Open AccessReview
Mucus-Pathogen Interactions in the Gastrointestinal Tract of Farmed Animals
Microorganisms 2018, 6(2), 55; https://doi.org/10.3390/microorganisms6020055 -
Abstract
Gastrointestinal infections cause significant challenges and economic losses in animal husbandry. As pathogens becoming resistant to antibiotics are a growing concern worldwide, alternative strategies to treat infections in farmed animals are necessary in order to decrease the risk to human health and increase
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Gastrointestinal infections cause significant challenges and economic losses in animal husbandry. As pathogens becoming resistant to antibiotics are a growing concern worldwide, alternative strategies to treat infections in farmed animals are necessary in order to decrease the risk to human health and increase animal health and productivity. Mucosal surfaces are the most common route used by pathogens to enter the body. The mucosal surface that lines the gastrointestinal tract is covered by a continuously secreted mucus layer that protects the epithelial surface. The mucus layer is the first barrier the pathogen must overcome for successful colonization, and is mainly composed of densely glycosylated proteins called mucins. The vast array of carbohydrate structures present on the mucins provide an important setting for host-pathogen interactions. This review summarizes the current knowledge on gastrointestinal mucins and their role during infections in farmed animals. We examine the interactions between mucins and animal pathogens, with a focus on how pathogenic bacteria can modify the mucin environment in the gut, and how this in turn affects pathogen adhesion and growth. Finally, we discuss analytical challenges and complexities of the mucus-based defense, as well as its potential to control infections in farmed animals. Full article
Open AccessOpinion
Bacteriophages as New Human Viral Pathogens
Microorganisms 2018, 6(2), 54; https://doi.org/10.3390/microorganisms6020054 -
Abstract
The pathogenesis of numerous human multifaceted devastating diseases, including a variety of neurodegenerative and autoimmune diseases, is associated with alterations in the gut microbiota; however, the underlying mechanisms are not completely understood. Our recent human metagenome and phagobiota proteome analyses and studies in
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The pathogenesis of numerous human multifaceted devastating diseases, including a variety of neurodegenerative and autoimmune diseases, is associated with alterations in the gut microbiota; however, the underlying mechanisms are not completely understood. Our recent human metagenome and phagobiota proteome analyses and studies in relevant animal models suggested that bacterial viruses might be implicated in the progression and maintenance of at least some pathologies, including those associated with protein misfolding. Here, for the first time, we propose the concept of bacteriophages as human pathogens. We suggest that bacterial viruses have different ways to directly and indirectly interact with eukaryotic cells and proteins, leading to human diseases. Furthermore, we suggest different causes of bacteriophages infection on the basis of the unique ways of interplay of phages, microbiota, and the human host. This concept opens a discussion of the role of bacteriophages as previously overlooked pathogenic factors and suggests that bacterial viruses have to be further explored as a diagnostic and treatment target for therapeutic intervention. Full article
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Open AccessReview
Mucus: An Underestimated Gut Target for Environmental Pollutants and Food Additives
Microorganisms 2018, 6(2), 53; https://doi.org/10.3390/microorganisms6020053 -
Abstract
Synthetic chemicals (environmental pollutants, food additives) are widely used for many industrial purposes and consumer-related applications, which implies, through manufactured products, diet, and environment, a repeated exposure of the general population with growing concern regarding health disorders. The gastrointestinal tract is the first
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Synthetic chemicals (environmental pollutants, food additives) are widely used for many industrial purposes and consumer-related applications, which implies, through manufactured products, diet, and environment, a repeated exposure of the general population with growing concern regarding health disorders. The gastrointestinal tract is the first physical and biological barrier against these compounds, and thus their first target. Mounting evidence indicates that the gut microbiota represents a major player in the toxicity of environmental pollutants and food additives; however, little is known on the toxicological relevance of the mucus/pollutant interplay, even though mucus is increasingly recognized as essential in gut homeostasis. Here, we aimed at describing how environmental pollutants (heavy metals, pesticides, and other persistent organic pollutants) and food additives (emulsifiers, nanomaterials) might interact with mucus and mucus-related microbial species; that is, “mucophilic” bacteria such as mucus degraders. This review highlights that intestinal mucus, either directly or through its crosstalk with the gut microbiota, is a key, yet underestimated gut player that must be considered for better risk assessment and management of environmental pollution. Full article
