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Outer membrane vesicles (OMVs) play vital roles in bacterial communication both intraspecifically and interspecifically. However, extracellular mechanisms of gut microbiota–derived OMVs in the intestine remain poorly understood. Here, we report that OMVs released from Akkermansia muciniphila are able to (i) restore disturbed balance of the gut microbiota by selectively promoting the proliferation of beneficial bacteria through membrane fusion, (ii) elicit mucosal immunoglobulin A response by translocating into Peyer’s patches and subsequently activating B cells and dendritic cells, and (iii) maintain the integrity of the intestinal barrier by entering intestinal epithelial cells to stimulate the expressions of tight junctions and mucus. We demonstrate that transplantation of gut microbiota–associated OMVs to the intestine can alleviate colitis and enhance anti–programmed cell death protein 1 therapy against colorectal cancer by regulating intestinal homeostasis. This work discloses the importance of gut microbiota–derived OMVs in intestinal ecology, providing an alternative target for disease intervention and treatment.

Despite the fact that oral microecologics are effective in modulating the gut microbiome, they always suffer from multiple insults during the journey from manufacture to arrival at the intestine. Inspired by the protective mechanism of mineralization, we describe a cytocompatible approach of biointerface mineralization that can generate an ultraresistant and self-removable coating on bacterial surface to solve these challenges. Mineral coating endows bacteria with robust resistances against manufacture-associated oxygen exposure, ultraviolet irradiation, and 75% ethanol. Following oral ingestion, the coating is able to actively neutralize gastric acid and release encapsulated bacteria through spontaneous yet rapid double-decomposition reaction. In addition to acid neutralization, the generated calcium ions can trigger micellar aggregation of bile acid, enabling dual exemptions from the insults of gastric acid and bile acid to achieve uncompromised bacterial viability. Further supported by the therapeutic efficacy of coated bacteria toward colitis mice, biointerface mineralization provides a versatile platform for developing next-generation living oral biotherapeutics.

Approaches capable of simultaneously treating cancer and preventing susceptible patients from lethal infections, such as coronavirus disease 2019, are highly desirable but have proven to be difficult. Here, dressing bacteria with a hybrid immunoactive nanosurface is reported to elicit dual anticancer and antiviral immunity. A combination of checkpoint blocking antibody and virus-specific antigen is covalently conjugated to polydopamine nanoparticles, which can be anchored onto bacterial surface, by a one-step in-situ polymerization of dopamine under a cell-friendly condition. By virtue of the ability to colonize and penetrate deep tumor tissue, dressed bacteria enable sustained release and expanded exposure of carried immunoactivators to stimulate immune cells. In addition to a carrier role, bacteria are able to further provoke innate immunity due to native immunogenicity of the pathogen-associated molecular patterns. Immunization with dressed bacteria promote the maturation and activation of antigen-presenting cells, which induces robust humoral and cellular immune responses in tumor-bearing mice. As evidenced by efficient production of viral antigen-specific immunoglobulin G antibody in serum and significantly suppressed tumor growth in different models, dressing bacteria with a hybrid immunoactive nanosurface paves an avenue to prepare next-generation therapeutics for synergistic treatment and prevention. This article is protected by copyright. All rights reserved.

Bacteriophages (phages) are widely explored as antimicrobials for treating infectious diseases due to their specificity and potency to infect and inhibit host bacteria. However, the application of phages to inhibit intracellular pathogens has been greatly restricted by inadequacy in cell entry and endosomal escape. Here, we describe the use of cationic polymers to selectively cap negatively charged phage head rather than positively charged tail by electrostatic interaction, resulting in charge-reversed phages with uninfluenced vitality. Given the positive surface charge and proton sponge effect of the nanocapping, capped phages are able to enter intestinal epithelial cells and subsequently escape from endosomes to lyse harbored pathogens. In a murine model of intestinal infection, oral ingestion of capped phages significantly reduces the translocation of pathogens to major organs, showing a remarkable inhibition efficacy. Our work proposes that simple synthetic nanocapping can manipulate phage bioactivity, offering a facile platform for preparing next-generation antimicrobials.

