Factors affecting rAAV titers during triple-plasmid transient transfection in HEK-293 cells.

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficiency utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhance fully packaged rAAV particle yield, we discovered cell-line-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.

Cloning, expression and characterisation of a novel mollusc α-1,2-Fucosyltransferase from Crassostrea gigas (CgFUT2).

Glycans containing fucose play crucial roles in cell biology, particularly in recognition processes. In humans, fucose found in H-blood group antigens is recognized by various pathogens, thereby influencing host-pathogen interactions. However, in invertebrate biology the specific functions of these modifications and the corresponding glycosyltransferases are not fully elucidated. Therefore, cloning these glycosyltransferases from different model systems will provide valuable insights into this process. Little is known about fucosyltransferases in molluscs. For this study, a sequence of the Pacific oyster, Crassostrea gigas, based on amino acid sequence homologies with rabbit and human α-1,2-fucosyltransferases, was chosen. The recombinant enzyme (350 amino acids) was able to transfer fucose from GDP-fucose to the galactose residue of type II disaccharides, terminal galactoses in complex N-glycan structures and several linear and branched galactans which were tested using a glycan microarray. The α-1,2-linkage formed was confirmed by NMR analysis. The enzyme was active in a broad pH-range, it was relatively stable upon storage conditions and its activity was not dependent on the presence of divalent cations. In this study, we were able to clone, express and characterise a novel α-1,2-fucosyltrasferase from Crassostrea gigas (CgFUT2).

Enhancing NA immunogenicity through novel VLP designs.

Current influenza virus vaccines poorly display key neuraminidase (NA) epitopes and do not robustly induce NA-reactive antibodies; instead, they focus on the induction of hemagglutinin (HA)-reactive antibodies. Next-generation influenza vaccines should be optimized in order to activate NA-reactive B cells and to induce a broadly cross-reactive and protective antibody response. We aimed at enhancing the immunogenicity of the NA on vaccines by two strategies: (i) modifying the HA:NA ratio of the vaccine preparation and (ii) exposing epitopes on the lateral surface or beneath the head of the NA by extending the NA stalk. The H1N1 glycoproteins from the influenza virus A/California/04/2009 strain were displayed on human immunodeficiency virus 1 (HIV-1) gag-based virus-like particles (VLP). Using the baculovirus insect cell expression system, we biased the quantity of surface glycoproteins employing two different promoters, the very late baculovirus p10 promoter and the early and late gp64 promoter. This led to a 1:1 to 2:1 HA:NA ratio, which was approximately double or triple the amount of NA as present on the wild-type influenza A virus (HA:NA ratio 3:1 to 5:1). Furthermore, by insertion of 15 amino acids from the A-New York/61/2012 strain (NY12) which prolongates the NA stalk (NA long stalk; NA-LS), we intended to improve the accessibility of the NA. Six different types of VLPs were produced and purified using a platform downstream process based on Capto-Core 700™ followed by Capto-Heparin™ affinity chromatography combined with ultracentrifugation. These VLPs were then tested in a mouse model. Robust titers of antibodies that inhibit the neuraminidase activity were elicited even after vaccination with two low doses (0.3 µg) of the H1N1 VLPs without compromising the anti-HA responses. In conclusion, our results demonstrate the feasibility of the two developed strategies to retain HA immunogenicity and improve NA immunogenicity as a future influenza vaccine candidate.

Different Neutralizing Antibody Responses of Heterologous Sera on Sheeppox and Lumpy Skin Disease Viruses.

Sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV) are the three members of the genus Capripoxvirus within the Poxviridae family and are the etiologic agents of sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD), respectively. LSD, GTP, and SPP are endemic in Africa and Asia, causing severe disease outbreaks with significant economic losses in livestock. Incursions of SPP and LSD have occurred in Europe. Vaccination with live attenuated homologous and heterologous viruses are routinely implemented to control these diseases. Using the gold standard virus neutralization test, we studied the ability of homologous and heterologous sera to neutralize the SPPV and LSDV. We found that LSD and SPP sera effectively neutralize their homologous viruses, and GTP sera can neutralize SPPV. However, while LSD sera effectively neutralizes SPPV, SPP and GTP sera cannot neutralize the LSDV to the same extent. We discuss the implications of these observations in disease assay methodology and heterologous vaccine efficacy.