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Open AccessReview
Chemical Elicitors of Antibiotic Biosynthesis in Actinomycetes
Microorganisms 2018, 6(2), 52; https://doi.org/10.3390/microorganisms6020052 -
Abstract
Whole genome sequencing of actinomycetes has uncovered a new immense realm of microbial chemistry and biology. Most biosynthetic gene clusters present in genomes were found to remain “silent” under standard cultivation conditions. Some small molecules—chemical elicitors—can be used to induce the biosynthesis of
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Whole genome sequencing of actinomycetes has uncovered a new immense realm of microbial chemistry and biology. Most biosynthetic gene clusters present in genomes were found to remain “silent” under standard cultivation conditions. Some small molecules—chemical elicitors—can be used to induce the biosynthesis of antibiotics in actinobacteria and to expand the chemical diversity of secondary metabolites. Here, we outline a brief account of the basic principles of the search for regulators of this type and their application. Full article
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Open AccessArticle
Development of Versatile Vectors for Heterologous Expression in Bacillus
Microorganisms 2018, 6(2), 51; https://doi.org/10.3390/microorganisms6020051 -
Abstract
The discovery of new enzymes for industrial application relies on a robust discovery pipeline. Such a pipeline should facilitate efficient molecular cloning, recombinant expression and functional screening procedures. Previously, we have developed a vector set for heterologous expression in Escherichia coli. Here,
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The discovery of new enzymes for industrial application relies on a robust discovery pipeline. Such a pipeline should facilitate efficient molecular cloning, recombinant expression and functional screening procedures. Previously, we have developed a vector set for heterologous expression in Escherichia coli. Here, we supplement the catalogue with vectors for expression in Bacillus. The vectors are made compatible with a versatile cloning procedure based on type IIS restriction enzymes and T4 DNA ligase, and encompass an effective counter-selection procedure and complement the set of vectors with options for secreted expression. We validate the system with expression of recombinant subtilisins, which are generally challenging to express in a heterologous system. The complementarity of the E. coli and Bacillus systems allows rapid switching between the two commonly used hosts without comprehensive intermediate cloning steps. The vectors described are not limited to the expression of certain enzymes, but could also be applied for the expression of other enzymes for more generalized enzyme discovery or development. Full article
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Open AccessReview
Antifungal Peptides of the AFP Family Revisited: Are These Cannibal Toxins?
Microorganisms 2018, 6(2), 50; https://doi.org/10.3390/microorganisms6020050 -
Abstract
The emergence and spread of pathogenic fungi resistant to currently used antifungal drugs represents a serious challenge for medicine and agriculture. The use of smart antimicrobials, so-called “dirty drugs” which affect multiple cellular targets, is one strategy to prevent resistance. Of special interest
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The emergence and spread of pathogenic fungi resistant to currently used antifungal drugs represents a serious challenge for medicine and agriculture. The use of smart antimicrobials, so-called “dirty drugs” which affect multiple cellular targets, is one strategy to prevent resistance. Of special interest is the exploitation of the AFP family of antimicrobial peptides, which include its founding member AFP from Aspergillus giganteus. This latter is a highly potent inhibitor of chitin synthesis and affects plasma membrane integrity in many human and plant pathogenic fungi. A transcriptomic meta-analysis of the afp-encoding genes in A. giganteus and A. niger predicts a role for these proteins during asexual sporulation, autophagy, and nutrient recycling, suggesting that AFPs are molecules important for the survival of A. niger and A. giganteus under nutrient limitation. In this review, we discuss parallels which exist between AFPs and bacterial cannibal toxins and provide arguments that the primary function of AFPs could be to kill genetically identical siblings. We hope that this review inspires computational and experimental biologists studying alternative explanations for the nature and function of antimicrobial peptides beyond the general assumption that they are mere defense molecules to fight competitors. Full article
