Bacterial Extracts Research Articles

LC-MS/MS-based profiling of bioactive metabolites of endophytic bacteria from Cannabis sativa and their anti-Phytophthora activity.

Protection of crop plants from phytopathogens through endophytic bacteria is a newly emerged area of biocontrol. In this study, endophytic bacteria were isolated from the rhizosphere of Cannabis sativa. Based on initial antimicrobial screening, three (03) bacteria Serratia marcescens MOSEL-w2, Enterobacter cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 were selected. Antimicrobial assays of these selected bacteria against Phytophthora parasitica revealed that E. cloacae MOSEL-w7 and Paenibacillus sp. MOSEL-w13 possessed strong activity against P. parasitica. All these bacterial extracts showed strong inhibition against P. parasitica at different concentrations (4-400µgmL-1). P. parasitica hyphae treated with ethyl acetate extract of E. cloacae MOSEL-w7 resulted in severe growth abnormalities compared to control. The extracts were further evaluated for in vivo detached-leaf assay against P. parasitica on the wild type tobacco. Application of 1% ethyl acetate bacterial extract of S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus sp. MOSEL-w13 reduced P. parasitica induced lesion sizes and lesion frequencies by 60-80%. HPLC based fractions of each extract also showed bioactivity against P. parasitica. A total of 24 compounds were found in the S. marcescens MOSEL-w2, 15 compounds in E. cloacae MOSEL-w7 and 20 compounds found in Paenibacillus sp. MOSEL-w13. LC-MS/MS analyses showed different bioactive compounds in the bacterial extracts such as Cotinine (alkylpyrrolidine), L-tryptophan, L-lysine, L-Dopa, and L-ornithine. These results suggest that S. marcescens MOSEL-w2, E. cloacae MOSEL-w7, and Paenibacillus MOSEL-w13 are a source of bioactive metabolites and could be used in combination with other biocontrol agents, with other modes of action for controlling diseases caused by Phytophthora in crops. They could be a clue for the broad-spectrum biopesticides for agriculturally significant crops.

Novel mechanisms of testosterone detoxification in UGT2B17 gene deletion carriers and androgen activation by gut microbiome

Background and objectives Testosterone metabolism by the highly polymorphic UDP-glucuronosyltransferase 2B17 (UGT2B17) is widely recognized, but the metabolic fate of testosterone in the carriers of UGT2B17 gene deletion is not well understood. Similarly, testosterone glucuronide (TG) is prone to secretion into intestinal lumen (1); however, the role of microbiome in activation of testosterone is not studied. Therefore, the aims of this study were: i) to elucidate the alteration in the liver proteome and its effect on steroid metabolism pathways (i.e., metabolic switching) in the UGT2B17 gene deletion, and ii) to assess the TG deconjugation by gut β-glucuronidases. Methods Human liver S9 fraction with gene deletion and high expression (n=3) were used. Untargeted proteomics data was acquired using Easy nano-LC 1200 coupled with the high-resolution QE-HF Orbitrap. The proteomic data were analyzed, processed and pathway mapping was performed using MaxQuant, Perseus and STRING, respectively. In vitro studies in liver S9 fraction of UGT2B17 gene deletion were carried out to investigate testosterone metabolism by aldo-keto reductases (AKRs). The glucuronidation pathway of 5β-dihydrotestosterone (5β-DHT) and 5β-tetrahydrotestosterone (5β-THT) were identified using recombinant UGT2B isoforms. These data were verified using testosterone incubations in human hepatocyte and serum levels of 5β-THTG after oral testosterone dosing (800 mg) in human. Activation mechanism TG to testosterone in the human gut was investigated by incubating TG with recombinant human gut microbial β-glucuronidases and human fecal extracts. Results A total of 1002 liver proteins were quantified in the S9 fraction, of which 14 and 39 proteins were exclusively from the deletion and high-expressor groups, respectively, whereas 949 proteins were common. The following pathways were upregulated in UGT2B17 deletion group: oxidation-reduction, drug metabolic, steroid metabolic, and fatty acid metabolic process. Notable proteins that were upregulated in the gene deletion groups were: AKR1D1, AKR1C4, AKR7DA3, AKR1A1, DHCR7, ADH6, ADH1C, ALDH1A1, ALDH9A1, and ALDH5A1 A deeper molecular pathway analysis revealed that the AKR1D1 and AKR1C4 that metabolize testosterone to inactive 5β-DHT and 5β-THT, respectively (2) (Figure 1) were significantly upregulated in UGT2B17 deletion group. UGT2B7 is primarily responsible for glucuronidation of 5β-THT (Figure 1). Finally, incubation with purified bacterial β-glucuronidases and human fecal extracts confirmed facile but variable deconjugation of TG to testosterone in human gut. Conclusion Novel mechanisms of testosterone metabolic switching in UGT2B17 gene deletion and testosterone activation by gut microbiome have been identified. These findings have a potential to provide deeper insights into the mechanisms of testosterone associated pathophysiological conditions, such obesity, insulin sensitivity, and cancer. References Li, C. Y. et al. J Steroid Biochem Mol Biol (2019). Rižner et al. Steroids (2014).