Increased efficacy of influenza virus vaccine candidate through display of recombinant neuraminidase on virus like particles.

Vaccination against influenza virus can reduce the risk of influenza by 40% to 60%, they rely on the production of neutralizing antibodies specific to influenza hemagglutinin (HA) ignoring the neuraminidase (NA) as an important surface target. Vaccination with standardized NA concentration may offer broader and longer-lasting protection against influenza infection. In this regard, we aimed to compare the potency of a NA displayed on the surface of a VLP with a soluble NA. The baculovirus expression system (BEVS) and the novel virus-free Tnms42 insect cell line were used to express N2 NA on gag-based VLPs. To produce VLP immunogens with high levels of purity and concentration, a two-step chromatography purification process combined with ultracentrifugation was used. In a prime/boost vaccination scheme, mice vaccinated with 1 µg of the N2-VLPs were protected from mortality, while mice receiving the same dose of unadjuvanted NA in soluble form succumbed to the lethal infection. Moreover, NA inhibition assays and NA-ELISAs of pre-boost and pre-challenge sera confirm that the VLP preparation induced higher levels of NA-specific antibodies outperforming the soluble unadjuvanted NA.

Determination, expression and characterization of an UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnT-I) from the Pacific oyster, Crassostrea gigas.

Molluscs are intermediate hosts for several parasites. The recognition processes, required to evade the host's immune response, depend on carbohydrates. Therefore, the investigation of mollusc glycosylation capacities is of high relevance to understand the interaction of parasites with their host. UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I (GnT-I) is the key enzyme for the biosynthesis of hybrid and complex type N-glycans catalysing the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the α-1,3 Man antenna of Man5GlcNAc2. Thereby, the enzyme produces a suitable substrate for further enzymes, such as α-mannosidase II, GlcNAc-transferase II, galactosyltransferases or fucosyltransferases. The sequence of GnT- I from the Pacific oyster, Crassostrea gigas, was obtained by homology search using the corresponding human enzyme as the template. The obtained gene codes for a 445 amino acids long type II transmembrane glycoprotein and shared typical structural elements with enzymes from other species. The enzyme was expressed in insect cells and purified by immunoprecipitation using protein A/G-plus agarose beads linked to monoclonal His-tag antibodies. GnT-I activity was determined towards the substrates Man5-PA, MM-PA and GnM-PA. The enzyme displayed highest activity at pH 7.0 and 30 °C, using Man5-PA as the substrate. Divalent cations were indispensable for the enzyme, with highest activity at 40 mM Mn2+, while the addition of EDTA or Cu2+ abolished the activity completely. The activity was also reduced by the addition of UDP, UTP or galactose. In this study we present the identification, expression and biochemical characterization of the first molluscan UDP-N-acetylglucosamine:α-1,3-D-mannoside ß-1,2-N-acetylglucosaminyltransferase I, GnT-I, from the Pacific oyster Crassostrea gigas.

Comprehensive Comparison of Baculoviral and Plasmid Gene Delivery in Mammalian Cells.

(1) Recombinant protein production in mammalian cells is either based on transient transfection processes, often inefficient and underlying high batch-to-batch variability, or on laborious generation of stable cell lines. Alternatively, BacMam, a transduction process using the baculovirus, can be employed. (2) Six transfecting agents were compared to baculovirus transduction in terms of transient and stable protein expression characteristics of the model protein ACE2-eGFP using HEK293-6E, CHO-K1, and Vero cell lines. Furthermore, process optimization such as expression enhancement using sodium butyrate and TSA or baculovirus purification was assessed. (3) Baculovirus transduction efficiency was superior to all transfection agents for all cell lines. Transduced protein expression was moderate, but an 18-fold expression increase was achieved using the enhancer sodium butyrate. Ultracentrifugation of baculovirus from a 3.5 L bioreactor significantly improved the transduction efficiency and protein expression. Stable cell lines were obtained with each baculovirus transduction, yet stable cell line generation after transfection was highly unreliable. (4) This study demonstrated the superiority of the BacMam platform to standard transfections. The baculovirus efficiently transduced an array of cell lines both transiently and stably and achieved the highest efficiency for all tested cell lines. The feasibility of the scale-up of baculovirus production was demonstrated and the possibility of baculovirus purification was successfully explored.

