Implementing Optogenetic-Controlled Bacterial Systems in Drosophila melanogaster for Alleviation of Heavy Metal Poisoning.

Drosophila melanogaster (fruit fly) is an animal model chassis in biological and genetic research owing to its short life cycle, ease of cultivation, and acceptability to genetic modification. While the D. melanogaster chassis offers valuable insights into drug efficacy, toxicity, and mechanisms, several obvious challenges such as dosage control and drug resistance still limit its utility in pharmacological studies. Our research combines optogenetic control with engineered gut bacteria to facilitate the precise delivery of therapeutic substances in D. melanogaster for biomedical research. We have shown that the engineered bacteria can be orally administered to D. melanogaster to get a stable density of approximately 28,000 CFUs/per fly, leading to no detectable negative effects on the growth of D. melanogaster. In a model of D. melanogaster exposure to heavy metal, these orally administered bacteria uniformly express target genes under green light control to produce MtnB protein for binding and detoxifying lead, which significantly reduces the level of oxidative stress in the intestinal tract of Pb-treated flies. This pioneering study lays the groundwork for using optogenetic-controlled bacteria in the model chassis D. melanogaster to advance biomedical applications.

Sequential delivery of photosensitizers and checkpoint inhibitors by engineered bacteria for enhanced cancer photodynamic immunotherapy.

Engineered bacteria-based cancer therapy has increasingly been considered to be a promising therapeutic strategy due to the development of synthetic biology. Wherein, engineering bacteria-mediated photodynamic therapy (PDT)-immunotherapy shows greater advantages and potential in treatment efficiency than monotherapy. However, the unsustainable regeneration of photosensitizers (PSs) and weak immune responses limit the therapeutic efficiency. Herein, we developed an engineered bacteria-based delivery system for sequential delivery of PSs and checkpoint inhibitors in cancer PDT-immunotherapy. The biosynthetic pathway of 5-aminolevulinic acid (5-ALA) was introduced into Escherichia coli, yielding a supernatant concentration of 172.19 mg/L after 10 h of growth. And another strain was endowed with the light-controllable releasement of anti-programmed cell death-ligand 1 nanobodies (anti-PD-L1). This system exhibited a collaborative effect, where PDT initiated tumor cell death and the released tumor cell fragments stimulated immunity, followed by the elimination of residual tumor cells. The tumor inhibition rate reached 74.97%, and the portion of activated T cells and inflammatory cytokines were reinforced. The results demonstrated that the engineered bacteria-based collaborative system could sequentially deliver therapeutic substance and checkpoint inhibitors, and achieve good therapeutic therapy. This paper will provide a new perspective for the cancer PDT-immunotherapy.

Short-branched alkyl sulfobetaine-passivated CsPbBr3 nanocrystals for efficient green light emitting diodes.

Inorganic cesium lead bromide nanocrystals (CsPbBr3 NCs) hold promising prospects for high performance green light-emitting diodes (LEDs) due to their exceptional color purity and high luminescence efficiency. However, the common ligands employed for passivating these indispensable NCs, such as long-chain organic ligands like oleic acid and oleylamine (OA/OAm), display highly dynamic binding and electronic insulating issues, thereby resulting in a low efficiency of the as-fabricated LEDs. Herein, we report a new zwitterionic short-branched alkyl sulfobetaine ligand, namely trioctyl(propyl-3-sulfonate) ammonium betaine (TOAB), to in situ passivate CsPbBr3 NCs via a feasible one-step solution synthesis, enabling efficiency improvement of CsPbBr3 NC-based LEDs. The zwitterionic TOAB ligand not only strengthened the surface passivation of CsPbBr3 NCs with a high photoluminescence quantum yield (PLQY) of 97%, but also enhanced the carrier transport in the fabricated CsPbBr3 NC thin films due to the short-branched alkyl design. Consequently, CsPbBr3 NCs passivated with TOAB achieved a green LED with an external quantum efficiency (EQE) of 7.3% and a maximum luminance of 5716 cd m-2, surpassing those of LEDs based on insulating long-chain ligand-passivated NCs. Our work provides an effective surface passivation ligand design to enhance the performance of CsPbBr3 NC-based LEDs.

