Elucidation of Gram-Positive Bacterial Iron(III) Reduction for Kaolinite Clay Refinement.

Recently, microbial-based iron reduction has been considered as a viable alternative to typical chemical-based treatments. The iron reduction is an important process in kaolin refining, where iron-bearing impurities in kaolin clay affects the whiteness, refractory properties, and its commercial value. In recent years, Gram-negative bacteria has been in the center stage of iron reduction research, whereas little is known about the potential use of Gram-positive bacteria to refine kaolin clay. In this study, we investigated the ferric reducing capabilities of five microbes by manipulating the microbial growth conditions. Out of the five, we discovered that Bacillus cereus and Staphylococcus aureus outperformed the other microbes under nitrogen-rich media. Through the biochemical changes and the microbial behavior, we mapped the hypothetical pathway leading to the iron reduction cellular properties, and found that the iron reduction properties of these Gram-positive bacteria rely heavily on the media composition. The media composition results in increased basification of the media that is a prerequisite for the cellular reduction of ferric ions. Further, these changes impact the formation of biofilm, suggesting that the cellular interaction for the iron(III)oxide reduction is not solely reliant on the formation of biofilms. This article reveals the potential development of Gram-positive microbes in facilitating the microbial-based removal of metal contaminants from clays or ores. Further studies to elucidate the corresponding pathways would be crucial for the further development of the field.

Planar AIEgens with Enhanced Solid-State Luminescence and ROS Generation for Multidrug-Resistant Bacteria Treatment.

Planar luminogens have encountered difficulties in overcoming intrinsic aggregation-caused emission quenching by intermolecular π-π stacking interactions. Although excited-state double-bond reorganization (ESDBR) can guide us on designing planar aggregation-induced emission (AIE) luminogens (AIEgens), its mechanism has yet been elucidated. Major challenges in the field include methods to efficiently restrict ESDBR and enhance AIE performance without using bulky substituents (e.g., tetraphenylethylene and triphenylamine). In this study, we rationally developed fluoro-substituent AIEgens with stronger intermolecular H-bonding interaction for restricted molecular motions and increased crystal density, leading to decreased nonradiative decay rate by one order of magnitude. The adjusted ESDBR properties also show a corresponding response to variation in viscosity. Furthermore, their aggregation-induced reactive oxygen species (ROS) generations have been discovered. The application of such planar AIEgen in treating multidrug-resistant bacteria has been demonstrated in a mouse model. The relationship between ROS generation and distinct E/Z-configurational stacking behaviors have been further understood, providing a design principle for synthesizing planar AIEgen-based photosensitizers.

Time-Dependent Photodynamic Therapy for Multiple Targets: A Highly Efficient AIE-Active Photosensitizer for Selective Bacterial Elimination and Cancer Cell Ablation.

Pathogen infections and cancer are two major human health problems. Herein, we report the synthesis of an organic salt photosensitizer (PS), called 4TPA-BQ, by a one-step reaction. 4TPA-BQ presents aggregation-induced emission features. Owing to the aggregation-induced reactive oxygen species generated and a sufficiently small ΔEST , 4TPA-BQ shows a satisfactorily high 1 O2 generation efficiency of 97.8 %. In vitro and in vivo experiments confirmed that 4TPA-BQ exhibited potent photodynamic antibacterial performance against ampicillin-resistant Escherichia coli with good biocompatibility in a short time (15 minutes). When the incubation duration persisted long enough (12 hours), cancer cells were ablated efficiently, leaving normal cells essentially unaffected. This is the first reported time-dependent fluorescence-guided photodynamic therapy in one individual PS, which achieves ordered and multiple targeting simply by varying the external conditions. 4TPA-BQ reveals new design principles for the implementation of efficient PSs in clinical applications.

Initial cyclic-di-GMP upregulation triggers sporadic cellular expansion leading to improved cellular survival.