The Distribution and Antibacterial Activity of Marine Sponge-Associated Bacteria in the Aegean Sea and the Sea of Marmara, Turkey.

The distribution and antibacterial activities of bacteria isolated from fifty-five marine sponge samples collected from the Aegean Sea and the Sea of Marmara were investigated in the period between 2011 and 2013. The antibacterial activities of the methanolic extracts of marine sponge-associated bacteria tested against six pathogenic bacteria: Staphylococcus aureus SA1 and SA2, Brucella melitensis B37, Vibrio vulnificus GK23, Escherichia coli O157:H7 (ATCC 35150), and Salmonella enterica subsp. enterica serovar typhi (ATCC 167), using disk diffusion tests and minimum inhibitory concentration technique. The bacteria isolated from sponges and ambient seawater samples were identified using VITEK 2 Compact 30 automated microbial identification system. All bacterial extracts were exhibited antibacterial activity with various MIC values ranging from 7.8mg/ mL to 1000mg/mL against all pathogenic bacteria tested. The antibacterial efficacy rates found to be higher in the Aegean Sea than the Sea of Marmara samples. Fifty-five sponge samples belonging to fifteen species and host twenty-two bacterial species belonging to seven classes in two different marine areas at varying rates were detected. The most common sponge-associated bacterium was recorded as Sphingomonas paucimobilis and Bacillus cereus in the Aegean Sea and the Sea of Marmara, respectively. The composition and counts of the sponge-associated bacteria were found significantly higher than the free-living bacteria in the ambient sea water sampling points of both two marine areas. The presence of high antibacterial potential of sponge-related bacteria obtained in this study provided data for further studies on marine-derived antimicrobial agents, including the effects of environmental differences.

The role of vaccine prophylaxis in the treatment of recurrent urinary tract infections in menopausal women

Recurrent urinary tract infections (RUTI) are one of the most common manifestations of the genitourinary menopausal syndrome and occur in more than 20% of peri- and post-menopausal women. The most common risk factor for RUTI in peri- and post-menopausal women is sex steroid deficiency. The urinary and genital tracts share a common embryonic origin due to the presence of α- and β-estrogen receptors, progesterone and androgen receptors in all structures of the urogenital tract: lower third of the ureters, urinary bladder, vascular plexus, urothelium, pelvic floor muscles, pelvic ligamentous apparatus. Patients with RUTI are treated in two stages: treatment of exacerbations and prevention of relapses. During exacerbations, short courses of antibiotic therapy are administered according to the sensitivity of the bacterial agent. If microbiological testing of urine samples is not possible, antibiotics are selected empirically, taking into account the most common pathogens of a UTI. Vaccine prophylaxis is the leading method of preventing a UTI recurrence. The efficacy and safety of E. coli bacterial extracts have been proven in numerous RCTs. The Uro-Vaxom vaccine reliably reduces the incidence of cystitis recurrence, reduces the need for antibacterial drugs, and therefore improves the quality of life of menopausal women with RUTIs. Vaccine prophylaxis in menopause can be administered as monotherapy or in combination with topical estrogen therapy, which also plays a positive role in the treatment of urinary tract infections.