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Open AccessArticle
A Sensitive and Rapid Method to Determine the Adhesion Capacity of Probiotics and Pathogenic Microorganisms to Human Gastrointestinal Mucins
Microorganisms 2018, 6(2), 49; https://doi.org/10.3390/microorganisms6020049 -
Abstract
Mucus is the habitat for the microorganisms, bacteria and yeast that form the commensal flora. Mucins, the main macromolecules of mucus, and more specifically, the glycans that cover them, play essential roles in microbial gastrointestinal colonization. Probiotics and pathogens must also colonize mucus
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Mucus is the habitat for the microorganisms, bacteria and yeast that form the commensal flora. Mucins, the main macromolecules of mucus, and more specifically, the glycans that cover them, play essential roles in microbial gastrointestinal colonization. Probiotics and pathogens must also colonize mucus to have lasting positive or deleterious effects. The question of which mucin-harboured glycan motifs favour the adhesion of specific microorganisms remains very poorly studied. In the current study, a simple test based on the detection of fluorescent-labeled microorganisms raised against microgram amounts of mucins spotted on nitrocellulose was developed. The adhesion of various probiotic, commensal and pathogenic microorganisms was evaluated on a panel of human purified gastrointestinal mucins and compared with that of commercially available pig gastric mucins (PGM) and of mucins secreted by the colonic cancer cell line HT29-MTX. The latter two proved to be very poor indicators of adhesion capacity on intestinal mucins. Our results show that the nature of the sialylated cores of O-glycans, determined by MALDI MS-MS analysis, potentially enables sialic acid residues to modulate the adhesion of microorganisms either positively or negatively. Other identified factors affecting the adhesion propensity were O-glycan core types and the presence of blood group motifs. This test should help to select probiotics with enhanced adhesion capabilities as well as deciphering the role of specific mucin glycotopes on microbial adhesion. Full article
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Open AccessArticle
Comparing the Recombinant Protein Production Potential of Planktonic and Biofilm Cells
Microorganisms 2018, 6(2), 48; https://doi.org/10.3390/microorganisms6020048 -
Abstract
Recombinant protein production in bacterial cells is commonly performed using planktonic cultures. However, the natural state for many bacteria is living in communities attached to surfaces forming biofilms. In this work, a flow cell system was used to compare the production of a
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Recombinant protein production in bacterial cells is commonly performed using planktonic cultures. However, the natural state for many bacteria is living in communities attached to surfaces forming biofilms. In this work, a flow cell system was used to compare the production of a model recombinant protein (enhanced green fluorescent protein, eGFP) between planktonic and biofilm cells. The fluorometric analysis revealed that when the system was in steady state, the average specific eGFP production from Escherichia coli biofilm cells was 10-fold higher than in planktonic cells. Additionally, epifluorescence microscopy was used to determine the percentage of eGFP-expressing cells in both planktonic and biofilm populations. In steady state, the percentage of planktonic-expressing cells oscillated around 5%, whereas for biofilms eGFP-expressing cells represented on average 21% of the total cell population. Therefore, the combination of fluorometric and microscopy data allowed us to conclude that E. coli biofilm cells can have a higher recombinant protein production capacity when compared to their planktonic counterparts. Full article
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Open AccessReview
Polyionic Tags as Enhancers of Protein Solubility in Recombinant Protein Expression
Microorganisms 2018, 6(2), 47; https://doi.org/10.3390/microorganisms6020047 -
Abstract
Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for
