Metaproteomic Analysis of Microbial Communities Affecting Fishery Products.

Fishery products are one of the main human nutritional sources, and due to the consumption increase, the quality of the derived products may be modified, during catching, technological processing, and storage. Detection and identification of pathogenic and spoilage microorganisms in fishery products is needed because the first may be involved in human diseases, while the second is responsible of significant economic losses. In this sense, liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method and computational analysis of MS data are useful tools for characterizing and identifying different microorganisms and to develop promising strategies for food science investigations. Moreover, in the past decade, metaproteomic methodologies have progressed for the study of microorganisms isolated from their natural samples and independently of the culture restrictions. Metaproteomics enables assessment of proteins and pathways from individual members of the consortium. Metaproteomics can provide a detailed understanding of which organisms occupy specific metabolic niches, how they interact, and how they utilize nutrients, and these insights can be obtained directly from environmental samples.According to that, the sample preparation of the fishery product, the LC-ESI-MS/MS dedicated method, and the MS data analysis were described in the present chapter to obtain the metaproteomic analysis of the respective microbiomes or microbial communities.

Shotgun proteomic analyses of Pseudomonas species isolated from fish products.

Numerous Pseudomonas species can infect aquatic animals, such as farmed rainbow trout, sea trout, sea bass, and sea bream, by causing disease or stress reactions. In aquaculture facilities, a number of Pseudomonas species have been isolated and identified as the main pathogens. The present study describes the characterization of 18 Pseudomonas strains, isolated from fish products using shotgun proteomics. The bacterial proteomes obtained were further analyzed to identify the main functional pathway proteins involved. In addition, this study revealed the presence of 1015 non-redundant peptides related to virulence factors. An additional 25 species-specific peptides were identified as putative Pseudomonas spp. biomarkers. The results constitute the largest dataset, described thus far for the rapid identification and characterization of Pseudomonas species present in edible fish; furthermore, these data can provide the basis for further research into the development of new therapies against these harmful pathogens.

Comprehensive shotgun proteomic characterization and virulence factors of seafood spoilage bacteria.

This article summarizes the characterization, by shotgun proteomics, of 11 bacterial strains identified as responsible for seafood spoilage. A total of 4455 peptide spectrum matches, corresponding to 4299 peptides and 3817 proteins were identified. Analyses of data determined the functional pathways they are involved in. The proteins identified were integrated into a protein-protein network that involves 371 nodes and 3016 edges. Those proteins are implicated in energy pathways, peptidoglycan biosynthesis, spermidine/putrescine metabolism. An additional 773 peptides were characterized as virulence factors, that participates in bacterial pathogenesis; while 14 peptides were defined as biomarkers, as they can be used to differentiate the bacterial species present. This report represents the most extensive proteomic repository available in the field of seafood spoilage bacteria; the data substantially advances the understanding of seafood decay, as well as provides fundamental bases for the recognition of the bacteria existent in seafood that cause spoilage during food processing/storage.

High-Resolution Comparative and Quantitative Proteomics of Biogenic-Amine-Producing Bacteria and Virulence Factors Present in Seafood.

The presence of biogenic amines (histamine, tyramine, putrescine, and cadaverine) in seafood is a significant concern for food safety. This review describes for the first time a shotgun quantitative proteomics strategy to evaluate and compare foodborne strains of bacteria that produce biogenic amines in seafoods. This approach recognized 35,621 peptide spectrum matches, belonging to 20,792 peptides, and 4621 proteins. It allowed the determination of functional pathways and the classification of the strains into hierarchical clusters. The study identified a protein-protein interaction network involving 1160 nodes/10,318 edges. Proteins were related to energy pathways, spermidine biosynthesis, and putrescine metabolism. Label-free quantitative proteomics allowed the identification of differentially regulated proteins in specific strains such as putrescine aminotransferase, arginine decarboxylase, and l-histidine-binding protein. Additionally, 123 peptides were characterized as virulence factors and 299 peptide biomarkers were selected to identify bacterial species in fish products. This study presents the most extensive proteomic repository and progress in the science of food biogenic bacteria and could be applied in the food industry for the detection of bacterial contamination that produces histamine and other biogenic amines during food processing/storage.

