Biology subjects

Hu, G.

Publications and source records attributed to Hu, G..

At least 91 records · Page 5Linked to original sources

bioRxiv2023

Preclinical studies with ground germinated barley (GGB) for oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice

Genetic deficiency of lysosomal acid maltase or acid -glucosidase (GAA) results in the orphan disease known as glycogen storage disease type II or acid maltase deficiency (AMD) or Pompe disease (PD), encompassing at least four clinical subtypes of varying severity. PD results from mutations in the GAA gene and deficient GAA activity, resulting in the accumulation of glycogen in tissues (primarily muscle) and characterized by progressive skeletal muscle weakness and respiratory insufficiency. The current approved enzyme replacement therapy (ERT) for PD is via intravenous infusion of a recombinant human GAA (rhGAA) secreted by CHO cells (Myozyme, Sanofi-Genzyme) given once every 2 weeks and has shown varying efficacy in patients. Although the current ERT has proven to be very efficient in rescuing cardiac abnormalities and extending the life span of infants, the response in skeletal muscle is variable. In late-onset patients, only mild improvements in motor and respiratory functions have been achieved and the current ERT is unsatisfactory in the reversal of skeletal muscle pathology. Additional challenges for ERT include insufficient targeting/uptake of enzyme into disease-relevant tissues, poor tolerability due to severe ERT-mediated anaphylactic and immunologic reactions and the prohibitively high cost of lifelong ERT ($250-500K/year adult patient). A consensus at a Nov.-2019 US Acid Maltase Deficiency Association conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment. Our objective is to develop an innovative and affordable approach via barley GAA (bGAA) from ground germinated barley (GGB) or liquid GGB (L-GGB) for Oral-ERT for PD or as a daily supplement to Myozyme. To this end, we hypothesize that a bGAA produced in germinated barley can be ingested daily that allows the maintenance of a therapeutic level of enzyme. We have shown in extensive preliminary data that GGB or L-GGB was (1) enzymatically active, (2) was taken up by GAA KO mice and human WBCs to reverse the enzyme defect that was blocked by mannose-6-phosphate, (3) hydrolyzed glycogen, (4) increased significant changes in the clinical phenotype towards the WT levels in GAA KO mice dose-dependently, (5) taken up by PD myoblasts, lymphoid/fibroblasts cells to reverse the defect, (6) bGAA was [~]70kD, (7) Km, Vmax, pH optima, inhibitors and kinetics was similar to human placental GAA and an rhGAA and (8) was strain specific.

genetics↗

bioRxiv2023

Bosutinib stimulates macrophage survival, phagocytosis and intracellular killing of bacteria

Host-acting compounds are emerging as potential alternatives to combat antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduces bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodelling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-{kappa}B activation which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and Tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, co-treatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infection through multiple mechanisms and could be used to boost host innate immunity to combat drug-resistant bacterial infections. SignificanceThis study shows that bosutinib, an FDA-approved chemotherapeutic, stimulates macrophage responses to antibiotic-resistant bacterial infection by enhancing phagocytosis and intracellular killing of bacteria and promoting survival of infected macrophages. These findings suggest that bosutinib could serve as an adjuvant therapy to combat drug resistant bacterial infections and opens the possibility to target Src kinases to boost innate immunity in general.

microbiology↗

bioRxiv2023

Predicting the dynamic interaction between intrinsically disordered proteins

Intrinsically disordered proteins (IDPs) participate in various biological processes. Interactions involving IDPs are usually dynamic and affected by their inherent conformation fluctuations. Comprehensive characterization of these interactions based on current techniques is challenging. Here, we present GSALIDP, a GraphSAGE-LSTM Network to capture the dynamic nature of IDP-involved interactions and predict their behaviors. This framework models multiple conformations of IDP as a dynamic graph which can effectively describe the fluctuation of its flexible conformation. The dynamic interaction between IDPs is studied and the datasets of IDP conformations and their interactions are obtained through atomistic molecular dynamic (MD) simulations. Residues of IDP are encoded through a series of features, including their frustration. GSALIDP can effectively predict the interaction sites of IDP and the contact residue pairs between IDPs. Its performance in predicting IDP interaction is on par with or even better than the conventional models in predicting the interaction of structural proteins. To the best of our knowledge, this is the first model to extend the protein interaction prediction to IDP-involved interactions.