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Since the introduction of recombinant protein expression in the second half of the 1970s, the growth of the biopharmaceutical field has been rapid and protein therapeutics has come to the foreground. Biophysical and structural characterisation of recombinant proteins is the essential prerequisite for their successful development and commercialisation as therapeutics. Despite the challenges, including low protein solubility and inclusion body formation, prokaryotic host systems and particularly Escherichia coli, remain the system of choice for the initial attempt of production of previously unexpressed proteins. Several different approaches have been adopted, including optimisation of growth conditions, expression in the periplasmic space of the bacterial host or co-expression of molecular chaperones, to assist correct protein folding. A very commonly employed approach is also the use of protein fusion tags that enhance protein solubility. Here, a range of experimentally tested peptide tags, which present specific advantages compared to protein fusion tags and the concluding remarks of these experiments are reviewed. Finally, a concept to design solubility-enhancing peptide tags based on a protein’s pI is suggested. Full article
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Open AccessReview
The Genus Wallemia—From Contamination of Food to Health Threat
Microorganisms 2018, 6(2), 46; https://doi.org/10.3390/microorganisms6020046 -
Abstract
The fungal genus Wallemia of the order Wallemiales (Wallemiomycotina, Basidiomycota) comprises the most xerotolerant, xerophilic and also halophilic species worldwide. Wallemia spp. are found in various osmotically challenged environments, such as dry, salted, or highly sugared foods, dry feed, hypersaline waters of solar
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The fungal genus Wallemia of the order Wallemiales (Wallemiomycotina, Basidiomycota) comprises the most xerotolerant, xerophilic and also halophilic species worldwide. Wallemia spp. are found in various osmotically challenged environments, such as dry, salted, or highly sugared foods, dry feed, hypersaline waters of solar salterns, salt crystals, indoor and outdoor air, and agriculture aerosols. Recently, eight species were recognized for the genus Wallemia, among which four are commonly associated with foods: W. sebi, W. mellicola, W. muriae and W. ichthyophaga. To date, only strains of W. sebi, W. mellicola and W. muriae have been reported to be related to human health problems, as either allergological conditions (e.g., farmer’s lung disease) or rare subcutaneous/cutaneous infections. Therefore, this allergological and infective potential, together with the toxins that the majority of Wallemia spp. produce even under saline conditions, defines these fungi as filamentous food-borne pathogenic fungi. Full article
Open AccessReview
Risk Mitigation for Immunocompromised Consumers of Mucormycete Spoiled and Fermented Foods: Germane Guidance and Remaining Needs
Microorganisms 2018, 6(2), 45; https://doi.org/10.3390/microorganisms6020045 -
Abstract
Mucoralean invasive fungal infections, while unusual among the general population, have a high mortality rate among immunocompromised individuals who become infected. They are also common spoilage organisms in cultured dairy products, some fresh produce, and baked goods. Additionally, Mucor and Rhizopus spp. are
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Mucoralean invasive fungal infections, while unusual among the general population, have a high mortality rate among immunocompromised individuals who become infected. They are also common spoilage organisms in cultured dairy products, some fresh produce, and baked goods. Additionally, Mucor and Rhizopus spp. are utilized in the production of traditional fermented foods including mold ripened cheeses and fermented soy products. The risk that consumption of these foods poses to immunocompromised consumers has been previously identified. However, actionable guidance on implementation of appropriate dietary restrictions and microbial specification targets for food manufacturers serving these populations is scarce and is limited by insufficient data regarding traceback analysis in cases of invasive fungal infections where food is the suspected transmission vector. Culture-dependent and molecular subtyping methods, including whole genome sequencing, will improve identification of the point source. In turn, the empirically determined information on root-cause can best direct the development of appropriate food safety policies and programs. Full article
Open AccessArticle
Binding of Helicobacter pylori to Human Gastric Mucins Correlates with Binding of TFF1
Microorganisms 2018, 6(2), 44; https://doi.org/10.3390/microorganisms6020044 -
Abstract
Helicobacter pylori binds to the gastric mucin, MUC5AC, and to trefoil factor, TFF1, which has been shown to interact with gastric mucin. We examined the interactions of TFF1 and H. pylori with purified gastrointestinal mucins from different animal species and from humans printed
