In Vitro and in Vivo Selection of Potentially Probiotic Lactobacilli From Nocellara del Belice Table Olives.

Table olives are increasingly recognized as a vehicle as well as a source of probiotic bacteria, especially those fermented with traditional procedures based on the activity of indigenous microbial consortia, originating from local environments. In the present study, we report characterization at the species level of 49 Lactic Acid Bacteria (LAB) strains deriving from Nocellara del Belice table olives fermented with the Spanish or Castelvetrano methods, recently isolated in our previous work. Ribosomal 16S DNA analysis allowed identification of 4 Enterococcus gallinarum, 3 E. casseliflavus, 14 Leuconostoc mesenteroides, 19 Lactobacillus pentosus, 7 L. coryniformis, and 2 L. oligofermentans. The L. pentosus and L. coryniformis strains were subjected to further screening to evaluate their probiotic potential, using a combination of in vitro and in vivo approaches. The majority of them showed high survival rates under in vitro simulated gastro-intestinal conditions, and positive antimicrobial activity against Salmonella enterica serovar Typhimurium, Listeria monocytogenes and enterotoxigenic Escherichia coli (ETEC) pathogens. Evaluation of antibiotic resistance to ampicillin, tetracycline, chloramphenicol, or erythromycin was also performed for all selected strains. Three L. coryniformis strains were selected as very good performers in the initial in vitro testing screens, they were antibiotic susceptible, as well as capable of inhibiting pathogen growth in vitro. Parallel screening employing the simplified model organism Caenorhabditis elegans, fed the Lactobacillus strains as a food source, revealed that one L. pentosus and one L. coryniformis strains significantly induced prolongevity effects and protection from pathogen-mediated infection. Moreover, both strains displayed adhesion to human intestinal epithelial Caco-2 cells and were able to outcompete foodborne pathogens for cell adhesion. Overall, these results are suggestive of beneficial features for novel LAB strains, which renders them promising candidates as starters for the manufacturing of fermented table olives with probiotic added value.

Combination of Metabolomic and Proteomic Analysis Revealed Different Features among Lactobacillus delbrueckii Subspecies bulgaricus and lactis Strains While In Vivo Testing in the Model Organism Caenorhabditis elegans Highlighted Probiotic Properties.

Lactobacillus delbrueckii represents a technologically relevant member of lactic acid bacteria, since the two subspecies bulgaricus and lactis are widely associated with fermented dairy products. In the present work, we report the characterization of two commercial strains belonging to L. delbrueckii subspecies bulgaricus, lactis and a novel strain previously isolated from a traditional fermented fresh cheese. A phenomic approach was performed by combining metabolomic and proteomic analysis of the three strains, which were subsequently supplemented as food source to the model organism Caenorhabditis elegans, with the final aim to evaluate their possible probiotic effects. Restriction analysis of 16S ribosomal DNA revealed that the novel foodborne strain belonged to L. delbrueckii subspecies lactis. Proteomic and metabolomic approaches showed differences in folate, aminoacid and sugar metabolic pathways among the three strains. Moreover, evaluation of C. elegans lifespan, larval development, brood size, and bacterial colonization capacity demonstrated that L. delbrueckii subsp. bulgaricus diet exerted beneficial effects on nematodes. On the other hand, both L. delbrueckii subsp. lactis strains affected lifespan and larval development. We have characterized three strains belonging to L. delbrueckii subspecies bulgaricus and lactis highlighting their divergent origin. In particular, the two closely related isolates L. delbrueckii subspecies lactis display different galactose metabolic capabilities. Moreover, the L. delbrueckii subspecies bulgaricus strain demonstrated potential probiotic features. Combination of omic platforms coupled with in vivo screening in the simple model organism C. elegans is a powerful tool to characterize industrially relevant bacterial isolates.

The loss of ATP2C1 impairs the DNA damage response and induces altered skin homeostasis: Consequences for epidermal biology in Hailey-Hailey disease.