molecular biology↗

bioRxiv2023

Continuity of Mitochondrial Budding: Insights from BS-C-1 Cells by in-situ Cryo-Electron Tomography

Mitochondrial division is a fundamental biological process that is crucial to cellular functionality and vitality. The prevailing hypothesis of Drp1 regulation with the involvement of ER and cytoskeleton does not account for all the observations. Following up our previous study in HeLa cells which led to the new hypothesis of mitochondrial division by budding, we employed in-situ Cryo-Electron Tomography (Cryo-ET) to visualize mitochondrial budding in intact healthy monkey kidney cells (BS-C-1 cells). Our findings reaffirm single and multiple mitochondrial budding, supporting the new hypothesis. Notably, the budding regions vary significantly in diameter and length, which may represent different stages of budding. More interestingly, no rings, or ring-like structures, or ER wrapping is presented in the budding regions suggesting mitochondrial budding is independent from Drp1 and ER. Meanwhile, we uncovered direct interactions between mitochondria and large vesicles, distinct from small mitochondrial-derived vesicles and extracellular mitovesicles. We propose these interacting vesicles may have mitochondrial origins.

biophysics↗

bioRxiv2023

Bioinformatics Illustrations Decoded by ChatGPT: The Good, The Bad, and The Ugly

Emerging studies underscore the promising capabilities of large language model-based chatbots in conducting fundamental bioinformatics data analyses. The recent feature of accepting image-inputs by ChatGPT motivated us to explore its efficacy in deciphering bioinformatics illustrations. Our evaluation with examples in cancer research, including sequencing data analysis, multimodal network-based drug repositioning, and tumor clonal evolution, revealed that ChatGPT can proficiently explain different plot types and apply biological knowledge to enrich interpretations. However, it struggled to provide accurate interpretations when quantitative analysis of visual elements was involved. Furthermore, while the chatbot can draft figure legends and summarize findings from the figures, stringent proofreading is imperative to ensure the accuracy and reliability of the content.

bioinformatics↗

bioRxiv2023

Cnot3 is required for male germ cell development and spermatogonial stem cell maintenance

The foundation of spermatogenesis and lifelong fertility is provided by spermatogonial stem cells (SSCs). SSCs divide asymmetrically to either replenish their numbers (self-renewal) or produce undifferentiated progenitors that proliferate before committing to differentiation. However, regulatory mechanisms governing SSC maintenance are poorly understood. Here, we show that the CCR4-NOT mRNA deadenylase complex subunit CNOT3 plays a critical role in maintaining spermatogonial populations in mice. Cnot3 is highly expressed in undifferentiated spermatogonia, and its deletion in spermatogonia resulted in germ cell loss and infertility. Single cell analyses revealed that Cnot3 deletion led to the de-repression of transcripts encoding factors involved in spermatogonial differentiation, including those in the glutathione redox pathway that are critical for SSC maintenance. Together, our study reveals that CNOT3 - likely via the CCR4-NOT complex - actively degrades transcripts encoding differentiation factors to sustain the spermatogonial pool and ensure the progression of spermatogenesis, highlighting the importance of CCR4-NOT-mediated post-transcriptional gene regulation during male germ cell development. HighlightsO_LICnot3 is predominantly expressed in undifferentiated spermatogonia, and its expression decreases in later stages. C_LIO_LIConditional deletion of Cnot3 leads to total germ cell loss and male infertility. C_LIO_LICNOT3 represses transcripts encoding factors involved in spermatogonial differentiation. C_LIO_LICNOT3 protects spermatogonial stem cells maintenance via GSH/redox pathway. C_LI Graphical AbstractCnot3 promotes SSC self-renewal by balancing ROS production via the regulation of GSH production O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/562256v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@3afef9org.highwire.dtl.DTLVardef@139cb19org.highwire.dtl.DTLVardef@1450c47org.highwire.dtl.DTLVardef@293300_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Proteolytic activation of fatty acid synthase signals pan-stress resolution