Adsorption of urease as part of a complex protein mixture onto soil and its implications for enzymatic activity

Understanding adsorption of important proteins as part of a complex protein mixture onto surfaces such as soil is critical for numerous environmental applications. In this study, we investigated the adsorption of urease as part of a total bacterial protein extract from Sporosarcina pasteurii onto soil mixtures, including sand and silt, and the resulting enzymatic activity. Proteomics analysis of the proteomes revealed that larger proteins were preferentially adsorbed onto the soil surfaces regardless of the type of soil; the median molecular weight of the proteins over-represented in the supernatant and soil fractions were 29.4 kDa and 36.6 kDa, respectively. In addition, the majority of total ureases (86.2–97.2%) was present in the soil fractions of soil mixtures containing silt. Adsorbed urease exhibited enzymatic activity up to 0.2 U/mg protein in the soil fractions at a substrate concentration of 1.67 mM. Additionally, urease activity in the soil correlated with the total amount of protein adsorbed and the amounts of adsorbed urease. Although the soil-adsorbed urease retained measurable activity, an overall loss of activity was observed in all samples. Understanding the dynamics of protein adsorption onto soil surfaces can aid in improved designs for engineering applications that utilize enzymatic activities.

Bacterial cellulose-rambutan leaf extract (Nephelium lappaceum L.) composite: preparation and characterization

The purpose of this research is to produce bacterial cellulose-rambutan leaf extract (BC-RLE) composite. The BC-RLE composite was prepared by immersing BC gel into RLE under various immersion time of 7, 14 and 21 days with/without using UV light radiation, and then, its physical (water content, and swelling), mechanical (tensile and compressive strength) and structural (functional group and degree of crystallinity) properties were characterized. The results showed that the water content of BC-RLE composites was lower than that of pure BC gel and decreased as increased immersion time of BC gel in RLE. Further, the UV light radiation is given during immersion of BC gel in RLE also reduced the water content of BC-RLE composites. However, the swelling degree of BC-RLE composites increased as increased immersion time, and a similar tendency was also shown by applying the UV light radiation. Mechanical properties, tensile, and compressive strength of BC-RLE composites increased as increased immersion time and by applying UV light radiation. The FTIR results showed that there was no effect of UV light radiation on the structure of BC-RLE composites. The Degree of crystallinity of BC-RLE composite decreased by applying the UV light radiation during immersion time.

Development of an NMR-Based Platform for the Direct Structural Annotation of Complex Natural Products Mixtures.

The development of new "omics" platforms is having a significant impact on the landscape of natural products discovery. However, despite the advantages that such platforms bring to the field, there remains no straightforward method for characterizing the chemical landscape of natural products libraries using two-dimensional nuclear magnetic resonance (2D-NMR) experiments. NMR analysis provides a powerful complement to mass spectrometric approaches, given the universal coverage of NMR experiments. However, the high degree of signal overlap, particularly in one-dimensional NMR spectra, has limited applications of this approach. To address this issue, we have developed a new data analysis platform for complex mixture analysis, termed MADByTE (Metabolomics and Dereplication by Two-Dimensional Experiments). This platform employs a combination of TOCSY and HSQC spectra to identify spin system features within complex mixtures and then matches spin system features between samples to create a chemical similarity network for a given sample set. In this report we describe the design and construction of the MADByTE platform and demonstrate the application of chemical similarity networks for both the dereplication of known compound scaffolds and the prioritization of bioactive metabolites from a bacterial prefractionated extract library.