Mutation of the Golgi Ca(2+)-ATPase ATP2C1 is associated with deregulated calcium homeostasis and altered skin function. ATP2C1 mutations have been identified as having a causative role in Hailey-Hailey disease, an autosomal-dominant skin disorder. Here, we identified ATP2C1 as a crucial regulator of epidermal homeostasis through the regulation of oxidative stress. Upon ATP2C1 inactivation, oxidative stress and Notch1 activation were increased in cultured human keratinocytes. Using RNA-seq experiments, we found that the DNA damage response (DDR) was consistently down-regulated in keratinocytes derived from the lesions of patients with Hailey-Hailey disease. Although oxidative stress activates the DDR, ATP2C1 inactivation down-regulates DDR gene expression. We showed that the DDR response was a major target of oxidative stress-induced Notch1 activation. Here, we show that this activation is functionally important because early Notch1 activation in keratinocytes induces keratinocyte differentiation and represses the DDR. These results indicate that an ATP2C1/NOTCH1 axis might be critical for keratinocyte function and cutaneous homeostasis, suggesting a plausible model for the pathological features of Hailey-Hailey disease.

Impact of a Complex Food Microbiota on Energy Metabolism in the Model Organism Caenorhabditis elegans.

The nematode Caenorhabditis elegans is widely used as a model system for research on aging, development, and host-pathogen interactions. Little is currently known about the mechanisms underlying the effects exerted by foodborne microbes. We took advantage of C. elegans to evaluate the impact of foodborne microbiota on well characterized physiological features of the worms. Foodborne lactic acid bacteria (LAB) consortium was used to feed nematodes and its composition was evaluated by 16S rDNA analysis and strain typing before and after colonization of the nematode gut. Lactobacillus delbrueckii, L. fermentum, and Leuconostoc lactis were identified as the main species and shown to display different worm gut colonization capacities. LAB supplementation appeared to decrease nematode lifespan compared to the animals fed with the conventional Escherichia coli nutrient source or a probiotic bacterial strain. Reduced brood size was also observed in microbiota-fed nematodes. Moreover, massive accumulation of lipid droplets was revealed by BODIPY staining. Altered expression of nhr-49, pept-1, and tub-1 genes, associated with obesity phenotypes, was demonstrated by RT-qPCR. Since several pathways are evolutionarily conserved in C. elegans, our results highlight the nematode as a valuable model system to investigate the effects of a complex microbial consortium on host energy metabolism.

Anti-Candida activity of 1-18 fragment of the frog skin peptide esculentin-1b: in vitro and in vivo studies in a Caenorhabditis elegans infection model.

Candida albicans represents one of the most prevalent species causing life-threatening fungal infections. Current treatments to defeat Candida albicans have become quite difficult, due to their toxic side effects and the emergence of resistant strains. Antimicrobial peptides (AMPs) are fascinating molecules with a potential role as novel anti-infective agents. However, only a few studies have been performed on their efficacy towards the most virulent hyphal phenotype of this pathogen. The purpose of this work is to evaluate the anti-Candida activity of the N-terminal 1-18 fragment of the frog skin AMP esculentin-1b, Esc(1-18), under both in vitro and in vivo conditions using Caenorhabditis elegans as a simple host model for microbial infections. Our results demonstrate that Esc(1-18) caused a rapid reduction in the number of viable yeast cells and killing of the hyphal population. Esc(1-18) revealed a membrane perturbing effect which is likely the basis of its mode of action. To the best of our knowledge, this is the first report showing the ability of a frog skin AMP-derived peptide (1) to kill both growing stages of Candida; (2) to promote survival of Candida-infected living organisms and (3) to inhibit transition of these fungal cells from the roundish yeast shape to the more dangerous hyphal form at sub-inhibitory concentrations.

Inhibition of microbial growth by carbon nanotube networks.

In the last years carbon nanotubes have attracted increasing attention for their potential applications in the biomedical field as diagnostic and therapeutic nano tools. Here we investigate the antimicrobial activity of different fully characterized carbon nanotube types (single walled, double walled and multi walled) on representative pathogen species: Gram-positive Staphylococcus aureus, Gram-negative Pseudomonas aeruginosa and the opportunistic fungus Candida albicans. Our results show that all the carbon nanotube types possess a highly significant antimicrobial capacity, even though they have a colony forming unit capacity and induction of oxidative stress in all the microbial species to a different extent. Moreover, scanning electron microscopy analysis revealed that the microbial cells were wrapped or entrapped by carbon nanotube networks. Our data taken together suggest that the reduced capacity of microbial cells to forming colonies and their oxidative response could be related to the cellular stress induced by the interactions of pathogens with the CNT network.