Chronic stress and inflammation are not only outcomes of pathological states but rather major drivers of many human diseases 1-4. Ideally, a given stress program is downregulated to basal levels upon restoration of homeostasis. Chronic responsiveness despite stress mitigation suggests a failure to sense the resolution of the initiating stressor. Here we show that a proteolytic cleavage event of fatty acid synthase (FASN) activates a global cue for stress resolution. FASN is well-established as the multifunctional enzyme catalyzing de novo biosynthesis of saturated fatty acid 5, 6. Surprisingly, our results demonstrate FASN functioning as a signaling molecule promoting an anti-inflammatory profile apart from fatty acid synthesis. Redox-dependent proteolytic cleavage of FASN by caspase activates a truncated C-terminal enzymatic fragment (FASN-CTF) that is sufficient to down-regulate multiple aspects of stress-responsiveness including gene expression and metabolic programs. Only a fraction of FASN is cleaved allowing for continued fat synthesis. FASN-CTF can signal stress resolution across tissues in a cell non-autonomous manner. Consistent with these findings, FASN processing is also seen in well-fed but not fasted mouse liver. As down-regulation of stress responsiveness is critical to health, our findings provide a potential pathway to control the magnitude for diverse aspects of stress responses.

molecular biology↗

bioRxiv2023

Genetic Screen Identified Prmt5 as a Neuroprotection Target against Cerebral Ischemia

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remains largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. We identified Prmt5 as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. Prmt5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that Prmt5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, we treated mice with middle cerebral artery occlusion (MCAO) with the Prmt5 inhibitor EPZ015666 and found that Prmt5 inhibition sustain protection against neuronal death in vivo. Together, our findings revealed a novel epigenetic mechanism of Prmt5 in cerebral ischemia and uncovered a potential target for neuroprotection.

cell biology↗

bioRxiv2023

Characterization of extracellular vesicles isolated from Sorghum bicolor reveals a conservation between monocot and eudicot extracellular vesicle proteomes

Plant extracellular vesicles (EVs) are membrane bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. In this paper, we described the isolation and characterization of extracellular vesicles from Sorghum bicolor. Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, Calcein-acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also being found in the EV proteome of Arabidopsis. These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

plant biology↗

bioRxiv2023

Coronary Artery Disease Risk Gene PRDM16 is Preferentially Expressed in Vascular Smooth Muscle Cells and a Potential Novel Regulator of Smooth Muscle Homeostasis

ObjectiveVascular smooth muscle cells (VSMCs) are the primary contractile component of blood vessels and can undergo phenotypic switching from a contractile to a synthetic phenotype in vascular diseases such as coronary artery disease (CAD). This process leads to decreased expression of SMC lineage genes and increased proliferative, migratory and secretory abilities that drive disease progression. Super-enhancers (SE) and occupied transcription factors are believed to drive expression of genes that maintain cell identify and homeostasis. The goal of this study is to identify novel regulator of VSMC homeostasis by screening for SE-regulated transcription factors in arterial tissues. Approach and ResultsWe characterized human artery SEs by analyzing the enhancer histone mark H3K27ac ChIP-seq data of multiple arterial tissues. We unexpectedly discovered the transcription factor PRDM16, a GWAS identified CAD risk gene with previously well-documented roles in brown adipocytes but with an unknown function in vascular disease progression, is enriched with artery-specific SEs. Further analysis of public bulk RNA-seq and scRNA-seq datasets, as well as qRT-PCR and Western blotting analysis, demonstrated that PRDM16 is preferentially expressed in arterial tissues and in contractile VSMCs but not in visceral SMCs, and down-regulated in phenotypically modulated VSMCs. To explore the function of Prdm16 in vivo, we generated Prdm16 SMC-specific knockout mice and performed histological and bulk RNA-Seq analysis of aortic tissues. SMC-deficiency of Prdm16 does not affect the aortic morphology but significantly alters expression of many CAD risk genes and genes involved in VSMC phenotypic modulation. Specifically, Prdm16 negatively regulates the expression of Tgfb2 that encodes for an upstream ligand of TGF-{beta} signaling pathway, potentially through binding to the promoter region of Tgfb2. These transcriptomic changes likely disrupt VSMC homeostasis and predispose VSMCs to a disease state. ConclusionsOur results suggest that the CAD risk gene PRDM16 is preferentially expressed in VSMCs and is a novel regulator of VSMC homeostasis. Future studies are warranted to investigate its role in VSMCs under pathological conditions such as atherosclerosis.