Molecular cloning, characterisation and molecular modelling of two novel T-synthases from mollusc origin.

The glycoprotein-N-acetylgalactosamine ß1,3-galactosyltransferase, known as T-synthase (EC 2.4.1.122), plays a crucial role in the synthesis of the T-antigen, which is the core 1 O-glycan structure. This enzyme transfers galactose from UDP-Gal to GalNAc-Ser/Thr. The T-antigen has significant functions in animal development, immune response, and recognition processes. Molluscs are a successful group of animals that inhabit various environments, such as freshwater, marine, and terrestrial habitats. They serve important roles in ecosystems as filter feeders and decomposers but can also be pests in agriculture and intermediate hosts for human and cattle parasites. The identification and characterization of novel carbohydrate active enzymes, such as T-synthase, can aid in the understanding of molluscan glycosylation abilities and their adaptation and survival abilities. Here, the T-synthase enzymes from the snail Pomacea canaliculata and the oyster Crassostrea gigas are identified, cloned, expressed, and characterized, with a focus on structural elucidation. The synthesized enzymes display core 1 ß1,3-galactosyltransferase activity using pNP-α-GalNAc as substrate and exhibit similar biochemical parameters as previously characterised T-synthases from other species. While the enzyme from C. gigas shares the same structural parameters with the other enzymes characterised so far, the T-synthase from P. canaliculata lacks the consensus sequence CCSD, which was previously considered indispensable.

Harnessing Attenuation-Related Mutations of Viral Genomes: Development of a Serological Assay to Differentiate between Capripoxvirus-Infected and -Vaccinated Animals.

Sheeppox, goatpox, and lumpy skin disease caused by the sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively, are diseases that affect millions of ruminants and many low-income households in endemic countries, leading to great economic losses for the ruminant industry. The three viruses are members of the Capripoxvirus genus of the Poxviridae family. Live attenuated vaccines remain the only efficient means for controlling capripox diseases. However, serological tools have not been available to differentiate infected from vaccinated animals (DIVA), though crucial for proper disease surveillance, control, and eradication efforts. We analysed the sequences of variola virus B22R homologue gene for SPPV, GTPV, and LSDV and observed significant differences between field and vaccine strains in all three capripoxvirus species, resulting in the truncation and absence of the B22R protein in major vaccines within each of the viral species. We selected and expressed a protein fragment present in wildtype viruses but absent in selected vaccine strains of all three species, taking advantage of these alterations in the B22R gene. An indirect ELISA (iELISA) developed using this protein fragment was evaluated on well-characterized sera from vaccinated, naturally and experimentally infected, and negative cattle and sheep. The developed wildtype-specific capripox DIVA iELISA showed >99% sensitivity and specificity for serum collected from animals infected with the wildtype virus. To the best of our knowledge, this is the first wildtype-specific, DIVA-capable iELISA for poxvirus diseases exploiting changes in nucleotide sequence alterations in vaccine strains.

Expression and Characterization of a ß-Galactosidase from the Pacific Oyster, Crassostrea gigas, and Evaluation of Strategies for Testing Substrate Specificity.