Bacterial second messenger c-di-GMP upregulation is associated with the transition from planktonic to sessile microbial lifestyle, inhibiting cellular motility, and virulence. However, in-depth elucidation of the cellular processes resulting from c-di-GMP upregulation has not been fully explored. Here, we report the role of upregulated cellular c-di-GMP in promoting planktonic cell growth of Escherichia coli K12 and Pseudomonas aeruginosa PAO1. We found a rapid expansion of cellular growth during initial cellular c-di-GMP upregulation, resulting in a larger planktonic bacterial population. The initial increase in c-di-GMP levels promotes bacterial swarming motility during the growth phase, which is subsequently inhibited by the continuous increase of c-di-GMP, and ultimately facilitates the formation of biofilms. We demonstrated that c-di-GMP upregulation triggers key bacterial genes linked to bacterial growth, swarming motility, and biofilm formation. These genes are mainly controlled by the master regulatory genes csgD and csrA. This study provides us a glimpse of the bacterial behavior of evading potential threats through adapting lifestyle changes via c-di-GMP regulation.

Visualizing the perturbation of cellular cyclic di-GMP levels in bacterial cells.

Cyclic di-GMP (c-di-GMP) has emerged as a prominent intracellular messenger that coordinates biofilm formation and pathogenicity in many bacterial species. Developing genetically encoded biosensors for c-di-GMP will help us understand how bacterial cells respond to environmental changes via the modulation of cellular c-di-GMP levels. Here we report the design of two genetically encoded c-di-GMP fluorescent biosensors with complementary dynamic ranges. By using the biosensors, we found that several compounds known to promote biofilm dispersal trigger a decline in c-di-GMP levels in Escherichia coli cells. In contrast, cellular c-di-GMP levels were elevated when the bacterial cells were treated with subinhibitory concentrations of biofilm-promoting antibiotics. The biosensors also revealed that E. coli cells engulfed by macrophages exhibit lower c-di-GMP levels, most likely as a response to the enormous pressures of survival during phagocytosis.

Targeted EpCAM-binding for the development of potent and effective anticancer proteins.

Protein-based cancer therapies are considered an alternative to conventional anticancer regimens, providing multifunctional properties while showing low toxicity. However, its widespread use is limited by absorption and instability issues, resulting in higher dosage requirements and a prolonged onset of bioactivity to elicit the desired response. Here, we developed a non-invasive antitumor treatment using designed ankyrin repeat protein (DARPin)-anticancer protein-conjugate that specifically targets the cancer biomarker, epithelial cell adhesion molecule (EpCAM). The DARPin-anticancer proteins bind to EpCAM-positive cancer cells and improve the in vitro anticancer efficacy by over 100-folds within 24 h, where the DARPin-tagged human lactoferrin fragment (drtHLF4) IC50 value is within the nanomolar range. Orally administered drtHLF4 was readily absorbed into the systemic flow of the HT-29 cancer murine model, exerting its anticancer effect on other tumors in the host body. Orally administered drtHFL4 cleared HT29-colorectal tumors using a single dose, whereas intratumoral injection cleared HT29-subcutaneous tumors within three doses. This approach addresses the limitations of other protein-based anticancer treatments by providing a non-invasive anticancer therapy with improved potency and tumor-specificity.

Functional divergence of FimX in PilZ binding and type IV pilus regulation.

Type IV pili (T4P) are polar surface structures that play important roles in bacterial motility, biofilm formation, and pathogenicity. The protein FimX and its orthologs are known to mediate T4P formation in the human pathogen Pseudomonas aeruginosa and some other bacterial species. It was reported recently that FimX(XAC2398) from Xanthomonas axonopodis pv. citri interacts with PilZ(XAC1133) directly through the nonenzymatic EAL domain of FimX(XAC2398). Here we present experimental data to reveal that the strong interaction between FimX(XAC2398) and PilZ(XAC1133) is not conserved in P. aeruginosa and likely other Pseudomonas species. In vitro and in vivo binding experiments showed that the interaction between FimX and PilZ in P. aeruginosa is below the measurable limit. Surface plasmon resonance assays further confirmed that the interaction between the P. aeruginosa proteins is at least more than 3 orders of magnitude weaker than that between the X. axonopodis pv. citri pair. The N-terminal lobe region of FimX(XAC2398) was identified as the binding surface for PilZ(XAC1133) by amide hydrogen-deuterium exchange and site-directed mutagenesis studies. Lack of several key residues in the N-terminal lobe region of the EAL domain of FimX is likely to account for the greatly reduced binding affinity between FimX and PilZ in P. aeruginosa. All together, the results suggest that the interaction between PilZ and FimX in Xanthomonas species is not conserved in P. aeruginosa due to the evolutionary divergence among the FimX orthologs. The precise roles of FimX and PilZ in bacterial motility and T4P biogenesis are likely to vary among bacterial species.