Enhanced Extracellular Synthesis of Gold Nanoparticles by Soluble Extracts from Escherichia coli Transformed with Rhizobium tropici Phytochelatin Synthase Gene

Phytochelatins, the enzymatic products of phytochelatin synthase, play a principal role in protecting the plants from heavy metal and metalloid toxicity due to their ability to scavenge metal ions. In the present study, we investigated the capacity of soluble intracellular extracts from E. coli cells expressing R. tropici phytochelatin synthase to synthesize gold nanoparticle. We discovered that the reaction mediated by soluble extracts from the recombinant E. coli cells had a higher yield of gold nanoparticles, compared to that from the control cells. The compositional and morphological properties of the gold nanoparticles synthesized by the intracellular extracts from recombinant cells and control cells were similar. In addition, this extracellular nanoparticle synthesis method produced purer gold nanoparticles, avoiding the isolation of nanoparticles from cellular debris when whole cells are used to synthesize nanoparticles. Our results suggested that phytochelatins can improve the efficiency of gold nanoparticle synthesis mediated by bacterial soluble intracellular extracts, and the potential of extracellular nanoparticle synthesis platform for the production of nanoparticles in large quantity and pure form is worth further investigation.

Application of Lactobacillus helveticus CNRZ 32 to Control the Microbial Contaminants in Cheese by Addition of Bacterial Extracts or by In Situ Production of Bioactive Metabolites

Microbial spoilage of cheese represents a major concern from both health and economic views. The addition of food preservatives is considered the most applied strategy to ensure food quality and to control microbial contamination. It well established that natural preservatives such as Nisin and Natamycin are of great effectiveness against a wide range of microbial concerns, but the lack of wide spectrum effect induces looking for more efficient alternatives. This research suggests alternative treatments to be evaluated side by side with both Nisin and Natamycin within real cheese models to control microbial contamination during the storage period. To evaluate this, two varieties of cheese were manufactured and inoculated with a set of pathogen and cheese spoiling microorganisms. Talaga cheese batches were separately treated with extract of MRS that previously fermented by Lactobacillus helveticus CNRZ 32, Nisin and Natamycin at Free State and Chitosan Nanoparticles-loaded state to become 6 treatments (T1 – T6) other than the control (C). The same treatments were applied to Karish cheese batches, in addition to inoculation of Lactobacillus helveticus CNRZ 32 1% (v/v) in the seventh batch. Upon microbiological analyses, results show that T2; the extract loaded on Chitosan Nanoparticles completely reduced the count of all pathogens and spoiling populations after two weeks of cold storage (2 – 6oC) in Talaga cheese. In the case of Karish cheese, the 7th batch treated with Lactobacillus helveticus CNRZ 32 inoculation had no pathogenic nor spoiler growth after one week of cold storage (2 – 6oC). These results suggest that Lactic acid bacteria especially Lactobacillus helveticus CNRZ 32 can efficiently ensure the safety and quality of cheese if applied in appropriate form.

Exploration of microbiome of medicinally important plants as biocontrol agents against Phytophthora parasitica.

In a preliminary plant-based microbiome study, diverse bacterial taxa were identified from different medicinal plants using 16S rRNA gene sequencing. Based on initial antimicrobial screening, eight (8) bacterial endophytes in six (6) different genera, Streptomyces, Pseudomonas, Enterobacter, Bacillus, Arthrobacter, and Delftia, from four important medicinal plants Dodonaea viscosa, Fagonia indica, Caralluma tuberculata, and Calendula arvensis were selected for further analyses. Antimicrobial assays revealed that Pseudomonas taiwanensis MOSEL-RD23 has strong anti-Phytophthora activity. Volatiles produced by P. taiwanensis MOSEL-RD23and Bacillus flexus MOSEL-MIC5 inhibited the growth of Phytophthora parasitica by more than 80%. Ethyl acetate extracts of Streptomyces alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, Enterobacter hormaechei MOSEL-FLS1, and Bacillus tequilensis MOSEL-FLS3, and Delftia lacustris MB322 displayed high potency against P. parasitica. All these bacterial extracts showed strong inhibition of more than 80% inhibition in vitro against P. parasitica at different concentrations (4-400µg/mL). Bacterial extracts showing strong antimicrobial activity were selected for bioactivity-driven fractionation and showed anti-Phytophthoral activity in multiple fractions and different peaks observed in UV-Vis spectroscopy. In the detached-leaf assay against P. parasitica on tobacco, 1% ethyl acetate bacterial extract of S. alboniger MOSEL-RD3, P. taiwanensis MOSEL-RD23, E. hormaechei MOSEL-FLS1, B. tequilensis MOSEL-FLS3, and D. lacustris MB322 reduced lesion sizes and lesion frequencies caused by P. parasitica by 68 to 81%. Overall, P. taiwanensis MOSEL-RD23 showed positive activities for all the assays. Analyzing the potential of bacterial endophytes as biological control agents can potentially lead to the formulation of broad-spectrum biopesticides for the sustainable production of crops.

