Recombinant Bacteria Research Articles

Recombinant Bacillus subtilis expressing functional peptide and its effect on blunt snout bream (Megalobrama amblycephala) in two state of stress

This study was conducted to investigate the effects of recombinant Bacillus subtilis CM66-P4' (secreting P4, which related to previous research in this laboratory) on the antioxidant capacity and immune function of blunt snout bream (Megalobrama amblycephala) through in vitro and in vivo experiment. The culture experiment was divided into 3 groups, including control group (CG, with no additional bacteria), original bacteria group (OBG, with 2×109 CFU/kg Bacillus subtilis CM66) and recombinant bacteria group (RBG, with 2×109 CFU/kg Bacillus subtilis CM66-P4'). After 8 weeks of feeding, a part of the fish were subjected to fishing stress, and the rest were subjected to starvation stress test. Blood samples were collected for the determination of immune and stress-related indexes. The hepatocytes were divided into control group (CG) and experiment group with P4 peptide (LTG and HTG). The cells were collected after starvation treatment and the expression of related genes was detected. The results showed as follows: compared with the CG group, the gene expressions of hepatocytic hsp60 and hsp70 in the LTG and HTG groups were significantly suppressed after 24 h starvation stress (P < 0.05). The content of MDA, the activities of AKP and ALT in OBG group were significantly changed after 30 days starvation (P < 0.05), while the indexes in RBG group had no significant change. The changes of plasma cortisol, malondialdehyde (MDA) and Immunoglobulin M (IgM) in CG and OBG groups were significantly changed at 4 h after fishing stress (P < 0.05), while the indexes in RBG group was not. In conclusion, this study confirmed that Bacillus subtilis CM66-P4' has great potential in preventing adverse effects of stress on aquatic livestock.

Immunoprotective effect of recombinant Lactobacillus plantarum expressing largemouth bass virus MCP on largemouth bass

Largemouth bass virus (LMBV) is an infectious pathogen that causes high mortality rates in largemouth bass, and outbreaks of this virus can significantly harm the aquaculture industry. Currently, no vaccine has been developed that can effectively prevent the transmission of LMBV. In this study, we constructed a recombinant Lactobacillus plantarum (L. plantarum) strain capable of expressing the MCP gene of LMBV and displaying this protein on its surface; then, we evaluated the immunoprotective effect of this recombinant bacterium on largemouth bass. Western blotting, immunofluorescence, and flow cytometry confirmed that MCP was successfully expressed and anchored on the surfaces of NC8 cells. Immunization of largemouth bass with NC8-pSIP409-pgsA'-MCP via the oral feeding route induced CD4, CD8, IL-1β, and IL-6 gene expression. In addition, NC8-pSIP409-pgsA'-MCP at different CFUs increased the survival of largemouth bass after LMBV infection; in particular, NC8-pSIP409-pgsA'-MCP (109 CFU) resulted in approximately 30% survival. NC8-pSIP409-pgsA'-MCP immunization alleviated the pathological changes in the liver and spleen, exerting a more advantageous protective effect. These data suggest that the recombinant L. plantarum strain NC8-pSIP409-pgsA'-MCP can increase the resistance of largemouth bass to LMBV infection and that this strain is a promising candidate oral vaccine for the prevention of LMBV infection.

Cloning and Expression of Pigeon-Derived Escherichia coli Type 1 Pilus Clusters and Analysis of Amino Acid Sequence Characteristics of Functional Proteins.

Type 1 pili, as an important virulence factor of E. coli, has certain homology between APEC and UPEC, but the homology degree is not clear enough. This study aims to compare the homology between them. The recombinant bacteria were constructed by homologous recombination. The pili were observed by TEM, and the hemagglutination characteristics were determined by MHSA. The complete gene sequence was determined by sequencing, and the amino acid sequences of the functional proteins of type 1 pili of APEC and UPEC were compared. TEM showed that they could express pili, which were slender, straight, and dense. Stable-pUC-fimBH has MHSA but stable-pUC-fimBG does not. The amino acid sequence similarity of FimB of NJ05 and UPEC was 98.8%, FimE was 99.4%, and the similarity between them was 51.5%. Compared with UPEC's type 1 pili FimC and FimD sequences, the similarity was 99.52% and 87.8%, respectively. The amino acid sequence of FimA of NJ05 was 89-96%, similar to UPEC, and the N-terminal and C-terminal amino acid sequences were exactly the same. The gene sequence and amino acid sequence similarity of FimH between them were both above 99%. The similarity of the pilus binding domain of FimH was 52.8%, but only 27.6% in the receptor binding domain. A few of the same amino acid residues were found in the corresponding regions of FimA, FimF, FimG, and FimH. The type 1 pili of APEC and UPEC come from the same origin, which is helpful to further reveal the pathogenic mechanism of E. coli infection in the poultry respiratory tract.