Despite bacterial-mediated biotherapies have been widely explored for treating different types of cancer, their implementation has been restricted by low treatment efficacy, due largely to the absence of tumor-specific accumulation following administration. Here, the conjugation of aptamers to bacterial surface is described by a simple and cytocompatible amidation procedure, which can significantly promote the localization of bacteria in tumor site after systemic administration. The surface density of aptamers can be easily adjusted by varying feed ratio and the conjugation is able to increase the stability of anchored aptamers. Optimal bacteria conjugated with an average of 2.8 × 105 aptamers per cell present the highest specificity to tumor cells in vitro, separately generating near 2- and 4-times higher accumulation in tumor tissue at 12 and 60 hours compared to unmodified bacteria. In both 4T1 and H22 tumor-bearing mouse models, aptamer-conjugated attenuated Salmonella show enhanced antitumor efficacy, along with highly activated immune responses inside the tumor. This work demonstrates how bacterial behaviors can be tuned by surface conjugation and supports the potential of aptamer-conjugated bacteria for both targeted intratumoral localization and enhanced tumor biotherapy., Bacteria-based therapy has shown promise for cancer treatment. To enhance tumor accumulation, here the authors describe the design of tumor specific aptamer-conjugated bacteria, to improve intratumor localization and enhance therapeutic efficacy.

Chirality is ubiquitous in biological systems, which is closely related to biological functions, life processes, and even the pathogenesis of diseases. However, the interface between the chirality of synthetic materials and organisms, particularly the immune system, remains poorly understood. Here, supramolecular chiral polymer micelles (SCPMs) are prepared by complexing antigenic proteins with chiral amino acid-modified polyethyleneimine. The introduction of chirality not only reduces the toxicity of cationic polymer, but also benefits cell uptake and antigen presentation. Especially, D-chirality presents the lowest cytotoxicity, while promoting the highest expression level of costimulatory molecules on dendritic cells compared to L-chirality and achirality. The superiority of D-chirality to stimulate dendritic cell maturation is supported by immunization with D-SCPMs, which achieves significant antigen-specific proliferation of T cells in the spleen, lymph nodes, and tumor of mice. Chirality-mediated antigen processing and presentation are demonstrated by D-SCPMs self-assembled from chiral alkaline histidine or neutral phenylalanine modified polyethyleneimine and tumor associated ovalbumin or severe acute respiratory syndrome coronavirus 2 spike 1 antigenic protein. Immunoactivation enabled by D-chirality opens a window to prepare potent nanotherapeutics for disease prevention and treatment.

The oral delivery of macromolecules is quite challenging due to environmental insults and biological barriers encountered along the gastrointestinal (GI) tract. Benefiting from their living characteristics, diverse bacterial species have been engineered as intelligent platforms to deliver various therapeutics. To tackle difficulties in oral delivery, innovative bacteria-based microdevices have been developed by virtue of advancements in synthetic biology and nanotechnology, with aims to overcome the instability and short half-life of macromolecules in the GI tract. In this review, we summarize the main classes of macromolecules that are produced and delivered through the oral ingestion of bacteria and bacterial derivatives. Furtherly, we discuss the engineering strategies and biomedical applications of these living microdevices in disease diagnosis, bioimaging, and treatment. Finally, we highlight the advantages as well as the limitations of these engineered bacteria used as platforms for the oral delivery of macromolecules and also propose their potential for clinical translation. The results summarized in this review article would contribute to the invention of next-generation bacteria-based systems for the oral delivery of macromolecules.

Disorders in the gut microbiota have been implicated in various diseases, such as inflammatory bowel diseases, diabetes, and cancers. Oral microecologics are of great importance due to their ability to directly intervene the gut microbiome as well as their noninvasiveness and low side effects, while have suffered from low bioavailability and a single therapeutic effect. Here, probiotics are coated with a therapeutic nanocoating for synergistically enhanced biotherapy, a method inspired by the robust protective and therapeutic effectiveness of silkworm cocoon. With its transition from a random coil to β-sheet conformation, silk fibroin can self-assemble onto the surface of bacteria. By a simple layer-by-layer procedure, an entire nanocoating can be formed along with a near quantitative coating ratio and almost uninfluenced bacterial viability. Thanks to its protective barrier role and innate pharmaceutical activity, silk fibroin nanocoating endows the coated bacteria with a markedly improved survival against gastric insults and a synergistically enhanced therapeutic effect in a murine model of intestinal mucositis. This work demonstrates how therapeutic elements can be combined with probiotics via a simple coating strategy and proposes an alternative to enhance bioavailability and treatment efficacy of oral microecologics.

Significance Staphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a type VII secretion system that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a type VII substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo., The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date, only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane-depolarizing activity is alleviated by coproduction of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologs at the tspA locus, suggestive of additional roles in intraspecies competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologs.

