Nádia Skorupa Parachin

New edge of antibiotic development: antimicrobial peptides and corresponding resistance

In the last years severe efforts turned into extensive research on development of novel antimicrobial compounds. Among them, antimicrobial peptides, commonly isolated from several organisms, have been considered part of innate immune system and also as potential antimicrobial drugs. AMPs have variable amino acid composition and size (ranging from less than 5–100 amino acid residues), commonly showing cationic and amphipathic properties. Nowadays about 2300 AMPs have been reported in the Antimicrobial Peptide Database (AMP database). Such research endeavor resulted in more than 100 peptide-based drugs available in the market with approximately 500–600 candidates in pre-clinical test development (Craik et al., 2013). Therefore the current issue encloses several aspects when studying within antimicrobial peptides field. Besides its antimicrobial activity, some AMPs also have antifungal activity, inmmunomodulatory and antitumural activities. For example defensins commonly found in plants, fungi, insect and mammalian cells are small cationic peptides of 45–54 amino acid residues with a conserved signature of cysteines, which can form three to four disulfide bridges. Those have been shown to have inmmunomodulatory activities that can be used as vaccine adjuvants or in the treatment of immune-depressed patients. Therefore, a better understanding of function and mechanism of action of such molecules is a great promise in anti-infective and immunomodulatory therapeutics (Silva et al., 2014). Defensins isolated from plants also have been considered as a biotechnological tool to improve crop production as recently reviewed (Lacerda et al., 2014). Among their advantages low effective fungicide concentrations and decreased environmental impact have been focused when compared to most common chemicals utilized for fungi control. Therefore many studies have focused in construction of genetic modified plants that over produce such molecules in order to reduce crop losses by fungi infection. Nevertheless it is important to emphasize that although there are many studies occurring at bench scale, indicating that genetic modified plants over producing defensins should appear in the market within the next years (Lacerda et al., not published). It is also know that antimicrobial peptides have other potential applications since some of them have also antitumoral and imunomodulator activities. Also know as anticancer peptides (ACP) and host defense peptides (HDP) they also represent important candidates as novel drugs and have been carefully addressed in the current issue. For instance, cationic peptides have been considered as anticancer agents for presenting numerous advantages over chemical agents such as higher specific cytotoxicity to tumor cells, lower side effects and easier absorption as recently listed (Mulder et al., 2013). Diverse studies have shown that cancer cells are more anionic than normal cells. Due this property, cationic AMPs seems to bind it faster and selectively resulting in cell death. A better understanding of ACP mechanism of action may result in novel pharmacies with optimized anticancer activity (Gaspar et al., 2013). Despite the great number of peptides available in databases, isolation of new molecules using classic purification techniques is crucial to identify novel molecules with diverse activities. Indeed a proteinase inhibitor isolated from J. curcas seed cake, named JcTI-I, was shown to have a potent activity against the human pathogenic bacteria S. aureus and S. enterica. Moreover it did present other relevant pharmacologically characteristics such as absence of hemolytic activity against human erythrocytes concomitant to pH and high salt concentration resistance (Costa et al., 2014). Moreover, microorganism have also been focused in this issue were the isolation and characterization of lipopeptide from Bacillus sp. were performed. This lipopetide shows strong fungicide activity (specially when this compound was self-assembled) (Roy et al., 2013). Lately not only nature has become a source of AMPs. Besides isolation of natural organisms, antimicrobial peptides might be improved or created using computational tools. This opens even more this so amazing field by creating infinite novel and remarkable possibilities. Recently a study screened the distribution of known motifs in prokaryotic extracellular and virulence proteins across a range of bacterial species in order to identify novel motifs in virulence proteins (Ruhanen et al., 2014). Such methodologies are able to generate thousands of novel molecules that require high-throughput in vitro and in vivo validation. In this sense it is also necessary to develop rapid assays that can be performed concomitant to million candidates. A recent method enabled the selection of AMPs directly on peptide microarrays allowing identification of AMPs that bind and are bactericide among those that bind but do not kill bacteria (Wimley, 2010). In another example, an affinity support might be applied for the isolation of AMPs that interact with lipopolysaccharides, the major cause of septic shock (Lopez-Abarrategui et al., 2013). Thus advances in peptide array discovery assays could provide a system to develop pathogen-specific antibiotics resulting in the discovery of target antibiotics (Diehnelt, 2013). In alignment with in silico models, several systemic strategies such as transcriptome and proteome have been utilized for understanding peptide interaction with its target, which may allow improvement or design new molecule properties (Tavares et al., 2013). These novel approaches could lead us to understand several compounds at the same time which may result in a rapid increased in AMP peptides available in the market for treating several diseases. Finally it is fundamental to understand mechanism of action of AMPs in order to improve its activity and predict possible resistance mechanisms. Although many AMP alters membrane permeability there are other action mechanisms such as synthesis inhibition of cell wall, protein synthesis or nucleic acids as reviewed in this issue (Guilhelmelli et al., 2013). Moreover it is also essential to clear elucidate host-pathogen relationship. A recent study hypothesized that Helmint defense peptides played a critical role in parasite interaction with its host (Robinson et al., 2013). This may be applied as immunomodulators agents since those molecules are able to interact with the host without cytotoxic or cytolytic effects. Overall the current issue highlights the relevance of such research topic with perspectives to develop entirely new molecules with vast application within health and agricultural field with c higher affinity for its target with concomitant reduction of side effects.