Co-expression of endoglucanase and cellobiohydrolase from yak rumen in lactic acid bacteria and its preliminary application in whole-plant corn silage fermentation.

Endoglucanase (EG) and cellobiohydrolase (CBH) which produced by microorganisms, have been widely used in industrial applications. In order to construct recombinant bacteria that produce high activity EG and CBH, in this study, eg (endoglucanase) and cbh (cellobiohydrolase) were cloned from the rumen microbial genome of yak and subsequently expressed independently and co-expressed within Lactococcus lactis NZ9000 (L. lactis NZ9000). The recombinant strains L. lactis NZ9000/pMG36e-usp45-cbh (L. lactis-cbh), L. lactis NZ9000/pMG36e-usp45-eg (L. lactis-eg), and L. lactis NZ9000/pMG36e-usp45-eg-usp45-cbh (L. lactis-eg-cbh) were successfully constructed and demonstrated the ability to secrete EG, CBH, and EG-CBH. The sodium carboxymethyl cellulose activity of the recombinant enzyme EG was the highest, and the regenerated amorphous cellulose (RAC) was the specific substrate of the recombinant enzyme CBH, and EG-CBH. The optimum reaction temperature of the recombinant enzyme CBH was 60°C, while the recombinant enzymes EG and EG-CBH were tolerant to higher temperatures (80°C). The optimum reaction pH of EG, CBH, and EG-CBH was 6.0. Mn2+, Fe2+, Cu2+, and Co2+ could promote the activity of CBH. Similarly, Fe2+, Ba2+, and higher concentrations of Ca2+, Cu2+, and Co2+ could promote the activity of EG-CBH. The addition of engineered strains to whole-plant corn silage improved the nutritional quality of the feed, with the lowest pH, acid detergent fiber (ADF), and neutral detergent fiber (NDF) contents observed in silage from the L. lactis-eg group (p < 0.05), and the lowest ammonia nitrogen (NH3-N), and highest lactic acid (LA) and crude protein (CP) contents in silage from the L. lactis-eg + L. lactis-cbh group (p < 0.05), while the silage quality in the L. lactis-cbh group was not satisfactory. Consequently, the recombinant strains L. lactis-cbh, L. lactis-eg, and L. lactis-eg-cbh were successfully constructed, which could successfully expressed EG, CBH, and EG-CBH. L. lactis-eg promoted silage fermentation by degrading cellulose to produce sugar, enabling the secretory expression of EG, CBH, and EG-CBH for potential industrial applications in cellulose degradation.

In Situ and Rapid Toxicity Assessment of Air Pollution by Self-Assembly Passive Colonization Hydrogel.

Air pollution is a leading environmental health risk factor, and in situ toxicity assessment is urgently needed. Bacteria-based bioassays offer cost-effective and rapid toxicity assessments. However, the application of these bioassays for air toxicity assessment has been challenging, due to the instability of bacterial survival and functionality when directly exposed to air pollutants. Here, we developed an approach employing self-assembly passive colonization hydrogel (SAPCH) for in situ air toxicity assessment. The SAPCH features a core-shell structure, enabling the quantitatively immobilization of bacteria on its shell while continuously provides nutrients from its core. An antimicrobial polyelectrolyte layer between the core and shell confines bacteria to the air-liquid interface, synchronizing bacterial survival with exposure to air pollutants. The SAPCH immobilized a battery of natural and recombinant luminescent bacteria, enabling simultaneous detection of various toxicological endpoints (cytotoxicity, genotoxicity and oxidative stress) of air pollutants within 2 h. Its sensitivity was 3-5 orders of magnitude greater than that of traditional liquid-phase toxicity testing, and successfully evaluating the toxicity of volatile organic compounds and combustion smoke. This study presents a method for in situ, rapid, and economical toxicity assessment of air pollution, making a significant contribution to future air quality monitoring and control.

Stability of a Mutualistic Escherichia coli Co-Culture During Violacein Production Depends on the Kind of Carbon Source.