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Helicobacter pylori binds to the gastric mucin, MUC5AC, and to trefoil factor, TFF1, which has been shown to interact with gastric mucin. We examined the interactions of TFF1 and H. pylori with purified gastrointestinal mucins from different animal species and from humans printed on a microarray platform to investigate whether TFF1 may play a role in locating H. pylori in gastric mucus. TFF1 bound almost exclusively to human gastric mucins and did not interact with human colonic mucins. There was a strong correlation between binding of TFF1 and H. pylori to human gastric mucins, and between binding of both TFF1 and H. pylori to gastric mucins with that of Griffonia simplicifolia lectin-II, which is specific for terminal non-reducing α- or β-linked N-acetyl-d-glucosamine. These results suggest that TFF1 may help to locate H. pylori in a discrete layer of gastric mucus and hence restrain their interactions with epithelial cells. Full article
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Open AccessBrief Report
Effect of UVC Radiation on Hydrated and Desiccated Cultures of Slightly Halophilic and Non-Halophilic Methanogenic Archaea: Implications for Life on Mars
Microorganisms 2018, 6(2), 43; https://doi.org/10.3390/microorganisms6020043 -
Abstract
Methanogens have been considered models for life on Mars for many years. In order to survive any exposure at the surface of Mars, methanogens would have to endure Martian UVC radiation. In this research, we irradiated hydrated and desiccated cultures of slightly halophilic
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Methanogens have been considered models for life on Mars for many years. In order to survive any exposure at the surface of Mars, methanogens would have to endure Martian UVC radiation. In this research, we irradiated hydrated and desiccated cultures of slightly halophilic Methanococcus maripaludis and non-halophilic Methanobacterium formicicum for various time intervals with UVC (254 nm) radiation. The survivability of the methanogens was determined by measuring methane concentrations in the headspace gas samples of culture tubes after re-inoculation of the methanogens into their growth-supporting media following exposure to UVC radiation. Hydrated M. maripaludis survived 24 h of UVC exposure, while in a desiccated condition they endured for 16 h. M. formicicum also survived UVC radiation for 24 h in a liquid state; however, in a desiccated condition, the survivability of M. formicicum was only 12 h. Some of the components of the growth media could have served as shielding agents that protected cells from damage caused by exposure to ultraviolet radiation. Overall, these results suggest that limited exposure (12–24 h) to UVC radiation on the surface of Mars would not necessarily be a limiting factor for the survivability of M. maripaludis and M. formicicum. Full article
Open AccessReview
Genetic Tools and Techniques for Recombinant Expression in Thermophilic Bacillaceae
Microorganisms 2018, 6(2), 42; https://doi.org/10.3390/microorganisms6020042 -
Abstract
Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to
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Although Escherichia coli and Bacillus subtilis are the most prominent bacterial hosts for recombinant protein production by far, additional species are being explored as alternatives for production of difficult-to-express proteins. In particular, for thermostable proteins, there is a need for hosts able to properly synthesize, fold, and excrete these in high yields, and thermophilic Bacillaceae represent one potentially interesting group of microorganisms for such purposes. A number of thermophilic Bacillaceae including B.methanolicus, B.coagulans, B.smithii, B.licheniformis, Geobacillus thermoglucosidasius, G. kaustophilus, and G. stearothermophilus are investigated concerning physiology, genomics, genetic tools, and technologies, altogether paving the way for their utilization as hosts for recombinant production of thermostable and other difficult-to-express proteins. Moreover, recent successful deployments of CRISPR/Cas9 in several of these species have accelerated the progress in their metabolic engineering, which should increase their attractiveness for future industrial-scale production of proteins. This review describes the biology of thermophilic Bacillaceae and in particular focuses on genetic tools and methods enabling use of these organisms as hosts for recombinant protein production. Full article
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Open AccessArticle
Regulation of Camphor Metabolism: Induction and Repression of Relevant Monooxygenases in Pseudomonas putida NCIMB 10007
Microorganisms 2018, 6(2), 41; https://doi.org/10.3390/microorganisms6020041 -
Abstract
For the first time, the differential rates of synthesis of all the key monooxygenases involved in the catabolism by Pseudomonas putida NCIMB 10007 of bicyclic (rac)-camphor to ∆2,5-3,4,4-trimethylpimelyl-CoA, the first aliphatic pathway intermediate, have been determined to help establish