ß-Galactosidases (EC 3.2.1.23) are exoglycosidases that catalyze the cleavage of glycoconjugates with terminal ß-D-galactose residues in ß1,3-, ß1,4- or ß1,6-linkage. Although this family of exoglycosidases has been extensively studied in vertebrates, plants, yeast, and bacteria, little information is available for mollusks. Mollusks are a diverse and highly successful group of animals that play many different roles in their ecosystems, including filter feeders and detritivores. Here, the first ß-galactosidase from the Pacific oyster, Crassostrea gigas was discovered, biochemically characterized, and compared to our previously characterized slug enzyme from Arion vulgaris (UniProt Ref. Nr.: A0A0B7AQJ9). Overall, the mussel enzyme showed similar biochemical parameters to the snail enzyme. The enzyme from C. gigas was most active in an acidic environment (pH 3.5) and at a reaction temperature of 50 °C. Optimal storage conditions were up to 37 °C. In contrast to the enzyme from A. vulgaris, the supplementation of cations (Ni2+, Co2+, Mn2+, Mg2+, Ca2+, Cu2+, Ba2+) increased the activity of the enzyme from C. gigas. Substrate specificity studies of the ß-galactosidases from the mussel, C. gigas, and the slug, A. vulgaris, revealed activity towards terminal ß1,3- and ß1,4-linked galactose residues for both enzymes. Using the same substrates in labeled and unlabeled form, we were able to detect the effect of labeling on the ß-galactosidase activity using MALDI-TOF MS, HPTLC, and HPLC. While lactose was cleaved by the enzymes in an unlabeled or labeled state, galacto-N-biose was not cleaved as soon as a 2-amino benzoic acid label was added. In this study we present the biochemical characterization of the first recombinantly expressed ß-galactosidase from the Pacific oyster, C. gigas, and we compare different analytical methods for the determination of ß-galactosidase activity using the enzyme from C. gigas and A. vulgaris.

Evaluation of an inducible knockout system in insect cells based on co-infection and CRISPR/Cas9.

Due to comparably high product titers and low production costs, the baculovirus/insect cell expression system is considered a versatile production platform in the biopharmaceutical industry. Its excellence in producing complex multimeric protein assemblies, including virus-like particles (VLPs), which are considered promising vaccine candidates to counter emerging viral threats, made the system even more attractive. However, the co-formation of budded baculovirus during VLP production poses a severe challenge to downstream processing. In order to reduce the amount of budded baculovirus in the expression supernatant we developed an inducible knockout system based on CRISPR/Cas9 and co-infection with two baculoviral vectors: one bringing along the Cas9 nuclease and the other one having incorporated the sequence for sgRNA expression. With our set-up high titer viruses can be generated separately, as only when both viruses infect cells simultaneously a knockout can occur. When budding essential genes gp64 and vp80 were targeted for knockout, we measured a reduction in baculovirus titer by over 90%. However, as a consequence, we also determined lower overall eYFP fluorescence intensity showing reduced recombinant protein production, indicating that further improvements in engineering as well as purification are required in order to ultimately minimize costs and timeframes for vaccine production utilizing the baculovirus/insect cell expression system.

Comprehensive mRNA-sequencing-based characterization of three HEK-293 cell lines during an rAAV production process for gene therapy applications.

Human embryonal kidney cells (HEK-293) are the most common host cells used for transient recombinant adeno-associated virus (rAAV) production in pharmaceutical industry. To better cover the expected gene therapy product demands in the future, different traditional strategies such as cell line sub-cloning and/or addition of chemical substances to the fermentation media have been used to maximize titers and improve product quality. A more effective and advanced approach to boost yield can be envisaged by characterizing the transcriptome of different HEK-293 cell line pedigrees with distinct rAAV productivity patterns to subsequently identify potential gene targets for cell engineering. In this work, the mRNA expression profile of three HEK-293 cell lines, resulting in various yields during a fermentation batch process for rAAV production, was investigated to gain basic insight into cell variability and eventually to identify genes that correlate with productivity. Mock runs using only transfection reagents were performed in parallel as a control. It finds significant differences in gene regulatory behaviors between the three cell lines at different growth and production stages. The evaluation of these transcriptomics profiles combined with collected in-process control parameters and titers shed some light on potential cell engineering targets to maximize transient production of rAAV in HEK-293 cells.

Expression and Characterisation of the First Snail-Derived UDP-Gal: Glycoprotein-N-acetylgalactosamine ß-1,3-Galactosyltransferase (T-Synthase) from Biomphalaria glabrata.