The L-tryptophan-derived purple pigment violacein (VIO) is produced in recombinant bacteria and studied for its versatile applications. Microbial synthetic co-cultures are gaining more importance as efficient factories for synthesizing high-value compounds. In this work, a mutualistic and cross-feeding Escherichia coli co-culture is metabolically engineered to produce VIO. The strains are genetically modified by auxotrophies in the tryptophan (TRP) pathway to enable a metabolic division of labor. Therein, one strain produces anthranilate (ANT) and the other transforms it into TRP and further to VIO. Population dynamics and stability depend on the choice of carbon source, impacting the presence and thus exchange of metabolites as well as overall VIO productivity. Four carbon sources (D-glucose, glycerol, D-galactose, and D-xylose) were compared. D-Xylose led to co-cultures which showed stable growth and VIO production, ANT-TRP exchange, and enhanced VIO production. Best titers were ∼126mgL-1 in shake flasks. The study demonstrates the importance and advantages of a mutualistic approach in VIO synthesis and highlights the carbon source's role in co-culture stability and productivity. Transferring this knowledge into an up-scaled bioreactor system has great potential in improving the overall VIO production.

Targeting N-acyl-homoserine lactones (AHLs) on the quorum quenching AiiA protein to eradicate biofilm formation in Vibrio parahaemolyticus

ABSTRACT AiiA can be targeted to quench N-acyl-homoserine lactones (AHLs) quorum sensing signaling molecules. In this paper, we extracted and purified AiiA protein by constructing recombinant engineering bacteria to analyze the effect of AiiA on the formation of Vibrio parahaemolyticus biofilm. The results suggest that AiiA has a particular potential in inhibiting the formation of biofilms and extracellular polymers. The ability of AiiA to inhibit biofilm increased and then decreased, with the lowest biofilm decomposition concentration of 8 μg/mL and the optimal inhibitor concentration of 85 μg/mL (86.9% biofilm inhibition). LC-MS results showed that when the concentration of C4-HSL in Vibrio parahaemolyticus ATCC33847 biofilm exceeded 3.70 μg/L, the content of quorum sensing signaling molecules reached a threshold and began to activate LuxR protein to regulate the expression of related genes. These results provide relevant experimental guidance for the clinical treatment of biofilm infections and the development of novel QS inhibitors.

Prokaryotic expression of DFP1 and DFP2 in Dermatophagoides farinae and their responses to temperature stress

The functions of highly expressed genes DFP1 and DFP2 in Dermatophagoides farinae remain unknown. DFP1 and DFP2 have been abundantly annotated and were up-regulated under temperature stress at 43 °C and –10 °C in our previous RNA-seq study, indicating that DFP1 and DFP2 may have temperature stress response function. Here, we amplified, cloned, and sequenced to obtain the complete coding sequences of DFP1 and DFP2 and predicted their protein characteristics using bioinformatics analysis. Then, prokaryotic expression systems were constructed and found that DFP1 was expressed in Escherichia coli Rosetta-gami 2 (DE3) but not BL21 (DE3); DFP2 was expressed in both BL21 (DE3) and Rosetta-gami 2 (DE3), with higher expression in BL21 (DE3). Finally, the growth curves of bacteria were drawn and indicated that the DFP1- and DFP2-pET32a carrying recombinant bacteria grew better than the respectiveonly pET32a carrying control bacteria after heat and cold stress. This study confirms for the first time that DFP1 and DFP2 respond to temperature stress at the protein level. The constructed prokaryotic expression systems will provide an experimental foundation for future antibody preparation for western blotting detection to confirm the temperature-stress response functions of DFP1 and DFP2.

Gene cloning, expression and performance validation of nitric oxide dismutase

Nitrous oxide (N2O) is a significant contributor to global warming and possesses an ozone-depleting impact nearly 298 times that of CO2. To reduce N2O emissions, the newly-discovered nod gene which can directly convert NO into N2 and O2 was successfully cloned from the anaerobic denitrification sludge. The recombinant plasmid containing the nod gene was built, and the expression of nod gene in Escherichia coli was determined, leading to the construction of recombinant engineering bacteria. Results showed that the recombinant engineering bacteria E. coli BL21 (DE3)-pET28a-nod could autonomously degrade NO, with a degradation rate of 72 % within 48 h, and could produce 2479.72 ppm of N2 and 75.12 mL of O2. The cumulative O2 production of the sludge sample and recombinant E. coli within 8 h was 1.75 mL and 8.45 mL, respectively. The cumulative O2 production of recombinant E. coli was at least 4.82 times higher than that of the sludge sample. The investigation proposed a new biodegradation pathway for nitrogen pollution.