Thermal adaptability of Kluyveromyces marxianus in recombinant protein production.

BACKGROUND: Kluyveromyces marxianus combines the ease of genetic manipulation and fermentation with the ability to efficiently secrete high molecular weight proteins, performing eukaryotic post-translational modifications. It is able to grow efficiently in a wide range of temperatures. The secretion performances were analyzed in the host K. marxianus L3 in the range between 5°C and 40°C by means of 3 different reporter proteins, since temperature appears a key parameter for production and secretion of recombinant proteins. RESULTS: The recombinant strains were able to grow up to 40°C and, along the tested temperature interval (5-40°C), the specific growth rates (µ) were generally lower as compared to those of the untransformed strain. Biomass yields were slightly affected by temperature, with the highest values reached at 15°C and 30°C. The secretion of the endogenous ß-fructofuranosidase, used as an internal control, was efficient in the range of the tested temperature, as evaluated by assaying the enzyme activity in the culture supernatants. The endogenous ß-fructofuranosidase production was temperature dependent, with the highest yield at 30°C. The heterologous proteins HSA, GAA and Sod1p were all successfully produced and secreted between 5°C and 40°C, albeit each one presented a different optimal production temperature (15, 40, 5-30°C for HSA, GAA and Sod1p, respectively). CONCLUSIONS: K. marxianus L3 has been identified as a promising and flexible cell factory. In a sole host, the optimization of growth temperatures for the efficient secretion of each individual protein can be carried out over a wide range of temperatures.

Graphite nanoplatelets and Caenorhabditis elegans: insights from an in vivo model.

We evaluated the toxicity of graphite nanoplatelets (GNPs) in the model organism Caenorhabditis elegans. The GNPs resulted nontoxic by measuring longevity as well as reproductive capability end points. An imaging technique based on Fourier transform infrared spectroscopy (FT-IR) mapping was also developed to analyze the GNPs spatial distribution inside the nematodes. Conflicting reports on the in vitro antimicrobial properties of graphene-based nanomaterials prompted us to challenge the host-pathogen system C. elegans-Pseudomonas aeruginosa to assess these findings through an in vivo model.

KlMID1, a relevant key player between endoplasmic reticulum homeostasis and mitochondrial dysfunction in Kluyveromyces lactis.

The interplay between calcium metabolism and glycosylation in yeast is largely unknown. In order to clarify this relationship, the effect of a mutation in the KlOCH1 gene, encoding the Golgi α-1,6-mannosyltransferase, on calcium homeostasis was studied in the yeast Kluyveromyces lactis. In particular, the role of the KlMID1 gene, encoding one of the components of the plasma membrane calcium channel (Cch1-Mid1), was investigated. Almost complete suppression of the phenotypes occurring in the mutant strain, ranging from oxidative stress to cell wall alteration, was observed by increased dosage of KlMID1. In addition, the N-glycosylation mutant showed increased calcium accumulation and decreased transcription of KlMID1 and KlCCH1. Moreover, the calcium alterations included an increased expressional profile for the KlPMC1 gene, encoding the vacuolar calcium ion pump. Furthermore, perturbation of endoplasmic reticulum (ER) homeostasis was observed in Kloch1-1 cells. Similarly, down-modulation of calcium signalling genes as well as altered mitochondrial functionality were induced in wild-type cells after treatment with DTT. However, no mitochondrial alteration occurred in the treated cells when KlMID1 was overexpressed. Our results suggest that the ER stress taking place in Kloch1-1 cells appears to be the primary cause of the KlMID1 down-modulation and its resulting effects on the expression of calcium homeostasis genes.

Oxidative stress activation of miR-125b is part of the molecular switch for Hailey-Hailey disease manifestation.