Study of the Antibacterial Activity of Rich Polyphenolic Extracts Obtained from Cytisus scoparius against Foodborne Pathogens.

Natural extracts containing high polyphenolic concentrations may act as good antimicrobials for their antibacterial and antibiofilm activity. The present research characterizes two hydro-organic extracts with high polyphenolic content, obtained from the shrub Cytisus scoparius as antipathogenic candidates. As a result of their own composition, both extracts, LE050 and PG050, have shown pronounced bioactivities with potential uses, especially in agricultural, livestock production, food manufacturing, and pharmaceutical industries. Polyphenolic compounds were extracted by using adjusted hydro-organic solvent mixtures. These extracts' in vitro antimicrobial activity was evaluated on Gram-positive and Gram-negative pathogenic bacteria, giving special attention to those involved in food contamination. Due to this, the biofilm dispersion was assessed on Listeria monocytogenes, Staphylococcus aureus and Pseudomonas aeruginosa. The extracts showed antimicrobial activity against the pathogenic species tested, presenting IC50 values between 0.625-20% v/v. Different behaviors have been detected between both extracts, probably linked to their distinct polyphenol composition, being LE050 extract the one with most promising bioactive applications. Finally, the results from the biofilm dispersion assays reveal that the extracts exhibit a good antibiofilm activity against the pathogenic bacteria tested.

Immunomodulatory effect of marine lipids on food allergy.

Seafood is highly enriched in n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFAs), particularly eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3), in contrast to the ultra-processed foods included in the modern Western diet that have high levels of n-6 linoleic acid (LA, 18:2 n-6), precursor for the pro-inflammatory n-6 arachidonic acid (ARA, 20:4 n-6). The capacity of marine lipids to reduce plasmatic triglycerides and blood pressure have been well-described. Moreover, recent studies have also raised evidence of a potential regulatory action of marine lipids on inflammation, the immune system, and food allergy (FA). FA is considered one of the main concerns to become life threatening in food safety. The prevalence of this emerging global problem has been increasing during the last two decades, especially in industrialized countries. About a 6-8% of young children and 2-4% of adults is estimated to be affected by FA. The main objective of the current study is to update the existing knowledge, but also the limitations, on the potential impact of marine lipids and their lipid mediators in regulating immunity, inflammation, and ultimately, food allergies. In particular, the focus is on the effect of marine lipids in modulating the key factors that control the sensitization and effector phases of FA, including gut microbiota (GM), inflammation, and immune system response. Results in animal models highlight the positive effect that consuming marine lipids, whether as a supplement or through seafood consumption, may have a relevant role in improving gut dysbiosis and inflammation, and preventing or reducing the severity of FA. However, more systematic studies in humans are needed to optimize such beneficial actions to each particular FA, age, and medical condition to reach an effective clinical application of marine lipids to improve FAs and their outcomes.

Exploring the Anti-Inflammatory Effect of Inulin by Integrating Transcriptomic and Proteomic Analyses in a Murine Macrophage Cell Model.

Inulin is a natural polysaccharide classified as a soluble fiber with demonstrated prebiotic activity. Prebiotics can reduce intestinal and systemic inflammation through modulation of the gut microflora and their metabolites. Additionally, extensive research is illuminating the role of macrophages in the interaction between gut microbiota and many systemic inflammatory diseases. In this study, the anti-inflammatory properties of inulin were evaluated using a murine macrophage cell model (RAW 264.7) of inflammation, and the immunomodulatory mechanism was investigated using omics technologies. The cells underwent comprehensive transcriptomic and proteomic analyses to identify the mechanisms responsible for the observed anti-inflammatory phenotype. Functional analyses of these omics results revealed two potential mechanisms that may lead to an overall reduction in cytokine and chemokine transcription: the inhibition of the NF-κB signaling pathway, leading to the downregulation of proinflammatory factors such as COX2, and the promotion of the phase II defense protein Hmox1 via the Nrf2 pathway. This study provides promising targets for research on immune modulation by dietary fibers and offers new strategies for the design of functional ingredients, foods, and nutraceutical products, which could ultimately lead to personalized nutrition and improved consumer health.