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For the first time, the differential rates of synthesis of all the key monooxygenases involved in the catabolism by Pseudomonas putida NCIMB 10007 of bicyclic (rac)-camphor to ∆2,5-3,4,4-trimethylpimelyl-CoA, the first aliphatic pathway intermediate, have been determined to help establish the relevant induction profile of each of the oxygen-dependent enzymes. The efficacy of both relevant substrates and pathway metabolites as inducers has been established. Further, inhibitors with characterised functionality have been used to indicate that the pertinent regulatory controls operate at the level of transcription of the corresponding genes. Full article
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Open AccessReview
Pediatric Methicillin-Resistant Staphylococcus aureus Osteoarticular Infections
Microorganisms 2018, 6(2), 40; https://doi.org/10.3390/microorganisms6020040 -
Abstract
Osteoarticular infections (OSI) are a significant cause of hospitalizations and morbidity in young children. The pediatric patient with OSI presents unique challenges in diagnosis and management due to higher morbidity, effect on growth plate with associated long-lasting sequelae, and challenges in early identification
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Osteoarticular infections (OSI) are a significant cause of hospitalizations and morbidity in young children. The pediatric patient with OSI presents unique challenges in diagnosis and management due to higher morbidity, effect on growth plate with associated long-lasting sequelae, and challenges in early identification and management. Methicillin-resistant Staphylococcus aureus (MRSA), first described in the 1960s, has evolved rapidly to emerge as a predominant cause of OSI in children, and therefore empiric treatment for OSI should include an antibiotic effective against MRSA. Characterizing MRSA strains can be done by antimicrobial susceptibility testing, detection of Panton–Valentine leukocidin (PVL) gene, staphylococcal cassette chromosome mec (SCCmec) typing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Worldwide, community-onset methicillin-resistant staphylococcal disease is widespread and is mainly associated with a PVL-producing clone, ST8/USA300. Many studies have implied a correlation between PVL genes and more severe infection. We review MRSA OSI along with the pertinent aspects of its pathogenesis, clinical spectrum, diagnosis, and current guidelines for management. Full article
Open AccessReview
The Role of Glycans in Bacterial Adhesion to Mucosal Surfaces: How Can Single-Molecule Techniques Advance Our Understanding?
Microorganisms 2018, 6(2), 39; https://doi.org/10.3390/microorganisms6020039 -
Abstract
Bacterial adhesion is currently the subject of increased interest from the research community, leading to fast progress in our understanding of this complex phenomenon. Resent research within this field has documented the important roles played by glycans for bacterial surface adhesion, either through
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Bacterial adhesion is currently the subject of increased interest from the research community, leading to fast progress in our understanding of this complex phenomenon. Resent research within this field has documented the important roles played by glycans for bacterial surface adhesion, either through interaction with lectins or with other glycans. In parallel with this increased interest for and understanding of bacterial adhesion, there has been a growth in the sophistication and use of sensitive force probes for single-molecule and single cell studies. In this review, we highlight how the sensitive force probes atomic force microscopy (AFM) and optical tweezers (OT) have contributed to clarifying the mechanisms underlying bacterial adhesion to glycosylated surfaces in general and mucosal surfaces in particular. We also describe research areas where these techniques have not yet been applied, but where their capabilities appear appropriate to advance our understanding. Full article
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Open AccessReview
Comparison of Yeasts as Hosts for Recombinant Protein Production
Microorganisms 2018, 6(2), 38; https://doi.org/10.3390/microorganisms6020038 -
Abstract
Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts
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Recombinant protein production emerged in the early 1980s with the development of genetic engineering tools, which represented a compelling alternative to protein extraction from natural sources. Over the years, a high level of heterologous protein was made possible in a variety of hosts ranging from the bacteria Escherichia coli to mammalian cells. Recombinant protein importance is represented by its market size, which reached $1654 million in 2016 and is expected to reach $2850.5 million by 2022. Among the available hosts, yeasts have been used for producing a great variety of proteins applied to chemicals, fuels, food, and pharmaceuticals, being one of the most used hosts for recombinant production nowadays. Historically, Saccharomyces cerevisiae was the dominant yeast host for heterologous protein production. Lately, other yeasts such as Komagataella sp., Kluyveromyces lactis, and Yarrowia lipolytica have emerged as advantageous hosts. In this review, a comparative analysis is done listing the advantages and disadvantages of using each host regarding the availability of genetic tools, strategies for cultivation in bioreactors, and the main techniques utilized for protein purification. Finally, examples of each host will be discussed regarding the total amount of protein recovered and its bioactivity due to correct folding and glycosylation patterns. Full article
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Open AccessArticle
Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity
Microorganisms 2018, 6(2), 37; https://doi.org/10.3390/microorganisms6020037 -
Abstract
Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm
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Protease inhibitors have a broad biotechnological application ranging from medical drugs to anti-microbial agents. The Inga laurina trypsin inhibitor (ILTI) previously showed a great in vitro inhibitory effect under the adherence of Staphylococcus species, being a strong candidate for use as an anti-biofilm agent. Nevertheless, this is found in small quantities in its sources, which impairs its utilization at an industrial scale. Within this context, heterologous production using recombinant microorganisms is one of the best options to scale up the recombinant protein production. Thus, this work aimed at utilizing Komagataella phaffii to produce recombinant ILTI. For this, the vector pPIC9K+ILTI was constructed and inserted into the genome of the yeast K. phaffii, strain GS115. The protein expression was highest after 48 h using methanol 1%. A matrix-assisted laser desorption ionization–time-of-flight (MALDI–TOF) analysis was performed to confirm the production of the recombinant ILTI and its activity was investigated trough inhibitory assays using the synthetic substrate Nα-Benzoyl-D,L-arginine p-nitroanilide hydrochloride (BAPNA). Finally, recombinant ILTI (rILTI) was used in assays, showing that there was no significant difference between native and recombinant ILTI in its inhibitory activity in biofilm formation. Anti-tumor assay against Ehrlich ascites tumor (EAT) cells showed that rILTI has a potential anti-tumoral effect, showing the same effect as Melittin when incubated for 48 h in concentrations above 25 µg/mL. All together the results suggests broad applications for rILTI. Full article
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Open AccessReview
Genotypic Features of Clinical and Bovine Escherichia coli O157 Strains Isolated in Countries with Different Associated-Disease Incidences
Microorganisms 2018, 6(2), 36; https://doi.org/10.3390/microorganisms6020036 -
Abstract
There is great geographical variation in the frequency of Escherichia coli O157 infections that correlates with important differences in the bovine reservoir of each country. Our group carried out a broad molecular characterization of human and bovine E. coli O157 strains circulating in
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There is great geographical variation in the frequency of Escherichia coli O157 infections that correlates with important differences in the bovine reservoir of each country. Our group carried out a broad molecular characterization of human and bovine E. coli O157 strains circulating in Argentina using different methodologies. Our data allows us to conclude that in Argentina, a high homogeneity is observed in both cattle and human strains, with almost exclusive circulation of strains belonging to the hypervirulent clade 8 described by Manning. The aim of this review was to compare the genetic background of E. coli O157 strains isolated in countries that have conducted similar studies, to try to correlate specific O157 genotypes with the incidence and severity of E. coli O157 associated diseases. The characteristics of the strains that cause disease in humans reflect the predominant genotypes in cattle in each of the countries analyzed. The main features clearly linked to high incidence or severity of E. coli O157 infections are lineage-specific polymorphism assay-6 lineage I/II, clade 8 strains and probably, clade 6 strains, the stx2a/stx2c genotype, the presence of q933 and q21 simultaneously, and putative virulence factor EC_3286. In countries with an absence of these features in O157 strains, the overall incidence of O157 disease is low. Argentina, where these characteristics are detected in most strains, shows the highest incidence of hemolytic uremic syndrome (HUS) worldwide. Full article