ISOLATION AND CHARACTERIZATION OF PIGMENT PRODUCING BACTERIA AND THEIR POSSIBLE USE IN TEXTILE INDUSTRY

The environment where we live is the habitat for various microorganisms, mostly bacteria which are used for various industrial applications like enzyme production, pharmaceutical production, fabric manufacturing and more. Colorants are used in lots of products including cosmetics, food and textiles. These colorants are synthetic and very harmful for humans. It is very harmful to environment because it contain toxic and non-biodegradable compounds. Now a days there is a growing demand for natural dyes which can be biodegradable and less harmful for the humans. Industry have lots of interest in microbial pigments, because they are more stable and soluble than pigments produced from plants and animals sources. It also can grow rapidly which will produce high yield throughout the year. Carotenoid are the very important compounds and are widely used in textile industry. Most of the fruits and vegetables contain carotenoids. These pigments have some limitations which include solubility, sensibility and instability on exposure to light, temperature and pH. The aim of the present study was to isolate pigment producing bacteria from three different soil samples. In this study yellow and orange pigmented bacteria were isolated and developed in to pure culture. Morphological and biochemical characterization was carried out and these isolates were observed for maximun pigment production at diffrent pH, Temperature and NaCl Concentrations. Yellow and Orange colour bacterial extracts of carotenoids pigments were extracted and used as natural colorants for dying of cloth. The present study indicated that pigment production was influenced by physical factors such as pH, Temperature and NaCl. Absorbance of carotenoids pigments were tested by using UV-Visible spectroscopy and these pigments shows a promising result in textile industry as natural colorants.

Comparative Proteomic Analyses Between Biofilm-Forming and Non-biofilm-Forming Strains of Corynebacterium pseudotuberculosis Isolated From Goats.

Caseous lymphadenitis (CLA) is a chronic disease that affects small ruminants and causes economic losses in the associated breeding system. The causative agent of CLA is Corynebacterium pseudotuberculosis, a Gram-positive bacterium that exhibits tropism for external and internal lymph nodes and induces abscess formation in the host. Bacterial communities often produce a biofilm matrix that serves various functions, including protection against hostile environmental conditions, antibiotics, and the host immune response. Although biofilm formation has been reported for C. pseudotuberculosis, not all strains demonstrate this property in culture. In this work, we report the first comparative proteomic analysis of one biofilm-forming (CAPJ4) and one biofilm-non-forming strain (CAP3W) of C. pseudotuberculosis isolated from goats. Bacterial whole cell protein extracts were obtained for mass spectrometry analyses. Using LC-MS/MS, our studies reveal three and four proteins exclusively found in the CAPJ4 and CAP3W proteome, respectively. In addition, label-free quantitative analysis identified 40 proteins showing at-least 2-fold higher values in CAPJ4 compared CAP3W proteome Notably, CAPJ4 differentially synthesized the penicillin-binding protein, which participates in the formation of peptidoglycans. CAPJ4 also exhibited upregulation of N-acetylmuramoyl-L-alanine amidase and galactose-1-phosphate uridylyltransferase, which are involved in biofilm formation and exopolysaccharide biosynthesis. Here, we demonstrate that biofilm formation in C. pseudotuberculosis is likely associated with specific proteins, some of which were previously shown to be associated with virulence and biofilm formation in other organisms. Our findings may drive studies related to the bacterial mechanisms involved in the biofilm formation, in addition to providing targets for the treatment of CLA.