Gut Microbiome Proteomics in Food Allergies.

Food allergies (FA) have dramatically increased in recent years, particularly in developed countries. It is currently well-established that food tolerance requires the strict maintenance of a specific microbial consortium in the gastrointestinal (GI) tract microbiome as alterations in the gut microbiota can lead to dysbiosis, causing inflammation and pathogenic intestinal conditions that result in the development of FA. Although there is currently not enough knowledge to fully understand how the interactions between gut microbiota, host responses and the environment cause food allergies, recent advances in '-omics' technologies (i.e., proteomics, genomics, metabolomics) and in approaches involving systems biology suggest future headways that would finally allow the scientific understanding of the relationship between gut microbiome and FA. This review summarizes the current knowledge in the field of FA and insights into the future advances that will be achieved by applying proteomic techniques to study the GI tract microbiome in the field of FA and their medical treatment. Metaproteomics, a proteomics experimental approach of great interest in the study of GI tract microbiota, aims to analyze and identify all the proteins in complex environmental microbial communities; with shotgun proteomics, which uses liquid chromatography (LC) for separation and tandem mass spectrometry (MS/MS) for analysis, as it is the most promising technique in this field.

The Role of the Gallbladder, the Intestinal Barrier and the Gut Microbiota in the Development of Food Allergies and Other Disorders.

The microbiota present in the gastrointestinal tract is involved in the development or prevention of food allergies and autoimmune disorders; these bacteria can enter the gallbladder and, depending on the species involved, can either be benign or cause significant diseases. Occlusion of the gallbladder, usually due to the presence of calculi blocking the bile duct, facilitates microbial infection and inflammation, which can be serious enough to require life-saving surgery. In addition, the biliary salts are secreted into the intestine and can affect the gut microbiota. The interaction between the gut microbiota, pathogenic organisms, and the human immune system can create intestinal dysbiosis, generating a variety of syndromes including the development of food allergies and autoimmune disorders. The intestinal microbiota can aggravate certain food allergies, which become severe when the integrity of the intestinal barrier is affected, allowing bacteria, or their metabolites, to cross the intestinal barrier and invade the bloodstream, affecting distal body organs. This article deals with health conditions and severe diseases that are either influenced by the gut flora or caused by gallbladder obstruction and inflammation, as well as putative treatments for those illnesses.

Proteomics Characterization of Food-Derived Bioactive Peptides with Anti-Allergic and Anti-Inflammatory Properties.

Bioactive peptides are found in foods and dietary supplements and are responsible for health benefits with applications in human and animal medicine. The health benefits include antihypertensive, antimicrobial, antithrombotic, immunomodulatory, opioid, antioxidant, anti-allergic and anti-inflammatory functions. Bioactive peptides can be obtained by microbial action, mainly by the gastrointestinal microbiota from proteins present in food, originating from either vegetable or animal matter or by the action of different gastrointestinal proteases. Proteomics can play an important role in the identification of bioactive peptides. High-resolution mass spectrometry is the principal technique used to detect and identify different types of analytes present in complex mixtures, even when available at low concentrations. Moreover, proteomics may provide the characterization of epitopes to develop new food allergy vaccines and the use of immunomodulating peptides to induce oral tolerance toward offending food allergens or even to prevent allergic sensitization. In addition, food-derived bioactive peptides have been investigated for their anti-inflammatory properties to provide safer alternatives to nonsteroidal anti-inflammatory drugs (NSAIDs). All these bioactive peptides can be a potential source of novel drugs and ingredients in food and pharmaceuticals. The following review is focused on food-derived bioactive peptides with antiallergic and anti-inflammatory properties and summarizes the new insights into the use of proteomics for their identification and quantification.