UDP-Gal: glycoprotein-N-acetylgalactosamine ß-1,3-galactosyltransferase (T-synthase, EC 2.4.1.122) catalyses the transfer of the monosaccharide galactose from UDP-Gal to GalNAc-Ser/Thr, synthesizing the core 1 mucin type O-glycan. Such glycans play important biological roles in a number of recognition processes. The crucial role of these glycans is acknowledged for mammals, but a lot remains unknown regarding invertebrate and especially mollusc O-glycosylation. Although core O-glycans have been found in snails, no core 1 ß-1,3-galactosyltransferase has been described so far. Here, the sequence of the enzyme was identified by a BlastP search of the NCBI Biomphalaria glabrata database using the human T-synthase sequence (NP_064541.1) as a template. The obtained gene codes for a 388 amino acids long transmembrane protein with two putative N-glycosylation sites. The coding sequence was synthesised and expressed in Sf9 cells. The expression product of the putative enzyme displayed core 1 ß-1,3-galactosyltransferase activity using pNP-α-GalNAc as the substrate. The enzyme showed some sequence homology (49.40% with Homo sapiens, 53.69% with Drosophila melanogaster and 49.14% with Caenorhabditis elegans) and similar biochemical parameters with previously characterized T-synthases from other phyla. In this study we present the identification, expression and characterisation of the UDP-Gal: glycoprotein-N-acetylgalactosamine ß-1,3-galactosyltransferase from the fresh-water snail Biomphalaria glabrata, which is the first cloned T-synthase from mollusc origin.

Quantitative proteomic analysis of extracellular vesicles in response to baculovirus infection of a Trichoplusia ni cell line.

Due to its outstanding suitability to produce complex biopharmaceutical products including virus-like particles and subunit vaccines, the baculovirus/insect cell expression system has developed into a highly popular production platform in the biotechnological industry. For high productivity, virus-cell communication and an efficient spreading of the viral infection are crucial, and, in this context, extracellular vesicles (EVs) might play a significant role. EVs are small particles, utilized by cells to transfer biologically active compounds such as proteins, lipids as well as nucleic acids to recipient cells for intracellular communication. Studies in mammalian cells showed that the release of EVs is altered in response to infection with many viruses, ultimately either limiting or fostering infection spreading. In this study we isolated and characterized EVs, from both uninfected and baculovirus infected Tnms42 insect cells. Via quantitative proteomic analysis we identified more than 3000 T. ni proteins in Tnms42 cell derived EVs, of which more than 400 were significantly differentially abundant upon baculovirus infection. Subsequent gene set enrichment analysis revealed a depletion of proteins related to the extracellular matrix in EVs from infected cultures. Our findings show a significant change of EV protein cargo upon baculovirus infection, suggesting a major role of EVs as stress markers. Our study might serve in designing new tools for process monitoring and control to further improve biopharmaceutical production within the baculovirus/insect cell expression system.

Increasing test specificity without impairing sensitivity: lessons learned from SARS-CoV-2 serology.

BACKGROUND: Serological tests are widely used in various medical disciplines for diagnostic and monitoring purposes. Unfortunately, the sensitivity and specificity of test systems are often poor, leaving room for false-positive and false-negative results. However, conventional methods were used to increase specificity and decrease sensitivity and vice versa. Using SARS-CoV-2 serology as an example, we propose here a novel testing strategy: the 'sensitivity improved two-test' or 'SIT²' algorithm. METHODS: SIT² involves confirmatory retesting of samples with results falling in a predefined retesting zone of an initial screening test, with adjusted cut-offs to increase sensitivity. We verified and compared the performance of SIT² to single tests and orthogonal testing (OTA) in an Austrian cohort (1117 negative, 64 post-COVID-positive samples) and validated the algorithm in an independent British cohort (976 negatives and 536 positives). RESULTS: The specificity of SIT² was superior to single tests and non-inferior to OTA. The sensitivity was maintained or even improved using SIT² when compared with single tests or OTA. SIT² allowed correct identification of infected individuals even when a live virus neutralisation assay could not detect antibodies. Compared with single testing or OTA, SIT² significantly reduced total test errors to 0.46% (0.24-0.65) or 1.60% (0.94-2.38) at both 5% or 20% seroprevalence. CONCLUSION: For SARS-CoV-2 serology, SIT² proved to be the best diagnostic choice at both 5% and 20% seroprevalence in all tested scenarios. It is an easy to apply algorithm and can potentially be helpful for the serology of other infectious diseases.

