Denise Schrama

Listeria monocytogenes dairy isolates show a different proteome response to sequential exposure to gastric and intestinal fluids.

The gastrointestinal system poses different stresses to the foodborne pathogen, Listeria monocytogenes, including the low pH of the stomach and the presence of bile and the high osmolality of the intestinal fluid. The present study evaluated how previous exposure of three L. monocytogenes dairy isolates (C882 and T8, serovar 4b isolates and A9 serovar 1/2a or 3b isolate) to a cheese-simulated medium (p H5.5 and 3.5% NaCl [w/v], adapted cultures) affected subsequent survival in a simulated gastrointestinal system. Listerial cultures exposed to the cheese-simulated medium at pH7.0, with no added NaCl, were considered non-adapted. To investigate the main events involved in listerial survival during the gastric and intestinal subsequent challenge, a proteomic approach was used. All L. monocytogenes strains were able to survive the deleterious effects of the gastrointestinal fluids and no significant differences were observed between adapted and non-adapted cells. However the L. monocytogenes strains showed a different protein pattern in response to the gastrointestinal stress. Data indicated that synthesis of stress related proteins is more pronounced in non-adapted cells. Although, a significant number of enzymes involved in glycolysis and energy production were also consistently over-produced by the three strains. These findings provided new insights into the means used by L. monocytogenes to overcome the gastrointestinal system and allow the pathogen to move to the next phase of the infectious process.

Nutritional mitigation of winter thermal stress in gilthead seabream associated metabolic pathways and potential indicators of nutritional state

A trial was carried out with gilthead seabream juveniles, aiming to investigate the ability of an enhanced dietary formulation (diet Winter Feed, WF, containing a higher proportion of marine-derived protein sources and supplemented in phospholipids, vitamin C, vitamin E and taurine) to assist fish in coping with winter thermal stress, compared to a low-cost commercial diet (diet CTRL). In order to identify the metabolic pathways affected by WF diet, a comparative two dimensional differential in-gel electrophoresis (2D-DIGE) analysis of fish liver proteome (pH 4–7) was undertaken at the end of winter. A total of 404 protein spots, out of 1637 detected, were differentially expressed between the two groups of fish. Mass spectrometry analysis of selected spots suggested that WF diet improved oxidative stress defense, reduced endoplasmic reticulum stress, enhanced metabolic flux through methionine cycle and phenylalanine/tyrosine catabolism, and induced higher aerobic metabolism and gluconeogenesis. Results support the notion that WF diet had a positive effect on fish nutritional state by partially counteracting the effect of thermal stress and underlined the sensitivity of proteome data for nutritional and metabolic profiling purposes. Intragroup variability and co-measured information were also used to pinpoint which proteins displayed a stronger relation with fish nutritional state. SIGNIFICANCE Winter low water temperature is a critical factor for gilthead seabream farming in the Mediterranean region, leading to a reduction of feed intake, which often results in metabolic and immunological disorders and stagnation of growth performances. In a recent trial, we investigated the ability of an enhanced dietary formulation (diet WF) to assist gilthead seabream in coping with winter thermal stress, compared to a standard commercial diet (diet CTRL). Within this context, in the present work, we identified metabolic processes that are involved in the stress-mitigating effect observed with diet WF, by undertaking a comparative analysis of fish liver proteome at the end of winter. This study brings information relative to biological processes that are involved in gilthead seabream winter thermal stress and shows that these can be mitigated through a nutritional strategy, assisting gilthead seabream to deal better with winter thermal conditions. Furthermore, the results show that proteomic information not only clearly distinguishes the two dietary groups from each other, but also captures heterogeneities that reflect intra-group differences in nutritional state. This was exploited in this work to refine the variable selection strategy so that protein spots displaying a stronger correlation with “nutritional state” could be identified as possible indicators of gilthead seabream metabolic and nutritional state. Finally, this study shows that gel-based proteomics seems to provide more reliable information than transmissive FT-IR spectroscopy, for the purposes of nutritional and metabolic profiling.