Probiotic self-coated with biofilms achieve strikingly enhanced oral availability and intestinal colonization in a porcine model., Transplanting beneficial bacteria to the gut microbiome can positively modulate the bacterial composition and remains of great interest in prevention and treatment. However, environmental assaults and rapid transit times in the gastrointestinal (GI) tract result in low oral bioavailability and limited intestinal colonization. Here, we describe a bioinspired strategy of self-coating with biofilms that endows the transplanted gut microbiota with superior resistance and adhesion capacity. Using clinical Bacillus subtilis as a model probiotic bacterium, biofilm-coated probiotics demonstrate substantially improved GI tract tolerance and mucoadhesion in mice and swine. In particular, coated probiotics exhibit a 125-fold higher oral bioavailability and a 17 times greater intestinal colonization than uncoated bacteria in the porcine model. With notable ability to survive and reside in the GI tract, coated bacteria further show a significantly enhanced decolonization effect in mice colonized with Staphylococcus aureus. Self-coating with biofilms suggests a robust platform for oral doses of gut microbiota.

The application of bacteria and bacteria-derived membrane vesicles (MVs) has promising potential to make a great impact on the development of controllable targeted drug delivery for combatting cancer. Comparing to most other traditional drug delivery systems, bacteria and their MVs have unique capabilities as drug carriers for cancer treatment. They can overcome physical barriers to target and accumulate in tumor tissues and initiate antitumor immune responses. Furtherly, they are able to be modified both genetically and chemically, to produce and transport anticancer agents into tumor tissues with improved safety and efficacy of cancer treatment but decreased cytotoxic effects to normal cells. In this review, we present some examples of tumor-targeting bacteria and bacteria-derived MVs for the delivery of anticancer drugs, including chemo-therapeutic, radio-therapeutic, photothermal-therapeutic, and immuno-therapeutic agents. We also discuss the advantages as well as the limitations of these tumor-targeting bacteria and their MVs used as platforms for controlled delivery of anticancer therapeutic agents, and further highlight their great potential on clinical translation.

Methods capable of distributing antitumor therapeutics uniformly and durably throughout an entire tumor would be of great significance in maximizing their treatment efficacy, but they have proven to be extremely challenging. Here, bacteria-mediated spatiotemporally controllable distribution of combination therapeutics in solid tumors is reported to reprogram the immune microenvironment for optimizing antitumor efficacy. By combining synthetic biology and interfacial chemistry, bacteria are inside and outside concurrently modified to express photothermal melanin and to attach immune checkpoint inhibitors on their surface. Due to the nature of bacteria to colonize the hypoxia intratumoral environment, both therapeutic agents can be distributed homogenously and lastingly in tumors during ex vivo human and in vivo mouse studies. Spatiotemporally controllable localization of melanin can repeatedly generate a moderate yet uniform heating of the tumor upon light exposure in a broad treatment window. Combination with similarly localized inhibitors elicits a dual photothermally stimulated and checkpoint-blockade-mediated immune activation effect, synergistically reprogramming the immunosuppressive tumor microenvironment. Therapeutic values are demonstrated by significantly inhibited tumor growth and prolonged survival of mice in both subcutaneous and orthotopic murine models. Colonization of dually modified bacteria paves an avenue for spatiotemporally controllable distribution of therapeutic drugs in solid tumors.

Bacteria have been extensively utilized for bioimaging, diagnosis and therapy given their unique characteristics including genetic manipulation, rapid proliferation and disease site targeting specificity. However, clinical translation of bacteria for these applications has been largely restricted by their unavoidable side effects and low treatment efficacies. Engineered bacteria for biomedical applications ideally need to generate only a low inflammatory response, show slow elimination by macrophages, low accumulation in normal organs, and almost unchanged inherent bioactivities. Here we describe a set of stealth bacteria, cell membrane coated bacteria (CMCB), meeting these requirement. Our findings are supported by evaluation in multiple mice models and ultimately demonstrate the potential of CMCB to serve as efficient tumor imaging agents. Stealth bacteria wrapped up with cell membranes have the potential for a myriad of bacterial-mediated biomedical applications., The use of engineered bacteria for biomedical applications is limited by side effects such as inflammatory response. Here the authors engineer cell membrane coated bacteria as in vivo tumor imaging agents, and show that these generate a lower inflammatory response and reduced macrophage clearance.