A programmable oral bacterial hydrogel for controllable production and release of nanovaccine for tumor immunotherapy.

Oral protein vaccines are mainly used to prevent the infection of intestinal pathogens in clinic due to their high safety and strong compliance. However, it is necessary to design the efficient delivery systems to overcome the harsh gastrointestinal environment in the application process. Here we established a programmable oral bacterial hydrogel system for spatiotemporally controllable production and release of nanovaccines. The system was divided into three parts: (1) Engineered bacteria were encapsulated in chitosan-sodium alginate microcapsules, which offered protection against the extreme acid conditions in the stomach. (2) Microcapsules were dissolved, and then engineered bacteria were released and colonized in the intestine. (3) The release of nanovaccines was controlled periodically by a synchronous lysis genetic circuit for tumor immunotherapy. Compared to control groups, tumor volume of subcutaneous tumor-bearing mice treated with bacterial microgels releasing optimized nanovaccine was almost inhibited by 75% and T cell response was activated at least 2-fold. We believe that this programmable bacterial hydrogel will offer a promising way for the application of oral nanovaccines.

Pseudohalogen Resurfaced CsPbBr3 Nanocrystals for Bright, Efficient, and Stable Green-Light-Emitting Diodes.

Lead halide perovskite nanocrystals (LHP NCs) are regarded as promising emitters for next-generation ultrahigh-definition displays due to their high color purity and wide color gamut. Recently, the external quantum efficiency (EQE) of LHP NC based light-emitting diodes (PNC LEDs) has been rapidly improved to a level required by practical applications. However, the poor operational stability of the device, caused by halide ion migration at the grain boundary of LHP NC thin films, remains a great challenge. Herein, we report a resurfacing strategy via pseudohalogen ions to mitigate detrimental halide ion migration, aiming to stabilize PNC LEDs. We employ a thiocyanate solution processed post-treatment method to efficiently resurface CsPbBr3 NCs and demonstrate that the thiocyanate ions can effectively inhibit bromide ion migration in LHP NC thin films. Owing to thiocyanate resurfacing, we fabricated LEDs with a high EQE of 17.3%, a maximum brightness of 48000 cd m-2, and an excellent operation half-life time.

Hydrogel-Encapsulated Engineered Microbial Consortium as a Photoautotrophic "Living Material" for Promoting Skin Wound Healing.

Genetically modified engineered microorganisms have been encapsulated in hydrogels and used as "living materials" for the treatment of skin diseases. However, their applications are often limited by the epidermal dry, nutrient-poor environment and cannot maintain functions stably for an expected sufficient time. To solve this problem, a photoautotrophic "living material" containing an engineered microbial consortium was designed and fabricated. The engineered microbial consortium comprised Synechococcus elongatus PCC7942 for producing sucrose by photosynthesis and another heterotrophic engineered bacterium (Escherichia coli or Lactococcus lactis) that can utilize sucrose for the growth and secretion of functional biomolecules. These engineered microorganisms in the "living material" were proved to function stably for a longer time than only individual microbes. Subsequently, CXCL12-secreting engineered L. lactis was used to construct the "living material", and its effect on promoting wound healing was verified in a full-thickness rat-skin defect model. The wounds treated by our hydrogel-encapsulated engineered microbial consortium (HeEMC) healed faster, with a wound area ratio of only 13.2% at day 14, compared to the remaining 62.6, 51.4, and 40.8% of the control, PEGDA, and PEGDA/CS groups, respectively. In conclusion, we established an efficient living material HeEMC to offer promising applications in the treatment of skin diseases.

Upconversion Optogenetic Engineered Bacteria System for Time-Resolved Imaging Diagnosis and Light-Controlled Cancer Therapy.