Rational design improves both thermostability and activity of a new D-tagatose 3-epimerase from Kroppenstedtia eburnean to produce D-allulose

D-allulose is a naturally occurring rare sugar and beneficial to human health. However, the efficient biosynthesis of D-allulose remains a challenge. Here, we mined a new D-tagatose 3-epimerase from Kroppenstedtia eburnean (KeDt3e) with high catalytic efficiency. Initially, crucial factors contributing to the low conversion of KeDt3e were identified through crystal structure analysis, density functional theory calculations (DFT), and molecular dynamics (MD) simulations. Subsequently, based on the mechanism, combining restructuring the flexible region, proline substitution based onconsensus sequence analysis, introducing disulfide bonds, and grafting properties, and reshaping the active center, the optimal mutant M5 of KeDt3e was obtained with enhanced thermostability and activity. The optimal mutant M5 exhibited an enzyme activity of 130.8 U/mg, representing a 1.2-fold increase; Tm value increased from 52.7 °C to 71.2 °C; and half-life at 55 °C extended to 273.7 min, representing a 58.2-fold improvement, and the detailed mechanism of performance improvement was analyzed. Finally, by screening the ribosome-binding site (RBS) of the optimal mutant M5 recombinant bacterium (G01), the engineered strain G08 with higher expression levels was obtained. The engineered strain G08 catalyzed 500 g/L D-fructose to produce 172.4 g/L D-allulose, with a conversion of 34.4% in 0.5 h and productivity of 344.8 g/L/h on a 1 L scale. This study presents a promising approach for industrial-scale production of D-allulose.

Application of Recombinant Lactic Acid Bacteria (LAB) Live Vector Oral Vaccine in the Prevention of F4+ Enterotoxigenic Escherichia coli.

Enterotoxigenic Escherichia coli (ETEC) causes severe diarrhea in piglets. The current primary approach for ETEC prevention and control relies on antibiotics, as few effective vaccines are available. Consequently, an urgent clinical demand exists for developing an effective vaccine to combat this disease. Here, we utilized food-grade Lactococcus lactis NZ3900 and expression plasmid pNZ8149 as live vectors, together with the secreted expression peptide Usp45 and the cell wall non-covalent linking motif LysM, to effectively present the mutant LTA subunit, the LTB subunit of heat-labile enterotoxin, and the FaeG of F4 pilus on the surface of recombinant lactic acid bacteria (LAB). Combining three recombinant LAB as a live vector oral vaccine, we assessed its efficacy in preventing F4+ ETEC infection. The results demonstrate that oral immunization conferred effective protection against F4+ ETEC infection in mice and piglets lacking maternal antibodies during weaning. Sow immunization during late pregnancy generated significantly elevated antibodies in colostrum, which protected piglets against F4+ ETEC infection during lactation. Moreover, booster immunization on piglets during lactation significantly enhanced their resistance to F4+ ETEC infection during the weaning stage. This study highlights the efficacy of an oral LAB vaccine in preventing F4+ ETEC infection in piglets by combining the sow immunization and booster immunization of piglets, providing a promising vaccination strategy for future prevention and control of ETEC-induced diarrhea in piglets.

Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9

BackgroundMucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917.ResultsThe HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) – rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/108 cfu.ConclusionsThe CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

Terminal-Enhanced Polymerization in the Biosynthesis of Polysialic Acid

Plasmids are commonly used tools in microbiology and molecular biology and have important and wide-ranging applications in the study of gene function, protein expression, and compound synthesis. The complex relationship between necessary antibiotic addition, compatibility between multiple plasmids, and the growth burden of host bacteria has plagued the wider use of compatibility plasmids. In this study, we constructed the terminal polymerization pathway of PSA by exogenously expressing the neuA, neuD, and neuS genes after the knockdown of Eschesrichia coli BL21 (DE3). Duet series vectors were utilized to regulate the expression level of neuA, neuD, and neuS genes to study the gene expression level, plasmid copy number growth burden, pressure of antibiotic addition, stability of compatible plasmids, and the level of expression stability of exogenous genes, as well as the effect on the biological reaction process. The results showed that the three genes, neuA, neuD, and neuS, were enhanced in the recombinant strain E. coli NA-05, with low copy, medium copy, and high copy, respectively. The effect of PSA synthesis under standard antibiotic pressure was remarkable. The results of this thesis suggest the use of a Duet series of compatible expression vectors to achieve the stable existence and co-expression of multiple genes in recombinant bacteria, which is a good reason for further research.