cell biology↗

bioRxiv2023

Enhanced niche colonisation and competition during bacterial adaptation to a fungus

Bacterial-fungal interactions (BFIs) influence microbial community performance of most ecosystems and elicit specific microbial behaviours, including stimulating specialised metabolite production. Using a simple BFI system encompassing the Gram-positive bacterium Bacillus subtilis and the black mould fungus Aspergillus niger, we established a co-culture experimental evolution method to investigate bacterial adaptation to the presence of a fungus. In the evolving populations, B. subtilis was rapidly selected for enhanced production of the lipopeptide surfactin and accelerated surface spreading ability, leading to inhibition of fungal expansion and acidification of the environment. These phenotypes were explained by specific mutations in the DegS-DegU two-component system. In the presence of surfactin, fungal hyphae exhibited bulging cells with delocalised secretory vesicles and RlmA-dependent cell wall stress induction. Increased surfactin production typically enhances the competitive success of bacteria against fungi, which likely explains the primary adaption path in the presence of A. niger. Significance statementExperimental evolution and co-cultivation of different microbes are important and useful techniques for discovering new traits and unravelling cryptic regulatory connections. We combined these methods by evolving the Gram-positive bacterium Bacillus subtilis in the presence of the black mould fungus Aspergillus niger that were previously shown to engage in an intricate and physical interaction. Both are ubiquitous, environmentally and industrially relevant model microbes in the colonisation of rhizo- and endosphere and in the production of enzymes. Our results demonstrate how laboratory adaptation can be exploited to improve biocontrol properties of bacteria.

microbiology↗

bioRxiv2023

Parallel genetic adaptation of Bacillus subtilis to different plant species

Plant growth-promoting rhizobacteria benefit plants by stimulating their growth or protecting them against phytopathogens. Rhizobacteria must colonise and persist on plant roots to exert their benefits. However, little is known regarding the processes by which rhizobacteria adapt to different plant species, or behave under alternating host plant regimes. Here, we used experimental evolution and whole-population whole-genome sequencing to analyse how Bacillus subtilis evolves on Arabidopsis thaliana and tomato seedlings, and under an alternating host plant regime, in a static hydroponic setup. We observed parallel evolution across multiple levels of biological organisation in all conditions, which was greatest for the two heterogeneous, multi-resource spatially-structured environments at the genetic level. Species-specific adaptation at the genetic level was also observed, possibly caused by the selection stress imposed by different host plants. Furthermore, a trade-off between motility and biofilm development was supported by mutational changes in motility- and biofilm-related genes. Finally, we identified several condition-specific and common targeted genes in different environments by comparing three different B. subtilis biofilm adaptation settings. The results demonstrate a common evolutionary pattern when B. subtilis is adapting to the plant rhizosphere in similar conditions, and reveal differences in genetic mechanisms between different host plants. These findings will likely support strain improvements for sustainable agriculture. Data summarySequencing data associated with this article are available in the CNGB Sequence Archive (CNSA) [1] of the China National GeneBank DataBase (CNGBdb) [2] under accession numbers CNP0002416 and CNP0003952. Strain data for the DK1042 ancestor are available under accession number CNP0002416. Impact statementFor rhizobacteria to benefit plant growth and protect against phytopathogens, bacteria must colonise and persist on plant roots. Understanding how rhizobacteria adapt to different plant species will assist strain development in sustainable agriculture. To explore the rhizobacterial adaptation process for different plant species and alternating host regimes, B. subtilis was experimentally evolved on A. thaliana or tomato roots, or an alternating host regime. Both parallel and species-specific adaptation was revealed at the genetic level. Analysis of the trade-off between motility and biofilm formation revealed several condition-specific and commonly targeted genes based on experimentally evolving B. subtilis biofilms.