Identification, Characterization, and Expression of a ß-Galactosidase from Arion Species (Mollusca).

ß-Galactosidases (ß-Gal, EC 3.2.1.23) catalyze the cleavage of terminal non-reducing ß-D-galactose residues or transglycosylation reactions yielding galacto-oligosaccharides. In this study, we present the isolation and characterization of a ß-galactosidase from Arion lusitanicus, and based on this, the cloning and expression of a putative ß-galactosidase from Arion vulgaris (A0A0B7AQJ9) in Sf9 cells. The entire gene codes for a protein consisting of 661 amino acids, comprising a putative signal peptide and an active domain. Specificity studies show exo- and endo-cleavage activity for galactose ß1,4-linkages. Both enzymes, the recombinant from A. vulgaris and the native from A. lusitanicus, display similar biochemical parameters. Both ß-galactosidases are most active in acidic environments ranging from pH 3.5 to 4.5, and do not depend on metal ions. The ideal reaction temperature is 50 °C. Long-term storage is possible up to +4 °C for the A. vulgaris enzyme, and up to +20 °C for the A. lusitanicus enzyme. This is the first report of the expression and characterization of a mollusk exoglycosidase.

Development and Optimization of Indirect ELISAs for the Detection of Anti-Capripoxvirus Antibodies in Cattle, Sheep, and Goat Sera.

Sheeppox (SPP), goatpox (GTP), and lumpy skin disease (LSD) are economically significant pox diseases of ruminants, caused by sheeppox virus (SPPV), goatpox virus (GTPV), and lumpy skin disease virus (LSDV), respectively. SPPV and GTPV can infect both sheep and goats, while LSDV mainly affects cattle. The recent emergence of LSD in Asia and Europe and the repeated incursions of SPP in Greece, Bulgaria, and Russia highlight how these diseases can spread outside their endemic regions, stressing the urgent need to develop high-throughput serological surveillance tools. We expressed and tested two recombinant truncated proteins, the capripoxvirus homologs of the vaccinia virus C-type lectin-like protein A34 and the EEV glycoprotein A36, as antigens for an indirect ELISA (iELISA) to detect anti-capripoxvirus antibodies. Since A34 outperformed A36 by showing no cross-reactivity to anti-parapoxvirus antibodies, we optimized an A34 iELISA using two different working conditions, one for LSD in cattle and one for SPP/GTP in sheep and goats. Both displayed sound sensitivities and specificities: 98.81% and 98.72%, respectively, for the LSD iELISA, and 97.68% and 95.35%, respectively, for the SPP/GTP iELISA, and did not cross-react with anti-parapoxvirus antibodies of cattle, sheep, and goats. These assays could facilitate the implementation of capripox control programs through serosurveillance and the screening of animals for trade.

CRISPRactivation-SMS, a message for PAM sequence independent gene up-regulation in Escherichia coli.

Governance of the endogenous gene regulatory network enables the navigation of cells towards beneficial traits for recombinant protein production. CRISPRactivation and interference provides the basis for gene expression modulation but is primarily applied in eukaryotes. Particularly the lack of wide-ranging prokaryotic CRISPRa studies might be attributed to intrinsic limitations of bacterial activators and Cas9 proteins. While bacterial activators need accurate spatial orientation and distancing towards the target promoter to be functional, Cas9-based CRISPR tools only bind sites adjacent to NGG PAM sequences. These circumstances hampered Cas9-guided activators from mediating the up-regulation of endogenous genes at precise positions in bacteria. We could overcome this limitation by combining the PAM independent Cas9 variant SpRY and a CRISPRa construct using phage protein MCP fused to transcriptional activator SoxS. This CRISPRa construct, referred to as SMS, was compared with previously reported CRISPRa constructs and showed up-regulation of a reporter gene library independent of its PAM sequence in Escherichia coli. We also demonstrated down-regulation and multi-gene expression control with SMS at non-NGG PAM sites. Furthermore, we successfully applied SMS to up-regulate endogenous genes, and transgenes at non-NGG PAM sites, which was impossible with the previous CRISPRa construct.

Production, Storage Stability, and Susceptibility Testing of Reuterin and Its Impact on the Murine Fecal Microbiome and Volatile Organic Compound Profile.