Synthesis of (R)-mellein by a partially reducing iterative polyketide synthase.

Mellein and the related 3,4-dihydroisocoumarins are a family of natural products with interesting biological properties. The mechanisms of dihydroisocoumarin biosynthesis remain largely speculative today. Here we report the synthesis of mellein by a partially reducing iterative polyketide synthase (PR-PKS) as a pentaketide product. Remarkably, despite the head-to-tail homology shared with several fungal and bacterial PR-PKSs, the mellein synthase exhibits a distinct keto reduction pattern in the synthesis of the pentaketide. We present evidence to show that the ketoreductase (KR) domain alone is able to recognize and differentiate the polyketide intermediates, which provides a mechanistic explanation for the programmed keto reduction in these PR-PKSs.

Elucidation of Iron(III) Bioleaching Properties of Gram-Positive Bacteria.

Microbial-based iron reduction is an emerging technology used as an alternative to conventional chemical-based iron reduction. The iron reduction in kaolin refinement is vital for enhancing its commercial value. Extensive studies on microbial-based iron reduction mainly focus on Gram-negative bacteria, whereas little is understood about Gram-positive bacteria's mechanism and potential application. This study aims to investigate the iron-reducing mechanism of two Gram-positive bacterial isolates, Bacillus cereus (B. cereus) and Staphylococcus aureus (S. aureus). By varying the growth environment of bacteria and monitoring the biochemical changes during the process of iron reduction, the results show that Gram-positive bacterial iron reduction performance depends on the medium composition, differing from Gram-negative bacteria-based reduction processes. Nitrogen-rich medium facilitates the microbial basification of the medium, where the alkaline conditions impact the microbial iron reduction process by altering the gene expression involved in intracellular pH homeostasis and microbial growth. This discovery will contribute to the mineral refining processes and promote the development of microbial-based bioprocesses for ore purification, while also laying the foundation for investigating other Gram-positive bacterial iron-reducing ability.

Measuring the Migration and Biofilm Formation of Various Bacteria.

As microbes that thrive in the host body primarily have adaptive abilities that facilitate their survival, methods for classifying and identifying their nature would be beneficial in facilitating their characterization. Currently, most studies focus only on one specific characterization method; however, the isolation and identification of microorganisms from the host is a continuous process and usually requires several combinatorial characterization methods. Herein, we describe methods of identifying the microbial biofilm-forming ability, the microbial respiration state, and their chemotaxis behavior. The methods are used to identify five microbes, three of which were isolated from the bone tissue of Sprague-Dawley (SD) rats (Corynebacterium stationis, Staphylococcus cohnii subsp. urealyticus, and Enterococcus faecalis) and two from the American Type Culture Collection (ATCC)-Staphylococcus aureus ATCC 25923 and Enterococcus faecalis V583. The microbes isolated from the SD rat bone tissue include the gram-positive microbes. These microbes have adapted to thrive under stressful and nutrient-limiting environments within the bone matrix. This article aims to provide the readers with the specific know-how of determining the nature and behavior of the isolated microbes within a laboratory setting.

Quantifying Bacterial Surface Swarming Motility on Inducer Gradient Plates.

Bacterial swarming motility is a common microbiological phenotype that bacterial communities use to migrate over semisolid surfaces. In investigations of induced swarming motility, specific concentration of an inducer may not be able to report events occurring within the optimal concentration range to elicit the desired responses from a species. Semisolid plates containing multiple concentrations are commonly used to investigate the response within an inducer concentration range. However, separate semisolid plates increase variations in medium viscosity and moisture content within each plate due to nonuniform solidification time. This paper describes a one-step method to simultaneously test surface swarming motility on a single gradient plate, where the isometrically arranged test wells allow the simultaneous acquisition of multiconcentration responses. In the present work, the surface swarming of Escherichia coli K12 and Pseudomonas aeruginosa PAO1 were evaluated in response to a concentration gradient of inducers such as resveratrol and arabinose. Periodically, the swarm morphologies were imaged using an imaging system to capture the entire surface swarming process. The quantitative measurement of the swarm morphologies was acquired using ImageJ software, providing analyzable information of the swarm area. This paper presents a simple gradient swarm plate method that provides qualitative and quantitative information about the inducers' effects on surface swarming, which can be extended to study the effects of other inducers on a broader range of motile bacterial species.