microbiology↗

bioRxiv2023

Magnesium ions mediate ligand binding and conformational transition of the SAM/SAH riboswitch

The SAM/SAH riboswitch binds S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) with similar affinities. Mg2+ is generally known to stabilize RNA structures by neutralizing phosphates, but how it contributes to ligand binding and conformational transition is understudied. Here, extensive molecular dynamics simulations (totaling 120 s) identified over 10 inner-shell Mg2+ ions in the SAM/SAH riboswitch. Six of them line the two sides of a groove to widen it and thereby pre-organize the riboswitch for ligand entry. They also form outer-shell coordination with the ligands and stabilize an RNA-ligand hydrogen bond, which effectively diminish the selectivity between SAM and SAH. One Mg2+ ion unique to the apo form maintains the Shine-Dalgarno sequence in an autonomous mode and thereby facilitates its release for ribosome binding. Mg2+ thus plays vital roles in SAM/SAH riboswitch function.

biophysics↗

bioRxiv2023

Empowering Beginners in Bioinformatics with ChatGPT

The impressive conversational and programming abilities of ChatGPT make it an attractive tool for facilitating the education of bioinformatics data analysis for beginners. In this study, we proposed an iterative model to fine-tune instructions for guiding a ChatGPT in generating code for bioinformatics data analysis tasks. We demonstrated the feasibility of the model by applying it to various bioinformatics topics. Additionally, we discussed practical considerations and limitations regarding the use of the model in chatbot-aided bioinformatics education.

bioinformatics↗

bioRxiv2023

TEAD1 is crucial for myelination, Remak bundles and functional regeneration of peripheral nerves

Previously we showed that the hippo pathway transcriptional effectors, YAP and TAZ, are essential for Schwann cells (SCs) to develop, maintain and regenerate myelin (Grove et al., 2017; Grove, Lee, Zhao, & Son, 2020). Although TEAD1 has been implicated as a partner transcription factor, the mechanisms by which it mediates YAP/TAZ regulation of SC myelination are unclear. Here, using conditional and inducible knockout mice, we show that TEAD1 is crucial for SCs to develop and regenerate myelin. It promotes myelination by both positively and negatively regulating SC proliferation, enabling Krox20/Egr2 to upregulate myelin proteins, and upregulating the cholesterol biosynthetic enzymes FDPS and IDI1. We also show stage-dependent redundancy of TEAD1 and that non-myelinating SCs have a unique requirement for TEAD1 to enwrap nociceptive axons in Remak bundles. Our findings establish TEAD1 as a major partner of YAP/TAZ in developmental myelination and functional nerve regeneration and as a novel transcription factor regulating Remak bundle integrity.

neuroscience↗

bioRxiv2022

Enhancing detectable fluorescence fluctuation for high-throughput and four-dimensional live-cell super-resolution imaging

Super-resolution (SR) imaging with high-throughput is invaluable to fast and high-precision profiling in a wide range of biomedical applications. However, prevalent SR methods require sophisticated acquisition devices and specific imaging control, and may cost a fairly long time on a single field-of-view. These essentially increase the construction difficulty, including challenges in imaging throughput, system establishment, and automation. Using the natural photophysics of fluorescence, fluctuation-based microscopy techniques can routinely break the diffraction limit with no need for additional optical components, but its long acquisition time still poses a challenge for high-throughput imaging or visualizing transient organelle dynamics. Here, we propose an SR method based on the Auto-Correlation with two-step Deconvolution (SACD) that reduces the number of frames required by maximizing the detectable fluorescence fluctuation behavior in each measurement, with further removal of tunable parameters by a Fourier ring correlation analysis. It only needs 20 frames for twofold lateral and axial resolution improvements, while the SR optical fluctuation imaging (SOFI) needs more than 1000 frames. By capturing raw images for [~]10 minutes, we record an SR image with [~]128 nm resolution that contains 2.4 gigapixels covering an area of [~]2.0 mm x 1.4 mm, including more than 2,000 cells. Beyond that, by applying continuity and sparsity joint constraint, the Sparse deconvolution-assisted SACD enables 4D live-cell SR imaging of events such as mitochondrial fission and fusion. Overall, as an open-sourced module, we anticipate SACD can offer direct access to SR, which may facilitate the biology studies of cells and organisms with high-throughput and low-cost.