A Zero-Valent Sulfur Transporter Helps Podophyllotoxin Uptake into Bacterial Cells in the Presence of CTAB.

Podophyllotoxin (PTOX) is naturally produced by the plant Podophyllum species. Some of its derivatives are anticancer drugs, which are produced mainly by using chemical semi-synthesis methods. Recombinant bacteria have great potential in large-scale production of the derivatives of PTOX. In addition to introducing the correct enzymes, the transportation of PTOX into the cells is an important factor, which limits its modification in the bacteria. Here, we improved the cellular uptake of PTOX into Escherichia coli with the help of the zero-valent sulfur transporter YedE1E2 in the presence of cetyltrimethylammonium bromide (CTAB). CTAB promoted the uptake of PTOX, but induced the production of reactive oxygen species. A protein complex (YedE1E2) of YedE1 and YedE2 enabled E. coli cells to resist CTAB by reducing reactive oxygen species, and YedE1E2 was a hypothetical transporter. Further investigation showed that YedE1E2 facilitated the uptake of extracellular zero-valent sulfur across the cytoplasmic membrane and the formation of glutathione persulfide (GSSH) inside the cells. The increased GSSH minimized oxidative stress. Our results indicate that YedE1E2 is a zero-valent sulfur transporter and it also facilitates CTAB-assisted uptake of PTOX by recombinant bacteria.

Both recombinant Bacillus subtilis Expressing PCV2d Cap protein and PCV2d-VLPs can stimulate strong protective immune responses in mice

Porcine circovirus type 2 (PCV2) is one of the most serious pathogens in pig herds worldwide. The Capsid protein (Cap), a structural protein of PCV2, is involved in the host's immune response; it induces neutralizing-antibody production and has good immunogenicity. The main PCV2 subtype currently prevalent in the Chinese pig herd is PCV2d. In this study, We constructed a recombinant Bacillus subtilis (B. subtilis) capable of secreting Cap protein, named pHT43-Cap/B. subtilis; we concentrated the supernatant of the recombinant bacteria and observed virus-like particles (VLPs) of PCV2d formed by Cap protein under transmission electron microscopy, named PCV2d-VLPs. The immunocompetence of the pHT43-Cap/B. subtilis and PCV2d-VLPs were then assessed by oral administration and by intramuscular injection into mice, respectively. The results showed that the levels of PCV2d-Cap protein-specific IgG in the serum and of PCV2d-Cap protein-specific sIgA in the small intestinal fluid of pHT43-Cap/B. subtilis immunized mice were elevated compared to the control group, both of them highly significant (p < 0.01), and the corresponding serum-specific IgG antibodies were effective in neutralizing PCV2d virulence. The virus load in the liver of the immunized mice was significantly lower than that in the control group (p < 0.01), as was the virus load in the spleen and lungs of the immunized mice (p < 0.05). In addition, the serum levels of PCV2d-Cap-specific IgG in mice immunized with PCV2d-VLPs by intramuscular injection were significantly elevated compared to the control group (p < 0.05), and the viral load in all tissues was significantly lower in immunized mice (p < 0.05). In conclusion, the recombinant bacterium pHT43-Cap/B. subtilis can induce effective mucosal and humoral immunity in mice, PCV2d-VLPs can induce humoral immunity in mice, and both vaccines have good immunogenicity; these results provide a theoretical and material basis for the development of a new vaccine against PCV2d.

Could Bioluminescent Bacteria be Used in the Search for New Plant-derived Antibacterial Substances?

