Elena Zanni

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.

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

ABSTRACT 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.

Zinc oxide microrods and nanorods: Different antibacterial activity and their mode of action against Gram-positive bacteria

The development of antibiotic resistance among pathogenic bacteria combined with increased implant-associated infections have determined a great interest towards new bactericidal materials containing various organic and inorganic substances. Among them, zinc oxide (ZnO) derived materials have received considerable attention due to their unique antibacterial, antifungal, and UV filtering properties as well as high catalytic and photochemical activities. In the present work, we investigate the antimicrobial properties against two Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) of ZnO microrods (MRs) and nanorods (NRs), produced in bulk quantities through simple and inexpensive methods. We demonstrate that the antimicrobial effect is strongly dependent on the rod size and dose. Scanning electron microscopy analysis revealed that the two investigated microbial types interact differently with the ZnO-MRs and NRs due to their different morphology. This resulted in different outcomes as reported by their respective Colony Forming Unit (CFU) capabilities. Moreover, Fourier Transform Infrared (FT-IR) spectroscopy revealed that changes in cell outer structures, i.e. membrane and exopolysaccharides (EPS), produced by the interaction with the ZnO structures, are responsible for the antimicrobial mechanism without the accumulation of reactive oxygen species. This was further strengthened by the increased survival observed in the case of bacterial cells treated in the presence of an osmotic support, like glycerol. In addition, Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis showed that reduced cell viability is not strictly correlated to increased zinc ion release in the suspension. We then concluded that ZnO-NRs have a superior antimicrobial effect against both S. aureus and B. subtilis at much lower doses when compared to ZnO-MRs. This is mainly due to the smaller diameter of the NRs, which promotes surface damaging and protein alteration of the cell wall. Finally, the lack of toxicity and the antimicrobial properties of ZnO-NRs versus S. aureus, validated in vivo using the nematode Caenorhabditis elegans as host infection model, confirm the promising exploitation of ZnO-NRs in biomedical applications.

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.

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

BackgroundProtein 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.ResultsHere 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.ConclusionsProper 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.

SOD1, a new Kluyveromyces lactis helper gene for heterologous protein secretion

ABSTRACT Bottlenecks in protein expression and secretion often limit the development of industrial processes. By manipulating chaperone and foldase levels, improvements in yeast secretion were found for a number of proteins. Recently, sustained endoplasmic reticulum stress, occurring due to recombinant protein production, was reported to cause oxidative stress in yeast. Saccharomyces cerevisiae cells are able to trigger an adaptive response to oxidative-stress conditions, resulting in the upregulation of both primary and secondary antioxidant defenses. SOD1 encodes for a superoxide dismutase that catalyzes the dismutation of superoxide anions (O2−) into oxygen and hydrogen peroxide. It is a Cu2+/Zn2+ metalloenzyme and represents an important antioxidant defense in nearly all aerobic and aerotolerant organisms. We found that overexpression of the Kluyveromyces lactis SOD1 (KlSOD1) gene was able to increase the production of two different heterologous proteins, human serum albumin (HSA) and glucoamylase from Arxula adeninivorans. In addition, KlSOD1 overexpression led to a significant decrease in the amount of reactive oxygen species (ROS) that originated during protein production. The yield of HSA also increased when K. lactis cells were grown in the presence of the antioxidant agent ascorbic acid and decreased when cells were challenged with menadione, a ROS generator compound. Moreover, we observed that, in high-osmolarity medium, cells overexpressing KlSOD1 showed higher growth rates than control cells. Our results thus further support the notion that the production of some heterologous proteins may be improved by manipulating genes involved in general stress responses.

Thermal adaptability of Kluyveromyces marxianus in recombinant protein production

BackgroundKluyveromyces 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.ResultsThe 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 β-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 β-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).ConclusionsK. 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.

Zinc Oxide Nanorods-Decorated Graphene Nanoplatelets: A Promising Antimicrobial Agent against the Cariogenic Bacterium Streptococcus mutans

Nanomaterials are revolutionizing the field of medicine to improve the quality of life due to the myriad of applications stemming from their unique properties, including the antimicrobial activity against pathogens. In this study, the antimicrobial and antibiofilm properties of a novel nanomaterial composed by zinc oxide nanorods-decorated graphene nanoplatelets (ZNGs) are investigated. ZNGs were produced by hydrothermal method and characterized through field-emission scanning electron microscopy (FE-SEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD) techniques. The antimicrobial activity of ZNGs was evaluated against Streptococcus mutans, the main bacteriological agent in the etiology of dental caries. Cell viability assay demonstrated that ZNGs exerted a strikingly high killing effect on S. mutans cells in a dose-dependent manner. Moreover, FE-SEM analysis revealed relevant mechanical damages exerted by ZNGs at the cell surface of this dental pathogen rather than reactive oxygen species (ROS) generation. In addition, inductively coupled plasma mass spectrometry (ICP-MS) measurements showed negligible zinc dissolution, demonstrating that zinc ion release in the suspension is not associated with the high cell mortality rate. Finally, our data indicated that also S. mutans biofilm formation was affected by the presence of graphene-zinc oxide (ZnO) based material, as witnessed by the safranin staining and growth curve analysis. Therefore, ZNGs can be a remarkable nanobactericide against one of the main dental pathogens. The potential applications in dental care and therapy are very promising.

Depletion of casein kinase I leads to a NAD(P)+/NAD(P)H balance-dependent metabolic adaptation as determined by NMR spectroscopy-metabolomic profile in Kluyveromyces lactis

BACKGROUND In the Crabtree-negative Kluyveromyces lactis yeast the rag8 mutant is one of nineteen complementation groups constituting the fermentative-deficient model equivalent to the Saccharomyces cerevisiae respiratory petite mutants. These mutants display pleiotropic defects in membrane fatty acids and/or cell walls, osmo-sensitivity and the inability to grow under strictly anaerobic conditions (Rag(-) phenotype). RAG8 is an essential gene coding for the casein kinase I, an evolutionary conserved activity involved in a wide range of cellular processes coordinating morphogenesis and glycolytic flux with glucose/oxygen sensing. METHODS A metabolomic approach was performed by NMR spectroscopy to investigate how the broad physiological roles of Rag8, taken as a model for all rag mutants, coordinate cellular responses. RESULTS Statistical analysis of metabolomic data showed a significant increase in the level of metabolites in reactions directly involved in the reoxidation of the NAD(P)H in rag8 mutant samples with respect to the wild type ones. We also observed an increased de novo synthesis of nicotinamide adenine dinucleotide. On the contrary, the production of metabolites in pathways leading to the reduction of the cofactors was reduced. CONCLUSIONS The changes in metabolite levels in rag8 showed a metabolic adaptation that is determined by the intracellular NAD(P)(+)/NAD(P)H redox balance state. GENERAL SIGNIFICANCE The inadequate glycolytic flux of the mutant leads to a reduced/asymmetric distribution of acetyl-CoA to the different cellular compartments with loss of the fatty acid dynamic respiratory/fermentative adaptive balance response.

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.