Bacteria have been widely exploited as bioagents for applications in diagnosis and treatment, benefitting from their living characteristics including colonization, rapid proliferation, and facile genetic manipulation. As such, bacteria being tailored to perform precisely in the right place at the right time to avoid potential side effects would be of great importance but has proven to be difficult. Here, a strategy of on-demand bacterial reactivation is described by individually restraining within a triggerable nanocoating. Upon reaching at a location of interest, nanocoatings can be triggered to dissolution in situ and subsequently decoat the bacteria which are able to recover their bioactivities as needed. It is demonstrated that gut microbiota coated with an enteric nanocoating can respond to gastrointestinal environments and reactivate in the intestine by a pH-triggered decoating. In virtue of this unique, coated bacteria remain inactive following oral administration to exempt acidic insults, while revive to restore therapeutic effects after gastric emptying. Consequently, improved oral availability and treatment efficacy are achieved in two mouse models of intestinal infection. Bacteria restrained by a triggerable nanocoating represent a smart therapeutic that can take effect when necessary. On-demand bacterial reactivation suggests a robust platform for the development of precision bacterial-mediated bioagents.

The type VII protein secretion system (T7SS) is conserved across Staphylococcus aureus strains and plays important roles in virulence and interbacterial competition. To date only one T7SS substrate protein, encoded in a subset of S. aureus genomes, has been functionally characterized. Here, using an unbiased proteomic approach, we identify TspA as a further T7SS substrate. TspA is encoded distantly from the T7SS gene cluster and is found across all S. aureus strains as well as in Listeria and Enterococci. Heterologous expression of TspA from S. aureus strain RN6390 indicates its C-terminal domain is toxic when targeted to the Escherichia coli periplasm and that it depolarizes the cytoplasmic membrane. The membrane depolarizing activity is alleviated by co-production of the membrane-bound TsaI immunity protein, which is encoded adjacent to tspA on the S. aureus chromosome. Using a zebrafish hindbrain ventricle infection model, we demonstrate that the T7SS of strain RN6390 promotes bacterial replication in vivo, and deletion of tspA leads to increased bacterial clearance. The toxin domain of TspA is highly polymorphic and S. aureus strains encode multiple tsaI homologues at the tspA locus, suggestive of additional roles in intra-species competition. In agreement, we demonstrate TspA-dependent growth inhibition of RN6390 by strain COL in the zebrafish infection model that is alleviated by the presence of TsaI homologues.Significance statementStaphylococcus aureus, a human commensal organism that asymptomatically colonizes the nares, is capable of causing serious disease following breach of the mucosal barrier. S. aureus strains encode a Type VII secretion system (T7SS) that is required for virulence in mouse infection models, and some strains also secrete a nuclease toxin by this route that has antibacterial activity. Here we identify TspA, widely found in Staphylococci and other pathogenic bacteria, as a T7 substrate. We show that TspA has membrane-depolarizing activity and that S. aureus uses TspA to inhibit the growth of a bacterial competitor in vivo.

The type VII protein secretion system (T7SS) plays a critical role in the virulence of human pathogens including Mycobacterium tuberculosis and Staphylococcus aureus. Here we report that the S. aureus T7SS secretes a large nuclease toxin, EsaD. The toxic activity of EsaD is neutralised during its biosynthesis through complex formation with an antitoxin, EsaG, which binds to its C-terminal nuclease domain. The secretion of EsaD is dependent upon a further accessory protein, EsaE, that does not interact with the nuclease domain, but instead binds to the EsaD N-terminal region. EsaE has a dual cytoplasmic/membrane localization and membrane-bound EsaE interacts with the T7SS secretion ATPase, EssC, implicating EsaE in targeting the EsaDG complex to the secretion apparatus. EsaD and EsaE are co-secreted whereas EsaG is found only in the cytoplasm and may be stripped off during the secretion process. Strain variants of S. aureus that lack esaD encode at least two copies of EsaG-like proteins most likely to protect themselves from the toxic activity of EsaD secreted by esaD+ strains. In support of this, a strain overproducing EsaD elicits significant growth inhibition against a sensitive strain. We conclude that T7SSs may play unexpected and key roles in bacterial competitiveness.

The cal Y gene, encoding metalloprotease camelysin in the Bacillus thuringiensis acrystalliferous strain XBU001, was amplified and sequenced. The camelysin from the cal Y sequence was 199 amino acids in size ( c . 22000Da). The temperature-sensitive plasmid pKESX was used to construct a metalloprotease camelysin-deficient strain of B. thuringiensis . The cal Y gene was replaced by an erythromycin-resistant gene in KCTF. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and MS analysis showed that the metalloprotease InhA was not expressed after knocking out the gene cal Y. The temperature-sensitive plasmid pKPC was used to construct a metalloprotease camelysin complementation strain KCTFC. The InhA protein was found in KCTFC. Analysis of the expression of InhA in the wild-type strain KCTF12, camelysin-deficient and complementation strains indicated that inh A expression depended on camelysin. Although camelysin did not directly regulate the expression of the InhA through binding to the promoter of the inh A, the results suggest that camelysin can positively regulate the expression of the InhA protein.