Proteomic Analysis Shows That Individual Listeria monocytogenes Strains Use Different Strategies in Response to Gastric Stress

Ingestion of contaminated dairy products, in particular soft cheese, is one of the major routes of infection by the human pathogen Listeria monocytogenes. During cheese processing, this foodborne pathogen is exposed to sublethal acid and osmotic stress conditions, which may induce tolerance responses and influence subsequent survival in the gastric tract. The aim of the current study was to evaluate the impact on a L. monocytogenes cheese isolate (serotype 4b) and two cheese dairy isolates (T8, serotype 4b, isolated from vat; and A9, serotype 1/2b or 3b, isolated from shelf stand) of exposure to sublethal conditions of pH and salt (5.5 and 3.5% [w/v] NaCl) in a cheese-simulated medium and further challenge with gastric stress. The bacterial cells exposed to pH 7.0 and no added salt were considered non-adapted. Via two-dimensional gel electrophoresis (2-DE), the proteomes of cheese-simulated medium and gastric challenged Listeria cells were compared. All L. monocytogenes isolates were able to survive the high acidity of gastric fluid (pH 2.5), and no significant differences were observed between adapted and non-adapted cells. However, the analysis of the intracellular proteome profiles revealed a significant intra-strain variation in the protein arsenal used to respond to the adaptation in the cheese-based medium and to the gastric stress. In cheese-based medium, the three strains produced different stress proteins. All three strains showed a higher abundance of carbohydrate proteins, but there was no overlap between them. Exposure to the gastric fluid induced the production of a group of proteins in T8 adapted and non-adapted cells that had not been detected previously in the cheese-based proteome. No such response was shown by A9 and C882 strains. Taken together, this study evidences the proteome tools used by adapted and non-adapted cells to cope with the hostile microenvironment of the stomach.

Adaptation of Listeria monocytogenes in a simulated cheese medium: effects on virulence using the Galleria mellonella infection model.

The aim of this study was to evaluate the effect of the acid and salt adaptation in a cheese‐based medium on the virulence potential of Listeria monocytogenes strains isolated from cheese and dairy processing environment using the Galleria mellonella model. Four L. monocytogenes strains were exposed to a cheese‐based medium in conditions of induction of an acid tolerance response and osmotolerance response (pH 5·5 and 3·5% w/v NaCl) and injected in G. mellonella insects. The survival of insects and the L. monocytogenes growth kinetics in insects were evaluated. The gene expression of hly, actA and inlA genes was determined by real‐time PCR. The adapted cells of two dairy strains showed reduced insect mortality (P < 0·05) in comparison with nonadapted cells. Listeria monocytogenes Scott A was the least virulent, whereas the cheese isolate C882 caused the highest insect mortality, and no differences (P > 0·05) was found between adapted and nonadapted cells. The gene expression results evidenced an overexpression of virulence genes in cheese‐based medium, but not in simulated insect‐induced conditions. Our results suggest that adaptation to low pH and salt in a cheese‐based medium can affect the virulence of L. monocytogenes, but this effect is strain dependent.

Enhanced dietary formulation to mitigate winter thermal stress in gilthead sea bream (Sparus aurata): a 2D-DIGE plasma proteome study

Low water temperatures during winter are common in farming of gilthead sea bream in the Mediterranean. This causes metabolic disorders that in extreme cases can lead to a syndrome called “winter disease.” An improved immunostimulatory nutritional status might mitigate the effects of this thermal metabolic stress. A trial was set up to assess the effects of two different diets on gilthead sea bream physiology and nutritional state through plasma proteome and metabolites. Four groups of 25 adult gilthead sea bream were reared during winter months, being fed either with a control diet (CTRL) or with a diet called “winter feed” (WF). Proteome results show a slightly higher number of proteins upregulated in plasma of fish fed the WF. These proteins are mostly involved in the immune system and cell protection mechanisms. Lipid metabolism was also affected, as shown both by plasma proteome and by the cholesterol plasma levels. Overall, the winter feed diet tested seems to have positive effects in terms of fish condition and nutritional status, reducing the metabolic effects of thermal stress.