Introduction. Currently, the search for new antibacterial substances is an urgent task due to the growing resistance of pathogens to existing antibiotics. One of the key directions in this area is the expansion of scientific research of medicinal plants, as new sources of therapeutic agents. This article examines the possibility of using highly sensitive bioluminescent test bacteria for these purposes, which can quickly detect non-specific antimicrobial activity and can be adapted to highly effective pharmaceutical screening technologies.Aim. To study the applicability of bioluminescent bacteria for the analysis of the antibacterial activity of biologically active substances (BAS) of plant origin.Materials and methods. BAS quercetin, 8-hydroxyquinoline, gallic acid and thymoquinone, which are often found in medicinal plant raw materials and with which its antibacterial properties are associated, were used in the work. Bacteria with constitutive bioluminescence Aliivibrio fischeri F1 and Escherichia coli (pXen7), as well as recombinant bioreporter strains with inducible luminescence were used as test-objects: E. coli (pRecA-lux), E. coli (pColD-lux), reacting to nucleic acid damage; E. coli (pKatG-lux) and E. coli (pSoxS-lux), sensitive to oxidative stress.Results and discussion. It was found that the nonspecific antimicrobial activity of the studied BAS is manifested in the inhibition of bacterial bioluminescence of test-strains with constitutive glowing. It was noted that the marine test-bacteria A. fischeri F1 have significantly greater sensitivity to the action of BAS, compared with the recombinant strain of E. coli (pXen7). It has been shown that their inhibitory effect begins at concentrations of 2 mcg/ml, and bactericidal activity occurs at concentrations of more than 20 mcg/ml. The results obtained are compared with the data on MIC and MBC of gram(+) and gram(–) pathogens. The study of the induction of bioluminescence of recombinant bioreporter strains showed that the antibacterial effect of the BAS is accompanied by oxidative stress. Also, quercetin caused activation of luminescence in E. coli (pRecA-lux) and E. coli (pColD-lux), which may indicate its participation in damage to nucleic acids. Analysis of the induction factors of bioreporter strains indicates that the revealed mechanisms of antibacterial activity are not major, but may be of a secondary nature.Conclusion. It has been shown that the intensity of the glow of natural and recombinant bioluminescent bacteria can be an indicator of the antibacterial activity of BAS of natural origin. The high sensitivity of A. fischeri F1 bacteria to the action of substances such as quercetin, 8-hydroxyquinoline, gallic acid and thymoquinone has been shown. Considering that bioluminescence analysis is a quantitative instrumental method, it can be easily adapted for high-throughput pharmaceutical screening. It has been shown that the luminescence intensity of natural and recombinant bioluminescent bacteria can be an indicator of the antibacterial activity of BAS of natural origin. The high sensitivity of A. fischeri F1 to the action of substances such as quercetin, 8-hydroxyquinoline, gallic acid and thymoquinone has been established. Taking in an account that bioluminescent analysis is a quantitative instrumental method, it can be easily adapted for high-throughput pharmaceutical screening.

Functional contacts for activation of urease from Helicobacter pylori: an integrated approach using evolutionary couplings, in-cell enzymatic assays, and computational docking

Introduction: Urease is an enzyme exploited by many virulent bacteria and fungi to infect the host and exert their virulence. The Gram-negative bacterium Helicobacter pylori relies on the activity of urease to infect the highly acidic human stomach. The activity of urease depends on the presence of a catalytic site containing two Ni(II) ions. In vivo, urease is initially synthesized as an inactive apo-enzyme and requires a post-translational activation process that involves the incorporation of the metal ions into its buried active site. In H. pylori, as well as in other bacteria, this activation process is mediated by four accessory proteins, named UreD, UreF, UreG, and UreE. Targeting the interactions between urease chaperones could potentially inhibit the activation of urease through blocking the Ni(II) ions incorporation, providing a route for the development of antimicrobial strategies against ureolytic pathogens.Methods: In this paper, an evolutionary couplings (EC) approach was adopted to determine the interaction surface between urease and UreD, the first protein that binds the enzyme, preparing it for the subsequent activation steps. Site-directed mutagenesis and an in-cell assay were used to detect urease activity in recombinant bacteria expressing the mutated operon. The obtained data were used to drive a protein-protein docking computational approach.Results and Discussion: The EC prediction retrieved ten pairs of residues lying at the interface between UreD and the urease subunit UreB, likely involved in contacts essential to build the protein complex. These contacts were largely confirmed experimentally, leading to the obtainment of a model for the urease-UreD complex that agrees well with the recently reported experimental cryo-EM structure. This work represents a proof of concept for the calculation of reliable models of protein interaction surfaces in the absence of experimental structures of critical assemblies.