The proteome of a new Bacillus thuringiensis subsp. kurstaki strain, 4.0718, from the middle vegetative ( T 1 ), early sporulation ( T 2 ), and late sporulation ( T 3 ) phases was analyzed using an integrated liquid chromatography (LC)-based protein identification system. The system comprised two-dimensional (2D) LC coupled with nanoscale electrospray ionization (ESI) tandem mass spectrometry (MS/MS) on a high-resolution hybrid mass spectrometer with an automated data analysis system. After deletion of redundant proteins from the different batches and B. thuringiensis subspecies, 918, 703, and 778 proteins were identified in the respective three phases. Their molecular masses ranged from 4.6 Da to 477.4 Da, and their isoelectric points ranged from 4.01 to 11.84. Function clustering revealed that most of the proteins in the three phases were functional metabolic proteins, followed by proteins participating in cell processes. Small molecular and macromolecular metabolic proteins were further classified according to the Kyoto Encyclopedia of Genes and Genome and BioCyc metabolic pathway database. Three protoxins (Cry2Aa, Cry1Aa, and Cry1Ac) as well as a series of potential intracellular active factors were detected. Many significant proteins related to spore and crystal formation, including sporulation proteins, help proteins, chaperones, and so on, were identified. The expression patterns of two identified proteins, CotJc and glutamine synthetase, were validated by Western blot analysis, which further confirmed the MS results. This study is the first to use shotgun technology to research the proteome of B. thuringiensis . Valuable experimental data are provided regarding the methodology of analyzing the B. thuringiensis proteome (which can be used to produce insecticidal crystal proteins) and have been added to the related protein database.

Double wires submerged arc welding is more and more widely applied. However, due to the immaturity of the welding process, the quality of double wires submerged arc welding is still not so good, and its application in the offshore platform engineering is limited. The paper adopt the simple flat plate welding, uses the finite element software ANSYS to simulates the temperature field of double wire submerged arc welding process. During the temperature field simulation process, double ellipsoid heat source model is used as a load heat source, composite convection coefficient is adopted to simulate heat loss, the element's birth and death is considered to simulate welding material injection, welding speed is assigned as a constant. At last, a good result of temperature field is obtained.

The calY gene, encoding metalloprotease camelysin in the Bacillus thuringiensis acrystalliferous strain XBU001, was amplified and sequenced. The camelysin from the calY sequence was 199 amino acids in size (c. 22 000 Da). The temperature-sensitive plasmid pKESX was used to construct a metalloprotease camelysin-deficient strain of B. thuringiensis. The calY gene was replaced by an erythromycin-resistant gene in KCTF. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and MS analysis showed that the metalloprotease InhA was not expressed after knocking out the gene calY. The temperature-sensitive plasmid pKPC was used to construct a metalloprotease camelysin complementation strain KCTFC. The InhA protein was found in KCTFC. Analysis of the expression of InhA in the wild-type strain KCTF12, camelysin-deficient and complementation strains indicated that inhA expression depended on camelysin. Although camelysin did not directly regulate the expression of the InhA through binding to the promoter of the inhA, the results suggest that camelysin can positively regulate the expression of the InhA protein.

To explore the feasibility of cloning of the hepatocyte receptor interacting with the Pre S1 protein of HBV by two hybrid system.Yeast expression plasmids encoding fusion proteins of full length or portions of Pre S1 of HBV and DNA binding domain of yeast protein GAL4 were constructed and used to transform yeast reporter strain SFY526. Reporter gene product ?galactosidase activity was assayed as a measure of transcription activation in yeast. Mammalian expression plasmid encoding fusion proteins of full length Pre S1 and DNA binding domain of GAL4 was constructed and used to cotransfect hepatoma cell line Huh?7 together with CAT reporter plasmid. Cell extracts were assayed for CAT activity by thin?layer chromatography.The fusion proteins of full length Pre S1 protein and GAL4 DNA binding domain present transcriptional activation function in yeast. The transcription activating sequence is localized to the 21 to 47 amino acids of Pre S1 protein Fusion proteins of full length Pre S 1 and GAL 4 DNA binding domain do not show transcriptional activation function in mammalian cells.The transcriptional activating sequence of HBVPre S1 protein in yeast overlaps the hepatocyte receptor binding site. The transcriptional activation function of HBV Pre S1 protein in yeast may prevent researchers?from using yeast two hybrid system to clone HBV receptor interacting with Pre S1 protein. However, the Pre S1 protein does not show transcriptional activation function in mammalian cells. Mammalian two?hybrid system may be a practical method to clone the HBV hepatocyte receptor interacting with Pre S1 protein.