Proteomic Characterization of Virulence Factors and Related Proteins in Enterococcus Strains from Dairy and Fermented Food Products.

Enterococcus species are Gram-positive bacteria that are normal gastrointestinal tract inhabitants that play a beneficial role in the dairy and meat industry. However, Enterococcus species are also the causative agents of health care-associated infections that can be found in dairy and fermented food products. Enterococcal infections are led by strains of Enterococcus faecalis and Enterococcus faecium, which are often resistant to antibiotics and biofilm formation. Enterococci virulence factors attach to host cells and are also involved in immune evasion. LC-MS/MS-based methods offer several advantages compared with other approaches because one can directly identify microbial peptides without the necessity of inferring conclusions based on other approaches such as genomics tools. The present study describes the use of liquid chromatography−electrospray ionization tandem mass spectrometry (LC−ESI−MS/MS) to perform a global shotgun proteomics characterization for opportunistic pathogenic Enterococcus from different dairy and fermented food products. This method allowed the identification of a total of 1403 nonredundant peptides, representing 1327 proteins. Furthermore, 310 of those peptides corresponded to proteins playing a direct role as virulence factors for Enterococcus pathogenicity. Virulence factors, antibiotic sensitivity, and proper identification of the enterococcal strain are required to propose an effective therapy. Data are available via ProteomeXchange with identifier PXD036435. Label-free quantification (LFQ) demonstrated that the majority of the high-abundance proteins corresponded to E. faecalis species. Therefore, the global proteomic repository obtained here can be the basis for further research into pathogenic Enterococcus species, thus facilitating the development of novel therapeutics.

The Use of Bacteriophages in Biotechnology and Recent Insights into Proteomics.

Phages have certain features, such as their ability to form protein-protein interactions, that make them good candidates for use in a variety of beneficial applications, such as in human or animal health, industry, food science, food safety, and agriculture. It is essential to identify and characterize the proteins produced by particular phages in order to use these viruses in a variety of functional processes, such as bacterial detection, as vehicles for drug delivery, in vaccine development, and to combat multidrug resistant bacterial infections. Furthermore, phages can also play a major role in the design of a variety of cheap and stable sensors as well as in diagnostic assays that can either specifically identify specific compounds or detect bacteria. This article reviews recently developed phage-based techniques, such as the use of recombinant tempered phages, phage display and phage amplification-based detection. It also encompasses the application of phages as capture elements, biosensors and bioreceptors, with a special emphasis on novel bacteriophage-based mass spectrometry (MS) applications.

Proteomic Characterization of Antibiotic Resistance in Listeria and Production of Antimicrobial and Virulence Factors.

Some Listeria species are important human and animal pathogens that can be found in contaminated food and produce a variety of virulence factors involved in their pathogenicity. Listeria strains exhibiting multidrug resistance are known to be progressively increasing and that is why continuous monitoring is needed. Effective therapy against pathogenic Listeria requires identification of the bacterial strain involved, as well as determining its virulence factors, such as antibiotic resistance and sensitivity. The present study describes the use of liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) to do a global shotgun proteomics characterization for pathogenic Listeria species. This method allowed the identification of a total of 2990 non-redundant peptides, representing 2727 proteins. Furthermore, 395 of the peptides correspond to proteins that play a direct role in Listeria pathogenicity; they were identified as virulence factors, toxins and anti-toxins, or associated with either antibiotics (involved in antibiotic-related compounds production or resistance) or resistance to toxic substances. The proteomic repository obtained here can be the base for further research into pathogenic Listeria species and facilitate the development of novel therapeutics for these pathogens.

Mastering the control of the Rho transcription factor for biotechnological applications.