Mucosal Vaccines against Bacterial and Viral Pathogens

The mucosal membranes of the human body play a crucial role in the development, maintenance, and regulation of barrier functions and immune homeostasis, representing an integral component of the overall immune system. Mucosal vaccines elicit immune processes in the lymphoid tissue associated with the mucosal membranes. A critical objective of mucosal immunization is the identification of an antigen delivery vector capable of ensuring optimal vaccine efficacy. The authors of this article have conducted extensive research on the probiotic properties of enterococci over an extended period. They employ a safe and beneficial probiotic strain, Enterococcus faecium L3, as a delivery vector for vaccine antigens. Initially, the gene encoding the pathogenicity factor Bac, derived from group B streptococci (Streptococcus agalactiae), was successfully integrated into the genome of the probiotic strain E. faecium L3. Intravaginal, oral, and intranasal mucosal immunization methods utilizing the L3-Bac+ probiotic, which expresses antigenic determinants of pathogenic streptococci, were found to confer protection against bacterial infection in laboratory animals. Subsequently, recombinant technologies were refined, leading to the development of a universal method for incorporating a region of interest from the gene into the structure of the major pili protein gene of E. faecium L3. Using this technology, candidate vaccines against various infections, including Streptococcus pneumoniae, influenza A virus, and SARS-CoV-2 following the onset of the Covid-19 pandemic, have been obtained and tested. In this study, alongside the presentation of our own data, the challenges associated with utilizing recombinant probiotic bacteria as vectors for vaccine antigen delivery are discussed.

High-precision targeting and destruction of cancer-associated PDGFR-β+ stromal fibroblasts through self-assembling, protein-only nanoparticles

The need for more effective and precision medicines for cancer has pushed the exploration of new materials appropriate for drug delivery and imaging, and alternative receptors for targeting. Among the most promising strategies, finding suitable cell surface receptors and targeting agents for cancer-associated platelet derived growth factor receptor β (PDGFR-β)+ stromal fibroblasts is highly appealing. As a neglected target, this cell type mechanically and biologically supports the growth, progression, and infiltration of solid tumors in non-small cell lung, breast, pancreatic, and colorectal cancers. We have developed a family of PDGFR-β-targeted nanoparticles based on biofabricated, self-assembling proteins, upon hierarchical and iterative selective processes starting from four initial candidates. The modular protein PDGFD-GFP-H6 is well produced in recombinant bacteria, resulting in structurally robust oligomeric particles that selectively penetrates into PDGFR-β+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-β ligand PDGFD. Upon in vivo administration, these GFP-carrying protein nanoparticles precisely accumulate in tumor tissues and enlighten them for IVIS observation. When GFP is replaced by a microbial toxin, selective tumor tissue destruction is observed associated with a significant reduction in tumor volume growth. The presented data validate the PDGFR-β/PDGFD pair as a promising toolbox for targeted drug delivery in the tumor microenvironment and oligomeric protein nanoparticles as a powerful instrument to mediate highly selective biosafe targeting in cancer through non-cancer cells. Statement of significanceWe have developed a transversal platform for nanoparticle-based drug delivery into cancer-associated fibroblasts. This is based on the engineered modular protein PDGFD-GFP-H6 that spontaneously self-assemble and selectively penetrates into PDGFR-β+ stromal fibroblasts in a dose-dependent manner, by means of the PDGFR-β ligand PDGFD. In vivo, these protein nanoparticles accumulate in tumor and when incorporating a microbial toxin, they destroy tumor tissues with a significant reduction in tumor volume, in absence of side toxicities. The data presented here validate the PDGFR-β/PDGFD pair as a fully versatile toolbox for targeted drug delivery in the tumor microenvironment intended as a synergistic treatment.

Bioluminescent monitoring of recombinant lactic acid bacteria and their products.

Lactic acid bacteria constitute a genetically diverse group of microorganisms with significant roles in the food industry, biotechnology, agriculture, and medicine. A core understanding of bacterial physiology in diverse environments is crucial to select and develop bacteria for industrial and medical applications. However, there is a lack of versatile tools to track (recombinant) protein production in lactic acid bacteria. In this study, we adapted a peptide-based bioluminescent tagging system that is functional across multiple genera and species. This system enables tracking of tagged proteins both in vitro and in situ, while it also can be used to enumerate recombinant bacteria from the mouse gastrointestinal tract with accuracy comparable to that of conventional plate counts. Our work expands the lactic acid bacteria genetic toolbox and will facilitate researchers in industry and academia with opportunities to monitor microbes and proteins under different physiologically relevant conditions.