Engineering bacteria can achieve targeted and controllable cancer therapy using synthetic biology technology and the characteristics of tumor microenvironment. Besides, the accurate tumor diagnosis and visualization of the treatment process are also vital for bacterial therapy. In this paper, a light control engineered bacteria system based on upconversion nanoparticles (UCNP)-mediated time-resolved imaging (TRI) was constructed for colorectal cancer theranostic and therapy. UCNP with different luminous lifetimes were separately modified with the tumor targeting molecule (folic acid) or anaerobic bacteria (Nissle 1917, EcN) to realize the co-localization of tumor tissues, thus improving the diagnostic accuracy based on TRI. In addition, blue light was used to induce engineered bacteria (EcN-pDawn-φx174E/TRAIL) lysis and the release of tumor apoptosis-related inducing ligand (TRAIL), thus triggering tumor cell death. In vitro and in vivo results indicated that this system could achieve accurate tumor diagnosis and light-controlled cancer therapy. EcN-pDawn-φx174E/TRAIL with blue light irradiation could inhibit 53% of tumor growth in comparison to that without blue light irradiation (11.8%). We expect that this engineered bacteria system provides a new technology for intelligent bacterial therapy and the construction of cancer theranostics.

Hydrogel microcapsules containing engineered bacteria for sustained production and release of protein drugs.

Subcutaneous administration of sustained-release formulations is a common strategy for protein drugs, which avoids first pass effect and has high bioavailability. However, conventional sustained-release strategies can only load a limited amount of drug, leading to insufficient durability. Herein, we developed microcapsules based on engineered bacteria for sustained release of protein drugs. Engineered bacteria were carried in microcapsules for subcutaneous administration, with a production-lysis circuit for sustained protein production and release. Administrated in diabetic rats, engineered bacteria microcapsules was observed to smoothly release Exendin-4 for 2 weeks and reduce blood glucose. In another example, by releasing subunit vaccines with bacterial microcomponents as vehicles, engineered bacterial microcapsules activated specific immunity in mice and achieved tumor prevention. The engineered bacteria microcapsules have potential to durably release protein drugs and show versatility on the size of drugs. It might be a promising design strategy for long-acting in situ drug factory.

BifA Triggers Phosphorylation of Ezrin to Benefit Streptococcus equi subsp. zooepidemicus Survival from Neutrophils Killing.

Streptococcus equi subsp. zooepidemicus (SEZ) ATCC35246 can invade the brain and cause severe neutrophils infiltration in brain tissue. This microorganism can survive and reproduce to an extremely high CFU burden (108-109/organ) under stressful neutrophils infiltration circumstances. The aim of this research is to explore the mechanism of the SEZ hypervirulent strain with its specific bifA gene which avoids being eliminated by neutrophils in the brain. We isolated the primary mouse neutrophils to treat SEZ WT and bifA gene defective (ΔBif) strains. The ΔBif strain had a weakened function of defending against neutrophils killing in vitro. The interaction between BifA and ezrin proteins in neutrophils were identified by co-IP and immunoblot. In neutrophils, the BifA interacts with ezrin and triggers the phosphorylation of ezrin at its Thr567 site in a PKC-dependent manner, then the excessive elevation of phosphorylated-ezrin recruits Dbl and activates Rac1. Since the Rac1 is closely relevant to several critical cellular functions, its abnormal activation will lead to neutrophils dysfunction and benefit to SEZ survival from neutrophils killing. Our findings reveal a novel consequence of BifA and ERM family protein (for ezrin, radixin, moesin) interaction, which happens between BifA and ezrin in neutrophils and contributes to SEZ survival in the brain. BifA should be considered as a potential target for drug development to prevent SEZ infection.

Light-Sensitive Lactococcus lactis for Microbe-Gut-Brain Axis Regulating via Upconversion Optogenetic Micro-Nano System.