Hailey-Hailey disease (HHD) is an autosomal dominant disorder characterized by suprabasal cutaneous cell separation (acantholysis) leading to the development of erosive and oozing skin lesion. Micro RNAs (miRNAs) are endogenous post-transcriptional modulators of gene expression with critical functions in health and disease. Here, we evaluated whether the expression of specific miRNAs may play a role in the pathogenesis of HHD. Here, we report that miRNAs are expressed in a non-random manner in Hailey-Hailey patients. miR-125b appeared a promising candidate for playing a role in HHD manifestation. Both Notch1 and p63 are part of a regulatory signalling whose function is essential for the control of keratinocyte proliferation and differentiation and of note, the expression of both Notch1 and p63 is downregulated in HHD-derived keratinocytes. We found that both Notch1 and p63 expression is strongly suppressed by miR-125b expression. Additionally, we found that miR-125b expression is increased by an oxidative stress-dependent mechanism. Our data suggest that oxidative stress-mediated induction of miR-125b plays a specific role in the pathogenesis of HHD by regulating the expression of factors playing an important role in keratinocyte proliferation and differentiation.

Anti-Pseudomonas activity of frog skin antimicrobial peptides in a Caenorhabditis elegans infection model: a plausible mode of action in vitro and in vivo.

The emergence of multidrug-resistant (MDR) microorganisms makes it increasingly difficult to treat infections. These infections include those associated with Pseudomonas aeruginosa, which are hard to eradicate, especially in patients with a compromised immune system. Naturally occurring membrane-active cationic antimicrobial peptides (CAMPs) serve as attractive candidates for the development of new therapeutic agents. Amphibian skin is one of the richest sources for such peptides, but only a few studies on their in vivo activities and modes of action have been reported. We investigated (i) the activity and mechanism underlying the killing of short CAMPs from frog skin (e.g., temporins and esculentin fragments) on an MDR clinical isolate of P. aeruginosa and (ii) their in vivo antibacterial activities and modes of action, using the minihost model of Caenorhabditis elegans. Our data revealed that in vivo, both temporin-1Tb and esculentin(1-18) were highly active in promoting the survival of Pseudomonas-infected nematodes, although temporin-1Tb did not show significant activity in vitro under the experimental conditions used. Importantly, esculentin(1-18) permeated the membrane of Pseudomonas cells within the infected nematode. To the best of our knowledge, this is the first report showing the ability of a CAMP to permeate the microbial membrane within a living organism. Besides shedding light on a plausible mode of action of frog skin CAMPs in vivo, our data suggest that temporins and esculentins would be attractive molecules as templates for the development of new therapeutics against life-threatening infections.

Secretion of Kluyveromyces lactis Cu/Zn SOD: strategies for enhanced production.

The Kluyveromyces lactis Cu/Zn SOD gene (SOD1) was fused with the toxin K1 signal sequence to obtain extracellular production of superoxide dismutase. Kluyveromyces marxianus L3 and K. lactis MW98-8C strains were transformed and compared as hosts for the secretion. The effects of the media composition were evaluated: In K. lactis, the highest volumetric activity was obtained in YKK synthetic medium in the presence of Cu(2+)/Zn(2+) cofactors (9.6 kU l(-1)). In K. marxianus, active SOD was produced only in YPD medium supplemented with Cu(2+) and Zn(2+) (8.8 kU l(-1)). In order to improve the production of secreted active SOD in K. lactis, the SOD1 copper carrier (CCS1) was overexpressed and targeted to the secretory apparatus. A positive effect was observed only when K. lactis was grown in a medium without Cu(2+)/Zn(2+) supplement. The best performing culture conditions for K. lactis and K. marxianus recombinant strains were successfully applied to two laboratory-scale fed-batch processes, and volumetric SOD activities increased up to 19.4 and 24.1 kU l(-1), respectively.

The Golgi alpha-1,6 mannosyltransferase KlOch1p of Kluyveromyces lactis is required for Ca2+/calmodulin-based signaling and for proper mitochondrial functionality.

