Microcin MccI47 selectively inhibits enteric bacteria and reduces carbapenem-resistant Klebsiella pneumoniae colonization in vivo when administered via an engineered live biotherapeutic

Background: The gastrointestinal (GI) tract is the reservoir for multidrug-resistant (MDR) pathogens, specifically carbapenem-resistant (CR) Klebsiella pneumoniae and other Enterobacteriaceae, which often lead to the spread of antimicrobial resistance genes, severe extraintestinal infections, and lethal outcomes. Selective GI decolonization has been proposed as a new strategy for preventing transmission to other body sites and minimizing spreading to susceptible individuals. Results: Here, we purify the to-date uncharacterized class IIb microcin I47 (MccI47) and demonstrate potent inhibition of numerous Enterobacteriaceae, including MDR clinical isolates, in vitro at concentrations resembling those of commonly prescribed antibiotics. We then genetically modify the probiotic bacterium Escherichia coli Nissle 1917 (EcN) to produce MccI47 from a stable multicopy plasmid by using MccI47 toxin production in a counterselection mechanism to engineer one of the native EcN plasmids, which renders provisions for inducible expression and plasmid selection unnecessary. We then test the clinical relevance of the MccI47-producing engineered EcN in a murine CR K. pneumoniae colonization model and demonstrate significant MccI47-dependent reduction of CR K. pneumoniae abundance after seven days of daily oral live biotherapeutic administration without disruption of the resident microbiota. Conclusions: This study provides the first demonstration of MccI47 as a potent antimicrobial against certain Enterobacteriaceae, and its ability to significantly reduce the abundance of CR K. pneumoniae in a preclinical animal model, when delivered from an engineered live biotherapeutic product. This study serves as the foundational step towards the use of engineered live biotherapeutic products aimed at the selective removal of MDR pathogens from the GI tract.

Tryptophan Production Maximization in a Fed-Batch Bioreactor with Modified E. coli Cells, by Optimizing Its Operating Policy Based on an Extended Structured Cell Kinetic Model

Hybrid kinetic models, linking structured cell metabolic processes to the dynamics of macroscopic variables of the bioreactor, are more and more used in engineering evaluations to derive more precise predictions of the process dynamics under variable operating conditions. Depending on the cell model complexity, such a math tool can be used to evaluate the metabolic fluxes in relation to the bioreactor operating conditions, thus suggesting ways to genetically modify the microorganism for certain purposes. Even if development of such an extended dynamic model requires more experimental and computational efforts, its use is advantageous. The approached probative example refers to a model simulating the dynamics of nanoscale variables from several pathways of the central carbon metabolism (CCM) of E. coli cells, linked to the macroscopic state variables of a fed-batch bioreactor (FBR) used for the tryptophan (TRP) production. The used E. coli strain was modified to replace the PTS system for glucose (GLC) uptake with a more efficient one. The study presents multiple elements of novelty: (i) the experimentally validated modular model itself, and (ii) its efficiency in computationally deriving an optimal operation policy of the FBR.

The DNA–carbon nanotube binding mode determines the efficiency of carbon nanotube-mediated DNA delivery to intact plants

Efficient delivery of DNA, RNA, and genome engineering machinery to plant cells will enable efforts to genetically modify plants for global food security, sustainable energy production, synthetic biology applications, and climate change resilience. For the delivery of functional genetic units into plant cells, charged nanoparticles, particularly carbon nanotubes (CNT), have attracted considerable interest. Although some success has been achieved using CNT-based approaches, the efficiency, batch reproducibility, and the limits of their applicability remain to be assessed. Here, we provide a mechanistic understanding of plasmid DNA-loaded CNTs based transfection of plant cells, and factors affecting the expression of the transformed plasmid. We show that transfection is inherently limited by the presence of the cell wall, Coulomb interactions between DNA and polymer coated CNT, and DNA size, whereas expression of the transformed plasmid is limited by relative gene-to-plasmid size and the intracellular accessibility of DNA. We further show that the formation of partially condensed DNA on the CNT surface is a prerequisite for successful transfection and expression. Furthermore, DNA does not detach completely from the CNT, so the accessibility of the transcription machinery to DNA is the key for transformation efficiency. This irreversible DNA plasmid binding and partial condensation limit the length of DNA that can be expressed, thus negatively affecting efficiency and reproducibility. Understanding the underlying mechanisms and limitations of CNT-mediated delivery of DNA through the plant cell wall is of considerable importance in guiding efforts to design nanomaterials for efficient transformation, trait engineering, and synthetic biology applications.