Whole genome sequencing of Streptomyces actuosus ISP-5337, Streptomyces sioyaensis B-5408, and Actinospica acidiphila B-2296 reveals secondary metabolomes with antibiotic potential

Streptomycetes are bacteria of biotechnological importance since they are avid producers of secondary metabolites, including antibiotics. Progress in genome mining has recently shown that Streptomyces species encode for many biosynthetic gene clusters which are mostly unexplored. Here, we selected three Actinomycetes species for whole genome sequencing that are known to produce potent thiopeptide antibiotics. Streptomyces actuosus biosynthesizes nosiheptide, Streptomyces sioyaensis produces siomycin, and Actinospica acidiphila is a member of the Actinomycete subfamily. Bioinformatic analyses demonstrated diverse secondary metabolomes with multiple antibiotic-encoding gene clusters. Detailed mass spectrometry analysis of metabolite extracts verified the active expression of nosiheptide and siomycin from S. actuosus and S. sioyaensis while fractionation of the bacterial extracts and subsequent challenge against Staphylococcus aureus demonstrated potent antibiotic activity of fractions containing these compounds. Whole genome sequencing of these species facilitates future bioengineering efforts for thiopeptides and characterization of relevant secondary metabolites.

Thiamine-producing lactic acid bacteria and their potential use in the prevention of neurodegenerative diseases.

Thiamine or vitamin B1, an essential micronutrient mainly involved in energy production, has a beneficial impact on the nervous system, and its deficiency can be associated with the development and progression of neurodegenerative diseases. The aim of this work was to select thiamine-producing lactic acid bacteria (LAB) and study their physiological effects using neuron cell cultures. In this study, 23 LAB able to produce thiamine were identified by growth in thiamine-free synthetic medium. Intra- and extracellular thiamine concentrations were determined using a microbiological method and results confirmed by HPLC techniques. A wide variation in vitamin production was found showing that this property was not only species specific but also a strain-dependent trait. Five of these strains were pre-selected for their capacity to produce higher concentrations of thiamine. Only the pre-treatment with the intracellular extract of Lactiplantibacillus (L.) plantarum CRL 1905 increased significantly neuronal survival in N2a cells' model of neurotoxicity (MPP+) with thiamine deficiency conditions (amprolium). Furthermore, amprolium-resistant variants of CRL 1905 were isolated by exposition of the strain to increasing concentrations of this toxic thiamine analogue. The variant A9 was able to increase more than 2 times the intracellular thiamine production of the original strain. A9 bacterial extract significantly prevented neuronal cell death and the increase of IL-6. The amprolium-resistant strain A9 showed a modulating and neuroprotective effect in an in vitro model of neurotoxicity constituting a potential bio-strategy to counteract thiamine deficiencies and thus prevent or treat neurodegenerative diseases. KEY POINTS: • LAB can produce thiamine in a species- and strain-dependant manner. • L. plantarum CRL 1905 significantly reduce MPP+-induced neurotoxicity in N2a cells. • Amprolium-resistant strain A9 has neuroprotective effect and prevents IL-6 increase.

Biopolymer production by halotolerant bacteria isolated from Caatinga biome.