The present review represents an update on the fundamental role played by the Rho factor, which facilitates the process of Rho-dependent transcription termination in the prokaryotic world; it also provides a summary of relevant mutations in the Rho factor and the insights they provide into the functions carried out by this protein. Furthermore, a section is dedicated to the putative future use of Rho (the 'taming' of Rho) to facilitate biotechnological processes and adapt them to different technological contexts. Novel bacterial strains can be designed, containing mutations in the rho gene, that are better suited for different biotechnological applications. This process can obtain novel microbial strains that are adapted to lower temperatures of fermentation, shorter production times, exhibit better nutrient utilization, or display other traits that are beneficial in productive Biotechnology. Additional important issues reviewed here include epistasis, the design of TATA boxes, the role of small RNAs, and the manipulation of clathrin-mediated endocytosis, by some pathogenic bacteria, to invade eukaryotic cells. KEY POINTS: • It is postulated that controlling the action of the prokaryotic Rho factor could generate major biotechnological improvements, such as an increase in bacterial productivity or a reduction of the microbial-specific growth rate. • The review also evaluates the putative impact of epistatic mechanisms on Biotechnology, both as possible responsible for unexpected failures in gene cloning and more important for the genesis of new strains for biotechnological applications • The use of clathrin-coated vesicles by intracellular bacterial microorganisms is included too and proposed as a putative delivery mechanism, for drugs and vaccines.

Proteomic Characterization of Bacteriophage Peptides from the Mastitis Producer Staphylococcus aureus by LC-ESI-MS/MS and the Bacteriophage Phylogenomic Analysis.

The present work describes LC-ESI-MS/MS MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analyses of tryptic digestion peptides from phages that infect mastitis-causing Staphylococcus aureus isolated from dairy products. A total of 1933 nonredundant peptides belonging to 1282 proteins were identified and analyzed. Among them, 79 staphylococcal peptides from phages were confirmed. These peptides belong to proteins such as phage repressors, structural phage proteins, uncharacterized phage proteins and complement inhibitors. Moreover, eighteen of the phage origin peptides found were specific to S. aureus strains. These diagnostic peptides could be useful for the identification and characterization of S. aureus strains that cause mastitis. Furthermore, a study of bacteriophage phylogeny and the relationship among the identified phage peptides and the bacteria they infect was also performed. The results show the specific peptides that are present in closely related phages and the existing links between bacteriophage phylogeny and the respective Staphylococcus spp. infected.

Shotgun Proteomics for Food Microorganism Detection.

Classical and culture-based methods for the identification and characterization of the biochemical properties of microorganisms are slow and labor-intensive. Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) has been used for the analysis of bacterial pathogen strain-specific diagnostic peptides allowing the characterization of bacterial strains.Here, we describe the analysis of tryptic digestion peptides by LC-ESI-MS/MS to search for specific biomarkers useful for the rapid identification of, on the one hand, the bacterial species and, on the other hand, the physiological and biochemical characteristics such as the expression of virulence factors, including toxins, immune-modulatory factors, and exoenzymes.

Animal and human RNA viruses: genetic variability and ability to overcome vaccines.

RNA viruses, in general, exhibit high mutation rates; this is mainly due to the low fidelity displayed by the RNA-dependent polymerases required for their replication that lack the proofreading machinery to correct misincorporated nucleotides and produce high mutation rates. This lack of replication fidelity, together with the fact that RNA viruses can undergo spontaneous mutations, results in genetic variants displaying different viral morphogenesis, as well as variation on their surface glycoproteins that affect viral antigenicity. This diverse viral population, routinely containing a variety of mutants, is known as a viral 'quasispecies'. The mutability of their virions allows for fast evolution of RNA viruses that develop antiviral resistance and overcome vaccines much more rapidly than DNA viruses. This also translates into the fact that pathogenic RNA viruses, that cause many diseases and deaths in humans, represent the major viral group involved in zoonotic disease transmission, and are responsible for worldwide pandemics.

Proteomic Characterization of Antibiotic Resistance, and Production of Antimicrobial and Virulence Factors in Streptococcus Species Associated with Bovine Mastitis. Could Enzybiotics Represent Novel Therapeutic Agents Against These Pathogens?