Genetic basis for hyper production of hyaluronic acid in natural and engineered microorganisms

Hyaluronic acid, or HA, is a rigid and linear biopolymer belonging to the class of the glycosaminoglycans, and composed of repeating units of the monosaccharides glucuronic acid and N-acetylglucosamine. HA has multiple important functions in the human body, due to its properties such as bio-compatibility, lubricity and hydrophilicity, it is widely applied in the biomedical, food, health and cosmetic fields. The growing interest in this molecule has motivated the discovery of new ways of obtaining it. Traditionally, HA has been extracted from rooster comb-like animal tissues. However, due to legislation laws HA is now being produced by bacterial fermentation using Streptococcus zooepidemicus, a natural producer of HA, despite it being a pathogenic microorganism. With the expansion of new genetic engineering technologies, the use of organisms that are non-natural producers of HA has also made it possible to obtain such a polymer. Most of the published reviews have focused on HA formulation and its effects on different body tissues, whereas very few of them describe the microbial basis of HA production. Therefore, for the first time this review has compiled the molecular and genetic bases for natural HA production in microorganisms together with the main strategies employed for heterologous production of HA.

Identification and characterization of putative xylose and cellobiose transporters in Aspergillus nidulans

BackgroundThe conversion of lignocellulosic biomass to biofuels (second-generation biofuel production) is an environmentally friendlier alternative to petroleum-based energy sources. Enzymatic deconstruction of lignocellulose, catalyzed by filamentous fungi such as Aspergillus nidulans, releases a mixture of mono- and polysaccharides, including hexose (glucose) and pentose (xylose) sugars, cellodextrins (cellobiose), and xylooligosaccharides (xylobiose). These sugars can subsequently be fermented by yeast cells to ethanol. One of the major drawbacks in this process lies in the inability of yeast, such as Saccharomyces cerevisiae, to successfully internalize sugars other than glucose. The aim of this study was, therefore, to screen the genome of A. nidulans, which encodes a multitude of sugar transporters, for transporters able to internalize non-glucose sugars and characterize them when introduced into S. cerevisiae.ResultsThis work identified two proteins in A. nidulans, CltA and CltB, with roles in cellobiose transport and cellulose signaling, respectively. CltA, when introduced into S. cerevisiae, conferred growth on low and high concentrations of cellobiose. Deletion of cltB resulted in reduced growth and extracellular cellulase activity in A. nidulans in the presence of cellobiose. CltB, when introduced into S. cerevisiae, was not able to confer growth on cellobiose, suggesting that this protein is a sensor rather than a transporter. However, we have shown that the introduction of additional functional copies of CltB increases the growth in the presence of low concentrations of cellobiose, strongly indicating CltB is able to transport cellobiose. Furthermore, a previously identified glucose transporter, HxtB, was also found to be a major xylose transporter in A. nidulans. In S. cerevisiae, HxtB conferred growth on xylose which was accompanied by ethanol production.ConclusionsThis work identified a cellobiose transporter, a xylose transporter, and a putative cellulose transceptor in A. nidulans. This is the first time that a sensor role for a protein in A. nidulans has been proposed. Both transporters are also able to transport glucose, highlighting the preference of A. nidulans for this carbon source. This work provides a basis for future studies which aim at characterizing and/or genetically engineering Aspergillus spp. transporters, which, in addition to glucose, can also internalize other carbon sources, to improve transport and fermentation of non-glucose sugars in S. cerevisiae.

Critical Aspects to be Considered Prior to Large-Scale Production of Peptides

Numerous peptides are available on the market as therapeutic drugs for regulating tumor growth, microorganism proliferation, immune response and/or metabolic disorders. Peptides are produced either by chemical synthesis or heterologous expression. Independent of the method chosen, there are challenges to transferring its production from the bench (~mg/L) to the industrial (~g/L) scale. Thus, the main scale-up pitfalls for the two methods of peptide production are reviewed here, including the advantages of each. Moreover, there will be a special focus on the main challenges for large-scale, heterologous production systems. Peptides that are currently available on the market are also described with an emphasis on how their process optimization has been designed in order to develop a cost-effective product.