Saline environments are extreme habitats with a high diversity of microorganisms source of a myriad of biomolecules. These microorganisms are assigned as extremophiles recognized to be producers of new natural compounds, which can be synthesized by helping to survive under harshness and extreme conditions. In Brazil, in the saline and semi-arid region of Areia Branca (Caatinga biome), halotolerant bacteria (able to growth at high NaCl concentrations) were isolated from rhizosphere of native plants Blutaparon portulacoides and Spergularia sp. and their biopolymer production was studied. A total of 25 bacterial isolates were identified at genus level based on 16S rRNA gene sequence analysis. Isolates were mainly Gram-positive bacteria from Bacillaceae, Staphylococcaceae, Microbacteriaceae, and Bacillales XII incertae sedis families, affiliates to Bacillus, Staphylococcus, Curtobacterium, and Exiguobacterium genera, respectively. One of the Gram-negative isolates was identified as member of the Pseudomonadaceae family, genus Pseudomonas. All the identified strains were halotolerant bacteria with optimum growth at 0.6-2.0M salt concentrations. Assays for biopolymer production showed that the halotolerant strains are a rich source of compounds as polyhydroxyalkanoates (PHA), biodegradable biopolymer, such as poly(3-hydroxybutyrate) (PHB) produced from low-cost substrates, and exopolysaccharides (EPS), such as hyaluronic acid (HA), metabolite of great interest to the cosmetic and pharmaceutical industry. Also, eight bacterial EPS extracts showed immunostimulatory activity, promising results that can be used in biomedical applications. Overall, our findings demonstrate that these biomolecules can be produced in culture medium with 0.6-2.0M NaCl concentrations, relevant feature to avoid costly production processes. This is the first report of biopolymer-producing bacteria from a saline region of Caatinga biome that showed important biological activities.

Process economics evaluation of cell-free synthesis for the commercial manufacture of antibody drug conjugates.

Continuous improvements of cell-free synthesis (CFS) systems have generated interest in adopting the technology for the manufacture of biologics. This paper provides an evaluation of the manufacturing cost-effectiveness of CFS for the commercial production of antibody-drug conjugates (ADCs). The evaluation was performed using an advanced techno-economic engine (TEE) built in Python. The TEE is programmed in an object-oriented environment capable of simulating a plethora of process flowsheets and predicting size and cost metrics for the process and the facility. A case study was formulated to compare the economics of whole bioprocesses based on either a CFS system or a mammalian cell system (CHO) for the manufacture of an ADC at a range of product demands. The analysis demonstrated the potential of CFS for the commercial manufacture of biologics and identified key cost drivers related to the system. The CFS system showed an approximately 80% increase in the cost of goods compared to CHO with a significant cost attributed to the in-house manufacture of the bacterial cell extract, necessary for the CFS reaction step in the process. A sensitivity and target analysis highlighted the need for further process improvements especially in the titer for the CFS process to become more competitive against well-established systems.

Synthetic mycobacterial diacyl trehaloses reveal differential recognition by human T cell receptors and the C-type lectin Mincle

The cell wall of Mycobacterium tuberculosis is composed of diverse glycolipids which potentially interact with the human immune system. To overcome difficulties in obtaining pure compounds from bacterial extracts, we recently synthesized three forms of mycobacterial diacyltrehalose (DAT) that differ in their fatty acid composition, DAT1, DAT2, and DAT3. To study the potential recognition of DATs by human T cells, we treated the lipid-binding antigen presenting molecule CD1b with synthetic DATs and looked for T cells that bound the complex. DAT1- and DAT2-treated CD1b tetramers were recognized by T cells, but DAT3-treated CD1b tetramers were not. A T cell line derived using CD1b-DAT2 tetramers showed that there is no cross-reactivity between DATs in an IFN-γ release assay, suggesting that the chemical structure of the fatty acid at the 3-position determines recognition by T cells. In contrast with the lack of recognition of DAT3 by human T cells, DAT3, but not DAT1 or DAT2, activates Mincle. Thus, we show that the mycobacterial lipid DAT can be both an antigen for T cells and an agonist for the innate Mincle receptor, and that small chemical differences determine recognition by different parts of the immune system.

Use of Capillary Zone Electrophoresis Coupled to Electrospray Mass Spectrometry for the Detection and Absolute Quantitation of Peptidoglycan-Derived Peptides in Bacterial Cytoplasmic Extracts.