Streptococcus spp. are major mastitis pathogens present in dairy products, which produce a variety of virulence factors that are involved in streptococcal pathogenicity. These include neuraminidase, pyrogenic exotoxin, and M protein, and in addition they might produce bacteriocins and antibiotic-resistance proteins. Unjustifiable misuse of antimicrobials has led to an increase in antibiotic-resistant bacteria present in foodstuffs. Identification of the mastitis-causing bacterial strain, as well as determining its antibiotic resistance and sensitivity is crucial for effective therapy. The present work focused on the LC-ESI-MS/MS (liquid chromatography-electrospray ionization tandem mass spectrometry) analysis of tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2706 non-redundant peptides belonging to 2510 proteins was identified and analyzed. Among them, 168 peptides were determined, representing proteins that act as virulence factors, toxins, anti-toxins, provide resistance to antibiotics that are associated with the production of lantibiotic-related compounds, or play a role in the resistance to toxic substances. Protein comparisons with the NCBI database allowed the identification of 134 peptides as specific to Streptococcus spp., while two peptides (EATGNQNISPNLTISNAQLNLEDKNK and DLWC*NM*IIAAK) were found to be species-specific to Streptococcus dysgalactiae. This proteomic repository might be useful for further studies and research work, as well as for the development of new therapeutics for the mastitis-causing Streptococcus strains.

Characterization of Bacteriophage Peptides of Pathogenic Streptococcus by LC-ESI-MS/MS: Bacteriophage Phylogenomics and Their Relationship to Their Host.

The present work focuses on LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analysis of phage-origin tryptic digestion peptides from mastitis-causing Streptococcus spp. isolated from milk. A total of 2,546 non-redundant peptides belonging to 1,890 proteins were identified and analyzed. Among them, 65 phage-origin peptides were determined as specific Streptococcus spp. peptides. These peptides belong to proteins such as phage repressors, phage endopeptidases, structural phage proteins, and uncharacterized phage proteins. Studies involving bacteriophage phylogeny and the relationship between phages encoding the peptides determined and the bacteria they infect were also performed. The results show how specific peptides are present in closely related phages, and a link exists between bacteriophage phylogeny and the Streptococcus spp. they infect. Moreover, the phage peptide M∗ATNLGQAYVQIM∗PSAK is unique and specific for Streptococcus agalactiae. These results revealed that diagnostic peptides, among others, could be useful for the identification and characterization of mastitis-causing Streptococcus spp., particularly peptides that belong to specific functional proteins, such as phage-origin proteins, because of their specificity to bacterial hosts.

Prokaryotic sigma factors and their transcriptional counterparts in Archaea and Eukarya.

RNA polymerases (RNAPs) carry out transcription in the three domains of life, Bacteria, Archaea, and Eukarya. Transcription initiation is highly regulated by a variety of transcription factors, whose number and subunit complexity increase during evolution. This process is regulated in Bacteria by the σ factor, while the three eukaryotic RNAPs require a complex set of transcription factors (TFs) and a TATA-binding protein (TBP). The archaeal transcription system appears to be an ancestral version of the eukaryotic RNAPII, requiring transcription factor B (TFB), TBP, and transcription factor E (TFE). The function of the bacterial sigma (σ) factor has been correlated to the roles played by the eukaryotic RNAP II and the archaeal RNAP. In addition, σ factors, TFB, and TFIIB all contain multiple DNA binding helix-turn-helix (HTH) structural motifs; although TFIIB and TFB display two HTH domains, while the bacterial σ factor spans 4 HTH motifs. The sequence similarities and structure alignments of the bacterial σ factor, eukaryotic TFIIB, and archaeal TFB evidence that these three proteins are homologs.Key Points• Transcription initiation is highly regulated by TFs.• Transcription is finely regulated in all domains of life by different sets of TFs.• Specific TFs in Bacteria, Eukarya and Archaea are homologs.