BACKGROUND: Protein N-glycosylation is a relevant metabolic pathway in eukaryotes and plays key roles in cell processes. In yeasts, outer chain branching is initiated in the Golgi apparatus by the alpha-1,6-mannosyltransferase Och1p. RESULTS: Here we report that, in Kluyveromyces lactis, this glycosyltransferase is also required to maintain functional mitochondria and calcium homeostasis. Cells carrying a mutation in KlOCH1 gene showed altered mitochondrial morphology, increased accumulation of ROS and reduced expression of calcium signalling genes such as calmodulin and calcineurin. Intracellular calcium concentration was also reduced in the mutant cells with respect to the wild type counterparts.Phenotypes that occur in cells lacking the alpha-1,6-mannosyltransferase, including oxidative stress and impaired mitochondria functionality, were suppressed by increased dosage of KlCmd1p. This, in turn, acts through the action of calcineurin. CONCLUSIONS: Proper functioning of the alpha-1,6-mannosyltransferase in the N-glycosylation pathway of K. lactis is required for maintaining normal calcium homeostasis; this is necessary for physiological mitochondria dynamics and functionality.

The apyrase KlYnd1p of Kluyveromyces lactis affects glycosylation, secretion, and cell wall properties.

The Kluyveromyces lactis ORF r_klactIV3,463 on chromosome IV, hereafter named KlYND1, encodes an endoapyrase that has nucleoside phosphatase activity with a lumenal orientation. The enzyme showed equally high activity towards GDP/UDP and ADP, and also showed activity, although to a lesser extent, towards GTP. No activity was detected with the other triphosphates and all monophosphates. The overexpression of KlYND1 in Klgda1Delta cells of K. lactis, devoid of the encoded GDPase/UDPase activity, suppressed the loss of O-glycosylation and cell wall-related defects described in such mutants, and suggests a partial overlap of function between the two genes, and therefore some redundancy. The overexpression of KlYND1 in wild-type cells enhanced the secretion of the recombinant human serum albumin and glucoamylase employed as reporters.

The Kluyveromyces lactis alpha1,6-mannosyltransferase KlOch1p is required for cell-wall organization and proper functioning of the secretory pathway.

Mutants of Kluyveromyces lactis denominated vga (vanadate glycosylation affected) bear various combinations of glycosylation and cell-wall defects. The vga3 mutation of K. lactis was mapped in the KlOCH1 gene, encoding the functional homologue of the Saccharomyces cerevisiaealpha1,6-mannosyltransferase. Quantitative analysis of cell-wall components indicated a noticeable increase of chitin and beta1,6-glucans and a severe decrease of mannoproteins in the mutant cells as compared with the wild-type counterparts. Fine-structure determination of the beta1,6-glucan polymer indicated that, in the vga3-1 strain, the beta1,6-glucans are shorter and have more branches than in the wild-type strain. This suggests that cell-wall remodelling changes take place in K. lactis in the presence of glycosylation defects. Moreover, the vga3 cells showed a significantly improved capability of secreting heterologous proteins. Such a capability, accompanied by the highly reduced N-glycosylation, may be of biotechnological interest, especially when hyper-glycosylation of recombinant products must be avoided.

The Golgi Ca2+-ATPase KlPmr1p function is required for oxidative stress response by controlling the expression of the heat-shock element HSP60 in Kluyveromyces lactis.

The Golgi P-type Ca2+-ATPase, Pmr1p, is the major player for calcium homeostasis in yeast. The inactivation of KlPMR1 in Kluyveromyces lactis leads to high pleiotropic phenotypes that include reduced glycosylation, cell wall defects, and alterations of mitochondrial metabolism. In this article we found that cells lacking KlPmr1p have a morphologically altered mitochondrial network and that mitochondria (m) from Klpmr1delta cells accumulate Ca2+ more slowly and reach a lower [Ca2+]m level, when exposed to [Ca2+] < 5 microM, than wild-type cells. The Klpmr1delta cells also exhibit traits of ongoing oxidative stress and present hyperphosphorylation of KlHog1p, the hallmark for the activation of stress response pathways. The mitochondrial chaperone KlHsp60 acts as a multicopy suppressor of phenotypes that occur in cells lacking the Ca2+-ATPase, including relief from oxidative stress and recovery of cell wall thickness and functionality. Inhibition of KlPMR1 function decreases KlHSP60 expression at both mRNA and protein levels. Moreover, KlPRM1 loss of function correlates with both decreases in HSF DNA binding activity and KlHSP60 expression. We suggest a role for KlPMR1 in HSF DNA binding activity, which is required for proper KlHSP60 expression, a key step in oxidative stress response.

Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p.

GDP-mannose is the mannosyl donor for the glycosylation reactions and is synthesized by GDP-mannose pyrophosphorylase from GTP and d-mannose-1-phosphate; in Saccharomyces cerevisiae this enzyme is encoded by the PSA1/VIG9/SRB1 gene. We isolated the Kluyveromyces lactis KlPSA1 gene by complementing the osmotic growth defects of S. cerevisiae srb1/psa1 mutants. KlPsa1p displayed a high degree of similarity with other GDP-mannose pyrophosphorylases and was demonstrated to be the functional homologue of S. cerevisiae Psa1p. Phenotypic analysis of a K. lactis strain overexpressing the KlPSA1 gene revealed changes in the cell wall assembly. Increasing the KlPSA1 copy number restored the defects in O-glycosylation, but not those in N-glycosylation, that occur in K. lactis cells depleted for the hexokinase Rag5p. Overexpression of GDP-mannose pyrophosphorylase also enhanced heterologous protein secretion in K. lactis as assayed by using the recombinant human serum albumin and the glucoamylase from Arxula adeninivorans.

Alterations of O-glycosylation, cell wall, and mitochondrial metabolism in Kluyveromyces lactis cells defective in KlPmr1p, the Golgi Ca(2+)-ATPase.

In yeast the P-type Ca(2+)-ATPase of the Golgi apparatus, Pmr1p, is the most important player in calcium homeostasis. In Kluyveromyces lactis KlPMR1 inactivation leads to pleiotropic phenotypes, including reduced N-glycosylation and altered cell wall morphogenesis. To study the physiology of K. lactis when KlPMR1 was inactivated microarrays containing all Saccharomyces cerevisiae coding sequences were utilized. Alterations in O-glycosylation, consistent with the repression of KlPMT2, were found and a terminal N-acetylglucosamine in the O-glycans was identified. Klpmr1Delta cells showed increased expression of PIRs, proteins involved in cell wall maintenance, suggesting that responses to cell wall weakening take place in K. lactis. We found over-expression of KlPDA1 and KlACS2 genes involved in the Acetyl-CoA synthesis and down-regulation of KlIDP1, KlACO1, and KlSDH2 genes involved in respiratory metabolism. Increases in oxygen consumption and succinate dehydrogenase activity were also observed in mutant cells. The described approach highlighted the unexpected involvement of KlPMR1 in energy-yielding processes.

Improved production of heterologous proteins by a glucose repression-defective mutant of Kluyveromyces lactis.

The secreted production of heterologous proteins in Kluyveromyces lactis was studied. A glucoamylase (GAA) from the yeast Arxula adeninivorans was used as a reporter protein for the study of the secretion efficiencies of several wild-type and mutant strains of K. lactis. The expression of the reporter protein was placed under the control of the strong promoter of the glyceraldehyde-3-phosphate dehydrogenase of Saccharomyces cerevisiae. Among the laboratory strains tested, strain JA6 was the best producer of GAA. Since this strain is known to be highly sensitive to glucose repression and since this is an undesired trait for biomass-oriented applications, we examined heterologous protein production by using glucose repression-defective mutants isolated from this strain. One of them, a mutant carrying a dgr151-1 mutation, showed a significantly improved capability of producing heterologous proteins such as GAA, human serum albumin, and human interleukin-1beta compared to the parent strain. dgr151-1 is an allele of RAG5, the gene encoding the only hexokinase present in K. lactis (a homologue of S. cerevisiae HXK2). The mutation in this strain was mapped to nucleotide position +527, resulting in a change from glycine to aspartic acid within the highly conserved kinase domain. Cells carrying the dgr151-1 allele also showed a reduction in N- and O-glycosylation. Therefore, the dgr151 strain may be a promising host for the production of heterologous proteins, especially when the hyperglycosylation of recombinant proteins must be avoided.