Engineered Spore-Forming Bacillus as a Microbial Vessel for Long-Term DNA Data Storage.

DNA data storage technology may supersede conventional chip or magnetic data storage medium, providing long-term stability, high density, and sustainable storage. Due to its error-correcting capability, DNA data stored in living organisms exhibits high fidelity in information replication. Here we report the development of a Bacillus chassis integrated with an inducible artificially assembled bacterial chromosome to facilitate random data access. We generated three sets of data in the form of DNA sequences using a rudimentary coding system accessible by the regulatory promoter. Sporulated Bacillus harboring the genes were used for long-term storage, where viability assays of spores were subjected to harsh environmental stresses to evaluate the data storage stability. The data accuracy remained above 99% after high temperature and oxidative stress treatment, whereas UV irradiation treatment provided above 96% accuracy. The developed Bacillus chassis and artificial chromosome facilitate the long-term storage of larger datum volume by using other DNA digital encoding and decoding programs.

CRISPR/Cas-Based Genome Editing for Human Gut Commensal Bacteroides Species.

Bacteroides is the most abundant genus in the human gut microbiome and has been increasingly used as model organisms for studying the function and ecology of the gut microbiome. However, genome editing tools for such commensal gut microbes are still lacking. Here we developed a versatile, highly efficient CRISPR/Cas-based genome editing tool that allows markerless gene deletion and insertion in human gut Bacteroides species. We constructed multiple CRISPR/Cas systems in all-in-one Bacteroides-E. coli shuttle plasmids and systematically evaluated the genome editing efficiency in Bacteroides thetaiotaomicron, including the mode of Cas protein expression (constitutive, inducible), different Cas proteins (FnCas12a, SpRY, SpCas9), and sgRNAs. Using the anhydrotetracycline (aTc)-inducible CRISPR/FnCas12a system, we successfully deleted large genomic fragments up to 50 kb to study the function of metabolic gene clusters. Furthermore, we demonstrated that CRISPR/FnCas12a can be broadly applied to engineer multiple human gut Bacteroides species, including Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, and Bacteroides vulgatus. We envision that CRISPR/Cas-based genome editing tools for Bacteroides will greatly facilitate mechanistic studies of the gut commensal and the development of engineered live biotherapeutics.

Rational design of fluorescent biosensor for cyclic di-GMP.

Messenger bagged: The design of a fluorophore-labeled protein biosensor for the bacterial messenger cyclic di-GMP is described. The biosensor responds to c-di-GMP with sub-micromolar sensitivity in a real-time fashion. The biosensor can be used for enzyme assays for diguanylate cyclases and c-di-GMP phosphodiesterases as well as the high-throughput screening of inhibitors.

Recent Development of Probiotic Bifidobacteria for Treating Human Diseases.

Bifidobacterium is a non-spore-forming, Gram-positive, anaerobic probiotic actinobacterium and commonly found in the gut of infants and the uterine region of pregnant mothers. Like all probiotics, Bifidobacteria confer health benefits on the host when administered in adequate amounts, showing multifaceted probiotic effects. Examples include B. bifidum, B. breve, and B. longum, common Bifidobacterium strains employed to prevent and treat gastrointestinal disorders, including intestinal infections and cancers. Herein, we review the latest development in probiotic Bifidobacteria research, including studies on the therapeutic impact of Bifidobacterial species on human health and recent efforts in engineering Bifidobacterium. This review article would provide readers with a wholesome understanding of Bifidobacteria and its potentials to improve human health.

Engineering of Human Lactoferrin for Improved Anticancer Activity.

