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.

Shotgun Proteomics Analysis, Functional Networks, and Peptide Biomarkers for Seafood-Originating Biogenic-Amine-Producing Bacteria.

Biogenic amine-producing bacteria are responsible for the production of basic nitrogenous compounds (histamine, cadaverine, tyramine, and putrescine) following the spoilage of food due to microorganisms. In this study, we adopted a shotgun proteomics strategy to characterize 15 foodborne strains of biogenic-amine-producing bacteria. A total of 10,673 peptide spectrum matches belonging to 4081 peptides and corresponding to 1811 proteins were identified. Relevant functional pathways were determined, and strains were differentiated into hierarchical clusters. An expected protein-protein interaction network was created (260 nodes/1973 interactions). Most of the determined proteins were associated with networks/pathways of energy, putrescine metabolism, and host-virus interaction. Additionally, 556 peptides were identified as virulence factors. Moreover, 77 species-specific peptide biomarkers corresponding to 64 different proteins were proposed to identify 10 bacterial species. This represents a major proteomic dataset of biogenic-amine-producing strains. These results may also be suitable for new treatments for food intoxication and for tracking microbial sources in foodstuffs.

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.

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.

Oral lichen planus: a microbiologist point of view.

Oral lichen planus (OLP) is a chronic disease of uncertain etiology, although it is generally considered as an immune-mediated disease that affects the mucous membranes and even the skin and nails. Over the years, this disease was attributed to a variety of causes, including different types of microorganisms. This review analyzes the present state of the art of the disease, from a microbiological point of view, while considering whether or not the possibility of a microbial origin for the disease can be supported. From the evidence presented here, OLP should be considered an immunological disease, as it was initially proposed, as opposed to an illness of microbiological origin. The different microorganisms so far described as putative disease-causing agents do not fulfill Koch's postulates; they are, actually, not the cause, but a result of the disease that provides the right circumstances for microbial colonization. This means that, at this stage, and unless new data becomes available, no microorganism can be envisaged as the causative agent of lichen planus.

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 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.

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.

Bacillus safensis subsp. osmophilus subsp. nov., isolated from condensed milk, and description of Bacillus safensis subsp. safensis subsp. nov.

A bacterial strain, designated BC09T, was isolated from a contaminated sample of condensed milk. Phylogenetic analyses based on 16S rRNA gene sequences placed strain BC09T into the genus Bacillus with its closest relatives being Bacillus safensis and Bacillus australimaris with 100 and 99.9 % similarity, respectively. Analysis of the gyrB gene confirmed the closeness of strain BC09T with respect to the species B. safensis since it presented 97.8 and 95.2 % similarity values, respectively, to the type strains of B. safensis and B. australimaris. DNA-DNA hybridization confirmed these results showing averages of 67 and 56 %, respectively, between strain BC09T and the type strains of B. safensis and B. australimaris. Average nucleotide identity blast values obtained for BC09T compared to the closest relative type strains were 95.7 and 67.6 %, respectively, and predicted DNA-DNA hybridization values were 93.1 and 51.9 %, respectively. However, strain BC09T differs from the type strains of its closest relatives in several phenotypic characteristics. MK-7 was the only menaquinone detected and iso-C15:0 and anteiso-C15:0 were the major fatty acids. The polar lipid profile consisted of diphosphatidylglycerol, phosphatidylglycerol, two unidentified phospholipids, two unidentifed glycolipids, three unidentified lipids and one unidentifed phosphoglycolipid. Meso-diaminopimelic acid was detected in the peptidoglycan. The G+C content was 40.9 mol%. Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain BC09T represents a new subspecies of B. safensis, for which the name Bacillus safensis subsp. osmophilus subsp. nov. is proposed. The type strain is BC09T (=LMG 30124T, =CECT 9344T).

Considerations on bacterial nucleoids.

The classic genome organization of the bacterial chromosome is normally envisaged with all its genetic markers linked, thus forming a closed genetic circle of duplex stranded DNA (dsDNA) and several proteins in what it is called as "the bacterial nucleoid." This structure may be more or less corrugated depending on the physiological state of the bacterium (i.e., resting state or active growth) and is not surrounded by a double membrane as in eukayotic cells. The universality of the closed circle model in bacteria is however slowly changing, as new data emerge in different bacterial groups such as in Planctomycetes and related microorganisms, species of Borrelia, Streptomyces, Agrobacterium, or Phytoplasma. In these and possibly other microorganisms, the existence of complex formations of intracellular membranes or linear chromosomes is typical; all of these situations contributing to weakening the current cellular organization paradigm, i.e., prokaryotic vs eukaryotic cells.