Applications of Proteomics in Aquaculture

Aquaculture is one of the fastest growing world industries due to the increased demand of fishery products for human consumption and capture restrictions as a result of aquatic ecosystems exploitation. Aquaculture is therefore an extremely competitive business with major challenges to keep a high quality farmed fish through a sustainable production system. These challenges imposed quite important changes in this more traditional market, namely at the level of integrating scientific knowledge and research. Proteomics presents itself as a powerful tool not only for a better understanding of the marine organisms biology but also to provide solutions to deal with changes and the increasing demand in the system’s production line to ensure the required supply. In this book chapter we will give an overview of aquaculture nowadays, its challenges and describe relevant proteomics studies in several areas of this industry. A brief description of the proteomics technical approaches applied to aquaculture will also be addressed.

Proteomics in Fish and Aquaculture Research

The demand for animal protein for human consumption is currently on the rise fueled mainly by an exponential increase of the world population. The higher demand of fishery products and capture restrictions as a result of wild fish stock exploitation made aquaculture an extremely important source of protein (mainly fish, shellfish, and algae) available in human diet. Production statistics database from FAO states a value of about 97.2 million tonnes, of which around 70.0 million tonnes of the total food fish and 27.0 million tonnes of aquatic plants. The awareness that nowadays competitiveness is extremely dependent on scientific knowledge and new technologies made the number of manuscripts published in this area to rise almost exponentially. Aquaculture faces many challenges in order to continuously deliver a high-quality farmed fish through a sustainable production system. In order to achieve this goal, new management strategies need to be addressed, and state-of-the-art technologies like proteomics have been applied to study many factors like welfare, safety, nutrition, and diseases, which are directly responsible for the end-product quality.

Effects of dietary lipid sources on hepatic nutritive contents, fatty acid composition and proteome of Nile tilapia (Oreochromis niloticus)

Dietary oils of varying fatty acid composition have been used in tilapia feeds; nevertheless, investigation of their effects on metabolism and physiological processes has been limited. Therefore, in this study, using proteomic technology, the effects of different dietary lipids (DLs) on hepatic lipid metabolism and physiological processes were investigaed in Nile tilapia (Oreochromis niloticus). Fish were fed with different DL, which included palm oil (PO), linseed oil (LO) and soybean oil (SBO) for 90 days. Growth performance appeared to be similar among the dietary group. Hepatic FA contents were reflected by DL. Dietary PO (source of saturated fatty acids; SFA) led to an increase in the hepatosomatic index as well as lipid and protein contents in liver. Dietary SBO (source of n-6 polyunsaturated fatty acids; n-6 PUFAs) increased hepatic carbohydrate contents, but decreased the hepatic protein. The proteomic analysis showed that these nutritive changes in the liver were mediated by several proteins involved in lipid, carbohydrate, and amino acid metabolism. Dietary SBO showed an increased accumulation in proteins related to oxidative stress, immune and inflammatory processes. Dietary LO (source of n-3 PUFAs) increased abundance of cytoskeleton-related proteins. qRT-PCR was performed to provide supportive information for the result of proteomic study. Similar effects of DLs on mRNA levels were observed for atp5b, krt18, and selenbp1. Combined together, dietary SFA could supply as energy reservoir for regular activites. Dietary n-3 and n-6 PUFAs led to induce vital metabolic and physiologic processes which would contribute to maintaining normal health and/or providing health-related benefits. SIGNIFICANCE: Dietary SBO increased hepatic carbohydrate contents, but decreased the hepatic protein. Different dietary lipid led to alter the abundance of several proteins (i.e., DHRS1, ATP5B, PLA2G12B, APO, AMY2A, GRP78, PRSS1, FAH, and PRSS36) involved in lipid, carbohydrate, and amino acid metabolism. Dietary SBO showed an increased accumulation in proteins (i.e., QDPR, CABC1, and PRDX6) that respond to oxidative stress, suggesting that n-6 PUFAs induce oxidative conditions. Dietary SBO led to increase the accumulation of proteins (i.e., NITR26, NCCRP1, and LCN) involved in immune and inflammatory processes. Dietary LO increased the abundant levels of cytoskeleton-related proteins (i.e., ACTB, AHNAK, ERC2, KRT18, and RILP1). Other proteins (i.e., SELENBP1, FAM46C, and ANC1) involved in other physiological processes were also modulated by DL. qRT-PCR was performed to provide supportive information of proteomic study. Similar effects of DLs on mRNA levels were observed for atp5b, krt18, and selenbp1.