Protease-digested lactoferrin fragments often exhibit improved therapeutic properties. However, there are limited studies investigating the anticancer properties of these fragments. The fragment with improved anticancer activities is an attractive alternative to chemotherapeutic drugs-presenting severe side effects. Herein, we report the isolation and characterization of recombinant engineered-lactoferrin (rtHLF4), exhibiting up to 100-fold improved anticancer activity compared to the full-length lactoferrin (flHLF). Further, rtHLF4 exerts its anticancer effect in a shorter duration. Through transcriptomic analysis of various cancer biomarkers, rtHLF4 was found to upregulate various pro-apoptotic markers and downregulate signaling proteins involved in angiogenesis and metastasis. We further determined that rtHLF4 showed no hemolytic activity at high concentrations. We believe that this anticancer protein can be further developed as a cancer treatment.

Impact of the Host-Microbiome on Osteomyelitis Pathogenesis.

The microbiome is a collection of genomes from microbiota, including all microorganisms in a niche, through direct and indirect interactions with the host. Certain microorganisms can exist in areas conventionally considered to be sterile, such as the bone matrix. Osseous microbiota dysbiosis caused by host-microbiome perturbation or external infections may ultimately lead to osteomyelitis, a bone inflammatory disorder. Our review covers the current discoveries on the impact of host-microbiome on osteomyelitis and some common osseous diseases. Some studies suggest that the microbiotas from both osseous and non-osseous tissues (e.g., blood or gut) impact the pathogenicity of osteomyelitis and other osseous diseases (e.g., rheumatoid arthritis). We believe that this review will provide readers with a better understanding on the role of the microbiome to the host's bone health.

Evaluation of the Anti-Inflammatory and Anti-Oxidative Effects of Therapeutic Human Lactoferrin Fragments.

Chronic inflammation is considered a pressing health issue that needs resolving. Inflammatory disease such as inflammatory bowel disease requires a long-term medical regimen to prevent disease progression. Conventionally, lactoferrin is used to treat mild gastrointestinal tract and skin inflammation. Protease-digested lactoferrin fragments often exhibit improved therapeutic properties compared to full-length lactoferrin (flHLF). However, there are no studies on the use of protease-digested lactoferrin fragments to treat inflammation. Herein, we assess the anti-inflammatory properties of engineered recombinant lactoferrin fragments (rtHLF4, rteHLF1, and rpHLF2) on non-malignant colonic fibroblast cells and colorectal cancer cells. We found that rtHLF4 is 10 times more effective to prevent inflammation compared to flHLF. These results were investigated by looking into the reactive oxygen species (ROS) production, angiogenesis activity, and cellular proliferation of the treated cells. We have demonstrated in this study the anti-inflammatory properties of the flHLF and the various lactoferrin fragments. These results complement the anti-cancer properties of these proteins that were demonstrated in an earlier study.

Engineered Live Biotherapeutics: Progress and Challenges.

The human microbiome plays an important role in human health, from metabolism to immunity. In the last few decades, advances in synthetic biology have enabled scientists to design and engineer live microorganisms for therapeutic purposes. In this review, major strategies for manipulating the microbiome are outlined, which include three emerging areas with promising therapeutic applications: engineered commensal bacteria, synthetic microbial consortia, and targeted modulation by phages. Furthermore, the applications of engineered live biotherapeutics in treating a variety of human diseases, including pathogenic infections, metabolic disorders, inflammatory bowel disease, and colorectal cancer, are highlighted. Finally, an overview of the challenges and opportunities in the future development of engineered live biotherapeutics is provided.

Advances in Bacterial Biofilm Management for Maintaining Microbiome Homeostasis.

Certain microbial biofilm in the human-microbiota community can negatively impact the host microbiome. This gives rise to various methods to prevent the formation of biofilms or to facilitate biofilm dispersal from surfaces and tissues in the host. Despite all these efforts, these persistent microbial biofilms on surfaces and in the host tissue can result in health problems to the host and its microbiome. It is the adaptive behavior of microbes within the biofilm that confers on these tenacious microbes the resistance to harsh environments, antibiotic treatments, and the ability to evade the host immune system. In this review, the approaches to combat microbial biofilm in the last decade are discussed. The biochemical pathway regulating biofilm formation is first discussed, followed by the discussion of the three approaches to combat biofilm formation: physical, chemical, and biological approaches. The advances in these approaches have given rise to methods of effectively dispersing the microbial biofilm and preventing the adherence of these microbial communities altogether. As there are numerous approaches to target biofilm, in this review the attempt is to provide insights on how these approaches have been used to modulate the host-microbiome by looking at the individual strengths and weaknesses.