Background: Probiotics are generally considered as safe, but infections may rarely occur in vulnerable patients. Alternatives to live microorganisms to manage dysbiosis may be of interest in these patients. Reuterin is a complex component system exhibiting broad spectrum antimicrobial activity and a possible candidate substance in these cases. Methods: Reuterin supernatant was cultured from Lentilactobacillus diolivorans in a bioreactor in a two-step process. Storage stability at -20°C and effect of repeated freeze-thaw cycles were assessed by high performance liquid chromatography (HPLC). Antimicrobial activity was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus (S.) aureus, Staphylococcus epidermidis, Streptococcus (S.) agalactiae, Propionibacterium acnes, and Pseudomonas aeruginosae. Male BALBc mice were gavage fed with reuterin supernatant (n = 10) or culture medium (n = 10). Fecal volatile organic compounds (VOC) were assessed by gas chromatography mass spectroscopy; the microbiome was examined by 16S rRNA gene sequencing. Results: The supernatant contained 13.4 g/L reuterin (3-hydroxypropionaldehyde; 3-HPA). 3-HPA content remained stable at -20°C for 35 days followed by a slow decrease of its concentration. Repeated freezing/thawing caused a slow 3-HPA decrease. Antimicrobial activity was encountered against S. aureus, S. epidermidis, and S. agalactiae. Microbiome analysis showed no differences in alpha and beta diversity markers. Linear discriminant effect size (LEfSe) analysis identified Lachnospiraceae_bacterium_COE1 and Ruminoclostridium_5_uncultured_Clostridiales_ bacterium (in the reuterin medium group) and Desulfovibrio_uncultured_ bacterium, Candidatus Arthromitus, Ruminococcae_NK4A214_group, and Eubacterium_xylanophilum_group (in the reuterin group) as markers for group differentiation. VOC analysis showed a significant decrease of heptane and increase of 3-methylbutanal in the reuterin group. Conclusion: The supernatant produced in this study contained acceptable amounts of 3-HPA remaining stable for 35 days at -20°C and exhibiting an antimicrobial effect against S. aureus, S. agalactiae, and S. epidermidis. Under in vivo conditions, the reuterin supernatant caused alterations of the fecal microbiome. In the fecal, VOC analysis decreased heptane and increased 3-methylbutanal were encountered. These findings suggest the high potential of the reuterin system to influence the intestinal microbiome in health and disease, which needs to be examined in detail in future projects.

(S)-Reutericyclin: Susceptibility Testing and In Vivo Effect on Murine Fecal Microbiome and Volatile Organic Compounds.

We aimed to assess the in vitro antimicrobial activity and the in vivo effect on the murine fecal microbiome and volatile organic compound (VOC) profile of (S)-reutericyclin. The antimicrobial activity of (S)-reutericyclin was tested against Clostridium difficile, Listeria monocytogenes, Escherichia coli, Enterococcus faecium, Staphylococcus aureus, Staphylococcus (S.) epidermidis, Streptococcus agalactiae, Pseudomonas aeruginosa and Propionibacterium acnes. Reutericyclin or water were gavage fed to male BALBc mice for 7 weeks. Thereafter stool samples underwent 16S based microbiome analysis and VOC analysis by gas chromatography mass spectrometry (GC-MS). (S)-reutericyclin inhibited growth of S. epidermidis only. Oral (S)-reutericyclin treatment caused a trend towards reduced alpha diversity. Beta diversity was significantly influenced by reutericyclin. Linear discriminant analysis Effect Size (LEfSe) analysis showed an increase of Streptococcus and Muribaculum as well as a decrease of butyrate producing Ruminoclostridium, Roseburia and Eubacterium in the reutericyclin group. VOC analysis revealed significant increases of pentane and heptane and decreases of 2,3-butanedione and 2-heptanone in reutericyclin animals. The antimicrobial activity of (S)-reutericyclin differs from reports of (R)-reutericyclin with inhibitory effects on a multitude of Gram-positive bacteria reported in the literature. In vivo (S)-reutericyclin treatment led to a microbiome shift towards dysbiosis and distinct alterations of the fecal VOC profile.