The long and winding road of reverse genetics in Trypanosoma cruzi

Trypanosomes are early divergent protists with distinctive features among eukaryotic cells. Together with Trypanosoma brucei and Leishmania spp., Trypanosoma cruzi has been one of the most studied members of the group. This protozoan parasite is the causative agent of Chagas disease, a leading cause of heart disease in the Americas, for which there is no vaccine or satisfactory treatment available. Understanding T. cruzi biology is crucial to identify alternative targets for antiparasitic interventions. Genetic manipulation of T. cruzi has been historically challenging. However, the emergence of CRISPR/Cas9 technology has significantly improved the ability to generate genetically modified T. cruzi cell lines. Still, the system alone is not sufficient to answer all biologically relevant questions. In general, current genetic methods have limitations that should be overcome to advance in the study of this peculiar parasite. In this brief historic overview, we highlight the strengths and weaknesses of the molecular strategies that have been developed to genetically modify T. cruzi, emphasizing the future directions of the field.

The Synergistic Effects of Rhamnolipids and Antibiotics Against Bacteria

Antibiotics are used to combat bacterial infections by slowing down and preventing the proliferation of bacteria. Antibiotic resistance is a threat to human health, attributed to its overuse and misuse. Altering the membrane permeability to induce antibiotic uptake may be an effective strategy used against both Gram-positive and Gram-negative infectious bacteria. Rhamnolipids (RLs) are biosurfactants produced by Pseudomonas aeruginosa. RLs surface-active properties operate by creating holes in bacterial cell membranes, increasing target cell permeability; allowing antibiotics to penetrate the cell. Rhamnolipids enhance the effect of antibiotics by targeting the intracellular machinery of bacteria. This project tested the susceptibility of bacteria when exposed to antibiotics with and without the addition of RLs, to quantitatively determine if RLs increase antibiotic potency. By analyzing the zones of inhibition data, the results demonstrated that RLs potentiated the antibiotics. Notably, kanamycin coupled with RLs had the most effect inhibiting bacterial growth. To further assess rhamnolipid biosynthesis, a BLAST search was performed exclusively on two genes, rhlA and rhlB. These genes code for the production of two proteins necessary for rhamnolipids. The search indicated a 48% correlation with putative proteins found in Burkholderia pseudomallei. Therefore, based on the experimental results and the BLAST analysis, further research should be conducted to explore the possible role of using rhamnolipids as antibiotic enhancers. Specifically, future experiments could focus on isolating the putative proteins of B.pseudomallei to genetically modify E.coli. Furthermore, isolated studies analyzing the genes of proteins to determine their role in the pathogenicity of Burkholderia species.

Recent achievements in CAR-T cell immunotherapy for glioblastoma treatment

Glioblastoma remains the most common and aggressive primary brain tumor today. Because of the neuroanatomical location of glioblastoma, conventional chemotherapy and radiation therapy have limited efficacy in patients with these tumors. Over the past decade, antitumor immunotherapy has become widespread among modern therapeutic approaches. The importance of immunotherapeutic methods lies in their ability to increase the effectiveness of cancer treatment and prevent relapses by enhancing the systemic and local immune response against tumor cells.One of the most promising directions in modern immunotherapy is CAR-T therapy, or adoptive cell therapy using genetically modified T-lymphocytes. The functional advantage of CAR-T therapy is its ability to genetically modify lymphocytes, leading to their activation in vitro.This review examines the key principles of CAR-T therapy and analyzes the published results of clinical trials for the treatment of glioblastoma using several modifications of CAR-T cells.

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube

Key message Biolistic delivery into pollen. Abstract In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.

Phytosterols and Novel Triterpenes Recovered from Industrial Fermentation Coproducts Exert In Vitro Anti-Inflammatory Activity in Macrophages

The unstoppable growth of human population that occurs in parallel with all manufacturing activities leads to a relentless increase in the demand for resources, cultivation land, and energy. In response, currently, there is significant interest in developing strategies to optimize any available resources and their biowaste. While solutions initially focused on recovering biomolecules with applications in food, energy, or materials, the feasibility of synthetic biology in this field has been demonstrated in recent years. For instance, it is possible to genetically modify Saccharomyces cerevisiae to produce terpenes for commercial applications (i.e., against malaria or as biodiesel). But the production process, similar to any industrial activity, generates biowastes containing promising biomolecules (from fermentation) that if recovered may have applications in different areas. To test this hypothesis, in the present study, the lipid composition of by-products from the industrial production of β-farnesene by genetically modified Saccharomyces cerevisiae are studied to identify potentially bioactive compounds, their recovery, and finally, their stability and in vitro bioactivity. The assayed biowaste showed the presence of triterpenes, phytosterols, and 1-octacosanol which were recovered through molecular distillation into a single fraction. During the assayed stability test, compositional modifications were observed, mainly for the phytosterols and 1-octacosanol, probably due to oxidative reactions. However, such changes did not affect the in vitro bioactivity in macrophages, where it was found that the obtained fraction decreased the production of TNF-α and IL-6 in lipopolysaccharide (LPS)-induced inflammation.