Production of a polar fish antimicrobial peptide in Escherichia coli using an ELP-intein tag

An important aspect related to infectious pathogens is their exceptional adaptability in developing resistance, which leads to a perpetual challenge in the discovery of antimicrobial drugs with novel mechanisms of action. Among them, antimicrobial peptides (AMPs) stand out as promising anti-infective molecules. In order to overcome the high costs associated with isolation from natural sources or chemical synthesis of AMPs we propose the expression of Pa-MAP 2, a polyalanine AMP. Pa-MAP 2 was fused to an ELP-intein tag where the ELP (Elastin-like polypeptide) was used to promote aggregation and fast and cost-effective isolation after expression, and the intein was used to stimulate a controlled AMP release. For these, the vector pET21a was used to produce Pa-MAP 2 fused to the N-termini region of a modified Mxe GyrA intein followed by 60 repetitions of ELP. Purified Pa-MAP 2 showed a MIC of 25μM against E. coli ATCC 8739. Batch fermentation demonstrated that Pa-MAP-2 can be produced in both rich and defined media at yields 50-fold higher than reported for other AMPs produced by the ELP-intein system, and in comparable yields to expression systems with protease or chemical cleavage.

Antibiotic combinations for controlling colistin-resistant Enterobacter cloacae

Enterobacter cloacae is a Gram-negative bacterium associated with high morbidity and mortality in intensive care patients due to its resistance to multiple antibiotics. Currently, therapy against multi-resistant bacteria consists of using colistin, in spite of its toxic effects at higher concentrations. In this context, colistin-resistant E. cloacae strains were challenged with lower levels of colistin combined with other antibiotics to reduce colistin-associated side effects. Colistin-resistant E. cloacae (ATCC 49141) strains were generated by serial propagation in subinhibitory colistin concentrations. After this, three colistin-resistant and three nonresistant replicates were isolated. The identity of all the strains was confirmed by MALDI-TOF MS, VITEK 2 and MicroScan analysis. Furthermore, cross-resistance to other antibiotics was checked by disk diffusion and automated systems. The synergistic effects of the combined use of colistin and chloramphenicol were observed via the broth microdilution checkerboard method. First, data here reported showed that all strains presented intrinsic resistance to penicillin, cephalosporin (except fourth generation), monobactam, and some associations of penicillin and β-lactamase inhibitors. Moreover, a chloramphenicol and colistin combination was capable of inhibiting the induced colistin-resistant strains as well as two colistin-resistant clinical strains. Furthermore, no cytotoxic effect was observed by using such concentrations. In summary, the data reported here showed for the first time the possible therapeutic use of colistin–chloramphenicol for infections caused by colistin-resistant E. cloacae.

Sugarcane Bagasse Hydrothermal Pretreatment Liquors as Suitable Carbon Sources for Hemicellulase Production by Aspergillus niger

The aim of this study was to valorize the hemicellulose-rich liquid fraction (liquor) arising from hydrothermal pretreatment of sugarcane bagasse (SCB) through its utilization as an unconventional, soluble carbon source for the production of hemicellulases, namely xylanases and α-L-arabinofuranosidases (ABFases), by Aspergillus niger DCFS11. Through the use of factorial design, pretreatment conditions producing liquors optimized for either early- or late-phase enzyme production were identified. Subsequent deep characterization of liquor components using liquid chromatography and mass spectrometry was performed to identify compounds likely responsible for hemicellulase induction. SCB liquors arising from various pretreatment configurations induced up to 2- and 8.6-fold higher xylanase and ABFase production, respectively, by A. niger DCFS11 than raw SCB substrate owing to the strong inducing potential of arabinosylated xylooligosaccharides and free arabinose solubilized during pretreatment. Notably, unlike the severe pretreatment conditions required for maximum cellulose saccharification and ethanol yields during biomass conversion, low severity and low biomass loading are required if enzyme production from liquor is desired at early-phase growth with no additional detoxification steps. This suggests that for effective application in biorefineries, separate or multi-step processes would be required to optimize both hemicellulase production by A. niger DCFS11 and cellulose digestion. This work demonstrates the potential of hydrothermal pretreatment of lignocellulosic substrates as a tool to increase the production of enzymes by filamentous fungi.

Comparison of Yeasts as Hosts for Recombinant Protein Production

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

Recombinant Inga Laurina Trypsin Inhibitor (ILTI) Production in Komagataella Phaffii Confirms Its Potential Anti-Biofilm Effect and Reveals an Anti-Tumoral Activity

1654 million in 2016 and is expected to reach

Effect of Pyruvate Decarboxylase Knockout on Product Distribution Using Pichia pastoris (Komagataella phaffii) Engineered for Lactic Acid Production

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.