biophysics↗

bioRxiv2022

Species and condition dependent mutational spectrum in experimentally evolved biofilms of Bacilli

Laboratory experimental evolution provides a powerful tool for studying microbial adaptation to different environments. To understand the differences and similarities of the dynamic evolutionary landscapes of two model species from the Bacillus genus as they adapt to abiotic and biotic surfaces, we revived the archived population samples from our four previous experimental evolution studies and performed longitudinal whole-population genome sequencing. Surprisingly, higher number of mutations, higher genotypic diversity, and higher evolvability were detected in the biotic conditions with smaller population size. Different adaptation strategies were observed in different environments within each species, with more diversified mutational spectrum detected in biotic conditions. The insertion sequences of Bacillus thuringiensis are critical for its adaptation to the plastic bead-attached biofilm environment, but insertion sequence mobility was a general phenomenon in this species independent of the selection condition. Additionally, certain parallel evolution has been observed across species and environments, particularly when two species adapt to the same environment at the same time. Further, our results suggest that the population size might be an important driver of evolution. Together, these results provide the first comprehensive mutational landscape of two bacterial species biofilms that is adapted to an abiotic and biotic surface. ImportanceBiofilm formation is a vital factor for the survival and adaptation of bacteria in diverse environmental niches. Experimental evolution combined with the advancement of whole-population genome sequencing provides us a powerful tool to understand the genomic dynamic of evolutionary adaptation to different environments, such as during biofilm development. Previous studies described the genetic and phenotypic changes of selected clones from experimentally evolved Bacillus thuringiensis and Bacillus subtilis that were adapted under abiotic and biotic biofilm conditions. However, the full understanding of the dynamic evolutionary landscapes was lacking. Further, the differences and similarities of adaptive mechanisms in B. thuringiensis and B. subtilis were not identified. To overcome these limitations, we performed longitudinal whole-population genome sequencing to study the underlying genetic dynamics at high resolution. Our study provides the first comprehensive mutational landscape of two bacterial species biofilms that is adapted to an abiotic and biotic surface.

microbiology↗

bioRxiv2022

Mitoxantrone Targets Both Host and Bacteria to Overcome Vancomycin Resistance in Enterococcus faecalis

Among Enterococci, intrinsic and acquired resistance to antibiotics such as {beta}-lactams and vancomycin critically limit treatment options for infection with these opportunistic pathogens. Antimicrobials that enhance the host immune response are emerging as alternative approaches, with the potential to overcome bacterial resistance. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria with a minimal inhibitory concentration (MIC) of ~1 g/ml through induction of reactive oxygen species and DNA damage. MTX synergises with vancomycin and lowers the vancomycin concentration required to kill VRE by over 140-fold. This synergy is specific to vancomycin-resistant, but not susceptible strains because vancomycin rendered the resistant strains more permeable to MTX and thus MTX-mediated DNA damage. In a murine wound infection model, MTX treatment effectively reduced VRE bacterial numbers by 120-fold and with further reduction when combined with vancomycin. Wounds treated with MTX had significantly higher numbers of macrophages and higher pro-inflammatory cytokines compared to untreated wounds. In addition, MTX augmented intracellular bacterial killing by both murine and human macrophages by upregulating the expression of lysosomal hydrolases cathepsins D and H, and {beta}-Hexosaminidase. These results show that MTX is a potent antibiotic against Gram-positive bacteria, synergizes with vancomycin, enhances macrophage recruitment and intracellular bactericidal activity, and represents a promising dual bacterium- and host-targeted therapeutic for overcoming vancomycin resistance. One sentence summaryMitoxantrone synergizes with vancomycin against vancomycin resistant bacterial strains via direct antibiotic activity and by augmenting both host macrophage recruitment to the site of infection and macrophage bactericidal activity.

microbiology↗