The discovery of the gut-brain axis has proven that brain functions can be affected by the gut microbiota's metabolites, so there are significant opportunities to explore new tools to regulate gut microbiota and thus work on the brain functions. Meanwhile, engineered bacteria as oral live biotherapeutic agents to regulate the host's healthy homeostasis have attracted much attention in microbial therapy. However, whether this strategy is able to remotely regulate the host's brain function in vivo has not been investigated. Here, we engineered three blue-light-responsive probiotics as oral live biotherapeutic agents. They are spatiotemporally delivered and controlled by the upconversion optogenetic micro-nano system. This micro-nano system promotes the small intestine targeting and production of the exogenous L. lactis in the intestines, which realizes precise manipulation of brain functions including anxiety behavior, Parkinson's disease, and vagal afferent. The noninvasive and real-time probiotic intervention strategy makes the communiation from the gut to the host more controllable, which will enable the potential for engineered microbes accurately and effectively regulating a host's health.

Precise Thermal Regulation of Engineered Bacteria Secretion for Breast Cancer Treatment In Vivo.

For the biomedical application of engineered bacteria, strictly regulating the function of engineered bacteria has always been the goal pursued. However, the existing regulation methods do not meet the needs of the in vivo application of engineered bacteria. Therefore, the exploration of the precise regulation of engineered bacteria is necessary. Herein, heat-sensitive engineered bacteria that can respond to thermal stimuli within 30 min were constructed, and the precise control of functions was verified in the intestines of various model organisms (including C. elegans, bees, and mice). Subsequently, heat-sensitive engineered bacteria were shown to colonize the mouse tumor microenvironment. Finally, thermal stimulation was proven to control engineered bacteria to produce the therapeutic protein tumor necrosis factor α (TNF-α) in the tumor. After three heat stimulation treatments, the growth of the tumor was significantly inhibited, suggesting that heat can be used as a strategy to precisely control engineered bacteria in vivo.

NIR light-responsive bacteria with live bio-glue coatings for precise colonization in the gut.

Recombinant bacterial colonization plays an indispensable role in disease prevention, alleviation, and treatment. Successful application mainly depends on whether bacteria can efficiently spatiotemporally colonize the host gut. However, a primary limitation of existing methods is the lack of precise spatiotemporal regulation, resulting in uncontrolled methods that are less effective. Herein, we design upconversion microgels (UCMs) to convert near-infrared light (NIR) into blue light to activate recombinant light-responsive bacteria (Lresb) in vivo, where autocrine "functional cellular glues" made of adhesive proteins assist Lresb inefficiently colonizing the gut. The programmable engineering platform is further developed for the controlled and effective colonization of Escherichia coli Nissle 1917 (EcN) in the gut. The colonizing bacteria effectively alleviate DSS-induced colitis in mice. We anticipate that this approach could facilitate the clinical application of engineered microbial therapeutics to accurately and effectively regulate host health.

PagC is involved in salmonella pullorum OMVs production and affects biofilm production.

The pagC gene is ubiquitously distributed in Salmonella, but there is limited information regarding its function. Pullorum disease (PD) is a septicemic disease caused by Salmonella Pullorum, which also harbors the pagC gene. In this study, we constructed an S. Pullorum pagC gene deletion strain and its complemented strain. First, we confirmed that the pagC gene does not participate in bacterial growth regulation or environmental pH adaptation. Interestingly, the results of subsequent analyses indicated that the pagC gene defect led to increased bacterial colonization in the intestine (especially in the cecum) and increased biofilm formation, while the number of outer-membrane vesicles (OMVs) in the bacterial culture decreased. Purified OMVs were able to reduce S. Pullorum biofilm formation in vitro. In addition, the results of a mass spectrometry analysis of purified OMVs indicated that some enzymes harbored by OMVs may be involved in biofilm degradation. Based on these results, we conclude that deletion of the pagC gene leads to reduced S. Pullorum OMVs production, which subsequently promotes biofilm stability, increases bacterial colonization in the intestine, and potentially inhibits the switch from sessile to planktonic growth.