Impacts of the combined exposure to seawater acidification and arsenic on the proteome of Crassostrea angulata and Crassostrea gigas

Proteomic analysis was performed to compare the effects of Arsenic (As), seawater acidification (Low pH) and the combination of both stressors (Low pH + As) on Crassostrea angulata and Crassostrea gigas juveniles in the context of global environmental change. This study aimed to elucidate if two closely related Crassostrea species respond similarly to these environmental stressors, considering both single and combined exposures, to infer if the simultaneous exposure to both stressors induced a differentiated response. Identification of the most important differentially expressed proteins between conditions revealed marked differences in the response of each species towards single and combined exposures, evidencing species-related differences towards each experimental condition. Moreover, protein alterations observed in the combined exposure (Low pH + As) were substantially different from those observed in single exposures. Identified proteins and their putative biological functions revealed an array of modes of action in each condition. Among the most important, those involved in cellular structure (Actin, Atlastin, Severin, Gelsolin, Coronin) and extracellular matrix modulation (Ependymin, Tight junction ZO-1, Neprilysin) were strongly regulated, although in different exposure conditions and species. Data also revealed differences regarding metabolic modulation capacity (ATP β, Enolase, Aconitate hydratase) and oxidative stress response (Aldehyde dehydrogenase, Lactoylglutathione, Retinal dehydrogenase) of each species, which also depended on single or combined exposures, illustrating a different response capacity of both oyster species to the presence of multiple stressors. Interestingly, alterations of piRNA abundance in C. angulata suggested genome reconfiguration in response to multiple stressors, likely an important mode of action related to adaptive evolution mechanisms previously unknown to oyster species, which requires further investigation. The present findings provide a deeper insight into the complexity of C. angulata and C. gigas responses to environmental stress at the proteome level, evidencing different capacities to endure abiotic changes, with relevance regarding the ecophysiological fitness of each species and competitive advantages in a changing environment.

Proteomics in Aquaculture: Quality and Safety

Aquaculture is a growing sector of the food industry that actively tries to integrate scientific knowledge into its management strategies. Aquaculture faces several key challenges including the provision of high-quality nutritional sources that serve to address both quality and safety, thus transforming production and management strategies to integrate sustainable principles. Proteomics has been established as a powerful and unbiased instrument in environmental monitoring and risk assessment. Proteomics in aquaculture has been applied to address welfare, nutrition, health, environmental quality, and safety. In this review, we will focus on the proteomics-based method developed and implemented for its application to elucidate the key challenges that face the fish and seafood industries: quality and safety. The latest methodological developments in high-throughput proteomics and metaproteomics are seeing rapid integration into aquaculture research contributing to the common goal of offering high quality food production processes and environmental sustainability.Abstract Aquaculture is a growing sector of the food industry that actively tries to integrate scientific knowledge into its management strategies. Aquaculture faces several key challenges including the provision of high-quality nutritional sources that serve to address both quality and safety, thus transforming production and management strategies to integrate sustainable principles. Proteomics has been established as a powerful and unbiased instrument in environmental monitoring and risk assessment. Proteomics in aquaculture has been applied to address welfare, nutrition, health, environmental quality, and safety. In this review, we will focus on the proteomics-based method developed and implemented for its application to elucidate the key challenges that face the fish and seafood industries: quality and safety. The latest methodological developments in high-throughput proteomics and metaproteomics are seeing rapid integration into aquaculture research contributing to the common goal of offering high quality food production processes and environmental sustainability.