Development of the myzozoan aquatic parasite Perkinsus marinus as a versatile experimental genetic model organism

The phylum Perkinsozoa is an aquatic parasite lineage that has devastating effects on commercial and natural mollusc populations, and also comprises parasites of algae, fish and amphibians. They are related to, and share much of their biology with, dinoflagellates and apicomplexans and thus offer excellent genetic models for both parasitological and evolutionary studies. Genetic transformation has been previously achieved for select Perkinsus spp. but with few tools for transgene expression and only limited selection efficacy. We thus sought to expand the power of experimental genetic tools for Perkinsus marinus — the principal perkinsozoan model to date. We constructed a modular plasmid assembly system that enables expression of multiple genes simultaneously. We developed an efficient selection system for three drugs, puromycin, bleomycin and blasticidin, that achieves transformed cell populations in as little as three weeks. We developed and quantified eleven new promoters of variable expression strength. Furthermore, we identified that genomic integration of transgenes is predominantly via non-homologous recombination and often involves transgene fragmentation including deletion of some introduced elements. To counter these dynamic processes, we show that bi-cistronic transcripts using the viral 2A peptides can couple selection systems to the maintenance of the expression of a transgene of interest. Collectively, these new tools and insights provide new capacity to efficiently genetically modify and study Perkinsus as an aquatic parasite and evolutionary model.

Co-production of polyhydroxybutyrate (PHB) and coenzyme Q10 (CoQ10) via no-sugar fermentation—a case by Methylobacterium sp. XJLW

Purpose To explore a competitive PHB-producing fermentation process, this study evaluated the potential for Methylobacterium sp. XJLW to produce simultaneously PHB and coenzyme Q10 (CoQ10) using methanol as sole carbon and energy source. Methods The metabolic pathways of PHB and CoQ10 biosynthesis in Methylobacterium sp. XJLW were first mined based on the genomic and comparative transcriptomics information. Then, real-time fluorescence quantitative PCR (RT-qPCR) was employed for comparing the expression level of important genes involved in PHB and CoQ10 synthesis pathways’ response to methanol and glucose. Transmission electron microscope (TEM), gas chromatography/mass spectrometry (GC-MS), nuclear magnetic resonance (NMR), Fourier transformation infrared spectrum (FT-IR), and liquid chromatography/mass spectrometry (LC-MS) methods were used to elucidate the yield and structure of PHB and CoQ10, respectively. PHB and CoQ10 productivity of Methylobacterium sp. XJLW were evaluated in Erlenmeyer flask for medium optimization, and in a 5-L bioreactor for methanol fed-batch strategy according to dissolved oxygen (DO) and pH control. Results Comparative genomics analysis showed that the PHB and CoQ10 biosynthesis pathways co-exist in Methylobacterium sp. XJLW. Transcriptomics analysis showed that the transcription level of key genes in both pathways responding to methanol was significantly higher than that responding to glucose. Correspondingly, strain Methylobacterium sp. XJLW can produce PHB and CoQ10 simultaneously with higher yield using cheap and abundant methanol than using glucose as sole carbon and energy source. The isolated products showed the structure characteristics same to that of standard PHB and CoQ10. The optimal medium and cultural conditions for PHB and CoQ10 co-production by Methylobacterium sp. XJLW was in M3 medium containing 7.918 g L-1 methanol, 0.5 g L-1 of ammonium sulfate, 0.1% (v/v) of Tween 80, and 1.0 g L-1 of sodium chloride, under 30 °C and pH 7.0. In a 5-L bioreactor coupled with methanol fed-batch process, a maximum DCW value (46.31 g L-1) with the highest yields of PHB and CoQ10, reaching 6.94 g L-1 and 22.28 mg L-1, respectively. Conclusion Methylobacterium sp. XJLW is potential for efficiently co-producing PHB and CoQ10 employing methanol as sole carbon and energy source. However, it is still necessary to further optimize fermentation process, and genetically modify strain pathway, for enhanced production of PHB and CoQ10 simultaneously by Methylobacterium sp. XJLW. It also suggests a potential strategy to develop efficiently co-producing other high-value metabolites using methanol-based bioprocess.