acids. The addition of unsaturated fatty acids (Tween 80) caused a significant (p = 0.01) decrease in acetate content. Similar results have been obtained in the presence of polyphenols (catechins and anthocyans): their mechanism of action is probably due to direct inhibition of the enzyme aldehyde dehydrogenase. Control experiments were performed with a low acetate producer strain, and a reduction in acetate content was detected. No differences in the glyceropyruvic metabolism of the two strains was evident.

Modulation of Na+/K+ pump in intact erythrocytes by cardioglycosides, steroid hormones and ouabain-like compounds.

1. Pure erythrocytes preparations, free from platelets and white cells, were incubated for a long time without hemolysis. 2. Dose-response experiments performed with (a) cardioglycosides (ouabain and K-strophantoside), (b) steroid hormones and their glucuronides (tetrahydrocortisol, oestradiol and the respective 3-glucuronic derivatives) and (c) ouabain-like compounds purified in our laboratory (0.7 kDa and 2-4 kDa respectively) emphasise a modulatory effect [activation of Na+ efflux rate and K+ uptake at very low ligand concentrations, inhibition at higher levels; maximum enhancement of cation transport: (a) and (b) 10-0.1 nM (+40-50%), (c) 1-0.01 nM (2.5-fold)]. 3. Binding experiments show upward-curved Scatchard graphs, with the Kd values of 50 nM and 18 microM and the Bmax values of 10.2 and 984.5 fmol/100 microliters RBC (red blood cells) respectively.

Serological diagnosis of hepatitis B and delta virus (HBV/HDV) coinfection.

Diagnosis of acute hepatitis B virus/hepatitis delta virus (HBV/HDV) coinfection is currently based on detection of anti-HD, however this antibody may be undetectable during the acute phase of hepatitis. To evaluate the entity of misdiagnosis of HBV/HDV coinfection in acute HBsAg-anti-HBc IgM positive hepatitis we examined sera from 245 consecutive patients obtained at admission and day 30, 60, 120, 210 and 400 of their follow-up. Anti-HD was detected in the serum of 26 out of 245 patients (10.6%). In 15% of cases it was present at admission, while in 92% it was found after 30 days. The combined detection of HDV-RNA, HDAg and IgM anti-HD in acute phase sera allowed a correct etiologic diagnosis in 69% of the cases. These findings suggest that the prevalence of HBV/HDV coinfection is underestimated when anti-HD is the only marker to be detected during the acute phase of disease. A correct etiologic diagnosis can only be made by testing acute phase sera for all the available markers of HDV. However, the best cost-effective procedure is to test any patient with HBV markers at presentation for anti-HD, 30-40 days after the onset of symptoms.