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.

The Interplay among Radiation Therapy, Antibiotics and the Microbiota: Impact on Cancer Treatment Outcomes.

Radiation therapy has been used for more than a century, either alone or in combination with other therapeutic modalities, to treat most types of cancer. On average, radiation therapy is included in the treatment plans for over 50% of all cancer patients, and it is estimated to contribute to about 40% of curative protocols, a success rate that may reach 90%, or higher, for certain tumor types, particularly on patients diagnosed at early disease stages. A growing body of research provides solid support for the existence of bidirectional interaction between radiation exposure and the human microbiota. Radiation treatment causes quantitative and qualitative changes in the gut microbiota composition, often leading to an increased abundance of potentially hazardous or pathogenic microbes and a concomitant decrease in commensal bacteria. In turn, the resulting dysbiotic microbiota becomes an important contributor to worsen the adverse events caused in patients by the inflammatory process triggered by the radiation treatment and a significant determinant of the radiation therapy anti-tumor effectiveness. Antibiotics, which are frequently included as prophylactic agents in cancer treatment protocols to prevent patient infections, may affect the radiation/microbiota interaction through mechanisms involving both their antimicrobial activity, as a mediator of microbiota imbalances, and their dual capacity to act as pro- or anti-tumorigenic effectors and, consequently, as critical determinants of radiation therapy outcomes. In this scenario, it becomes important to introduce the use of probiotics and/or other agents that may stabilize the healthy microbiota before patients are exposed to radiation. Ultimately, newly developed methodologies may facilitate performing personalized microbiota screenings on patients before radiation therapy as an accurate way to identify which antibiotics may be used, if needed, and to inform the overall treatment planning. This review examines currently available data on these issues from the perspective of improving radiation therapy outcomes.

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.

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.

FPG1, a gene involved in foam formation in Saccharomyces cerevisiae.

Foam formation in fermentations conducted by Saccharomyces cerevisiae, either at the beginning of the fermentation process or at the end in the case of sparkling wines, is due, to a large extent, to cell wall mannoproteins, which provide hydrophobicity to the yeast cells and favour their floating index as well as stabilization of the foam. The foam may be an undesirable by-product if it accumulates on top of the fermentation tanks, but its formation is a good property in either beer or sparkling wines. It is therefore important to know the yeast genes involved in foam formation, in order to suppress or potentiate their expression according to the end product to be obtained. The present study identified and characterized, for the first time in an oenological S. cerevisiae strain, a gene involved in foam formation, named FPG1 (foam-promoting gene). The protein encoded by FPG1 is a mannoprotein precursor present in the cell wall and somewhat homologous to Awa1p, a foaming protein described in a sake S. cerevisiae strain. A foamless strain was prepared by FPG1 deletion, and a foam hyper-producing strain was also constructed, thus allowing the conclusion that Fpg1p is a mannoprotein involved in yeast frothing.

Proteins influencing foam formation in wine and beer: the role of yeast.

This review focuses on the role of proteins in the production and maintenance of foam in both sparkling wines and beer. The quality of the foam in beer but especially in sparkling wines depends, among other factors, on the presence of mannoproteins released from the yeast cell walls during autolysis. These proteins are hydrophobic, highly glycosylated, and their molecular masses range from 10 to 200 kDa--characteristics that allow mannoproteins to surround and thus stabilize the gas bubbles of the foam. Both the production and stabilization of foam also depend on other proteins. In wine, these include grape-derived proteins such as vacuolar invertase; in beer, barley-derived proteins, such as LTP1, protein Z, and hordein-derived polypeptides, are even more important in this respect than mannoproteins.

A new disruption vector (pDHO) to obtain heterothallic strains from both Saccharomyces cerevisiae and Saccharomyces pastorianus.

Yeasts are responsible for several traits in fermented beverages, including wine and beer, and their genetic manipulation is often necessary to improve the quality of the fermentation product. Improvement of wild-type strains of Saccharomyces cerevisiae and Saccharomyces pastorianus is difficult due to their homothallic character and variable ploidy level. Homothallism is determined by the HO gene in S. cerevisiae and the Sc-HO gene in S. pastorianus. In this work, we describe the construction of an HO disruption vector (pDHO) containing an HO disruption cassette and discuss its use in generating heterothallic yeast strains from homothallic Saccharomyces species.