Peptidoglycan (PGN) is an essential structure found in the bacterial cell wall. During the bacterial life cycle, PGN continuously undergoes biosynthesis and degradation to ensure bacterial growth and division. The resulting PGN fragments (muropeptides and peptides), which are generated by the bacterial autolytic system, are usually transported into the cytoplasm to be recycled. On the other hand, PGN fragments can act as messenger molecules involved in the bacterial cell wall stress response as in the case of β-lactamase induction in the presence of β-lactam antibiotic or in triggering mammalian innate immune response. During their cellular life, bacteria modulate their PGN degradation by their autolytic system or their recognition by the mammalian innate immune system by chemically modifying their PGN. Among these modifications, the amidation of the ε-carboxyl group of meso-diaminopimelic acid present in the PGN peptide chain is frequently observed. Currently, the detection and quantitation of PGN-derived peptides is still challenging because of the difficulty in separating these highly hydrophilic molecules by RP-HPLC as these compounds are eluted closely after the column void volume or coeluted in many cases. Here, we report the use of capillary zone electrophoresis coupled via an electrospray-based CE-MS interface to high-resolution mass spectrometry for the quantitation of three PGN peptides of interest and their amidated derivatives in bacterial cytoplasmic extracts. The absolute quantitation of the tripeptide based on the [13C,15N] isotopically labeled standard was also performed in crude cytoplasmic extracts of bacteria grown in the presence or absence of a β-lactam antibiotic (cephalosporin C). Despite the high complexity of the samples, the repeatability of the CZE-MS quantitation results was excellent, with relative standard deviations close to 1%. The global reproducibility of the method including biological handling was better than 20%.

Gut microbiota impact on the peripheral immune response in non-alcoholic fatty liver disease related hepatocellular carcinoma

The gut microbiota is reported to modulate the immune response in hepatocellular carcinoma (HCC). Here, we employ metagenomic and metabolomic studies to characterise gut microbiota in patients with non-alcoholic fatty liver disease (NAFLD) related cirrhosis, with or without HCC, and evaluate its effect on the peripheral immune response in an ex vivo model. We find that dysbiosis characterises the microbiota of patients with NAFLD-cirrhosis, with compositional and functional shifts occurring with HCC development. Gene function of the microbiota in NAFLD-HCC supports short chain fatty acid production, and this is confirmed by metabolomic studies. Ex vivo studies show that bacterial extracts from the NAFLD-HCC microbiota, but not from the control groups, elicit a T cell immunosuppressive phenotype, characterised by expansion of regulatory T cells and attenuation of CD8 + T cells. Our study suggest that the gut microbiota in NAFLD-HCC is characterised by a distinctive microbiome/metabolomic profile, and can modulate the peripheral immune response.

Suppression of Fusarium dry rot of potato using beneficial bacterial treatments

Fusarium dry rot affects up to 60% of potato tubers during storage. Pathogen resistance development to synthetic chemical fungicides is a main contributing factor. Previous work has demonstrated the use of bacterial antagonists against plant pathogens. In this study, bacteria isolated from disease suppressive composts were tested to evaluate their antagonistic activity against F. sambucinum in vitro, as well as to assess their ability to reduce Fusarium dry rot of potato tubers. To identify the potential implication of antifungal compounds in their inhibitory activity, bacterial extracts were produced and assayed against F. sambucinum mycelial growth and conidial germination. In vitro results showed that all tested bacteria suppressed F. sambucinum mycelial growth over six days of incubation. When testing the bacteria against potato dry rot, 30 of 32 bacteria reduced the symptoms of the disease at the higher bacterial concentration tested. The combined in vitro and in vivo studies indicated Bacillus subtilis B9–8, Pseudomonas moraviensis F9–6, Pseudomonas koreensis F9–9, and Pseudomonas gessardii M9–16 were the most effective strains. Extracts from these four bacteria were able to inhibit mycelial growth and/or conidial germination of the fungal mold. Results from this study suggests that multiple antagonistic bacteria may be useful in controlling Fusarium dry rot on potato tubers. Extracts from the four most effective bacteria indicated that antibiosis was a main mode of action against F. sambucinum.