Phage resistance in Klebsiella pneumoniae and bidirectional effects impacting antibiotic susceptibility.

OBJECTIVES: Bacteriophage (phage) therapy is a promising anti-infective option to combat antimicrobial resistance. However, the clinical utilization of phage therapy has been severely compromised by the potential emergence of phage resistance. Although certain phage resistance mechanisms can restore bacterial susceptibility to certain antibiotics, a lack of knowledge of phage resistance mechanisms hinders optimal use of phages and their combination with antibiotics. METHODS: Genome-wide transposon screening was performed with a mutant library of Klebsiella pneumoniae MKP103 to identify phage pKMKP103_1-resistant mutants. Phage-resistant phenotypes were evaluated by time-kill kinetics and efficiency of plating assays. Phage resistance mechanisms were investigated with adsorption, one-step growth, and mutation frequency assays. Antibiotic susceptibility was determined with broth microdilution and population analysis profiles. RESULTS: We observed a repertoire of phage resistance mechanisms in K pneumoniae, such as disruption of phage binding (fhuA::Tn and tonB::Tn), extension of the phage latent period (mnmE::Tn and rpoN::Tn), and increased mutation frequency (mutS::Tn and mutL::Tn). Notably, in contrast to the prevailing view that phage resistance re-sensitizes antibiotic-resistant bacteria, we observed a bidirectional steering effect on bacterial antibiotic susceptibility. Specifically, rpoN::Tn increased susceptibility to colistin while mutS::Tn and mutL::Tn increased resistance to rifampicin and colistin. DISCUSSION: Our findings demonstrate that K pneumoniae employs multiple strategies to overcome phage infection, which may result in enhanced or reduced antibiotic susceptibility. Mechanism-guided phage steering should be incorporated into phage therapy to better inform clinical decisions on phage-antibiotic combinations.

Transcriptomic interplay between Acinetobacter baumannii , human macrophage and polymyxin.

Optimization of antibiotic therapy has been hindered by our dearth of understanding on the mechanism of the host-pathogen-drug interactions. Here, we employed dual RNA-sequencing to examine transcriptomic perturbations in response to polymyxin B in a co-culture infection model of Acinetobacter baumannii and human macrophages. Our findings revealed that polymyxin B treatment induced significant transcriptomic response in macrophage-interacting A. baumannii , exacerbating bacterial oxidative stress, disrupting metal homeostasis, affecting osmoadaptation, triggering stringent stress response, and influencing pathogenic factors. Moreover, infected macrophages adapt heme catabolism, coagulation cascade, and hypoxia-inducible signaling to confront bacterial invasion. Disrupting rcnB , ompW , and traR/dksA genes in A. baumannii impairs metal homeostasis, osmotic stress defense and stringent responses, thereby enhancing antibacterial killing by polymyxin. These findings shed light on the global stress adaptations at the network level during host-pathogen-drug interactions, revealing promising therapeutic targets for further investigation. IMPORTANCE: In the context of the development of bacterial resistance during the course of antibiotic therapy, the role of macrophages in shaping bacterial response to antibiotic killing remains enigmatic. Herein we employed dual RNA-sequencing and an in vitro tripartite model to delve into the unexplored transcriptional networks of the Acinetobacter baumannii -macrophage-polymyxin axis. Our findings uncovered the potential synergy between macrophages and polymyxin B which appear to act in co-operation to disrupt multiple stress tolerance mechanisms in A. baumannii . Notably, we discovered the critical roles of bacterial nickel/cobalt homeostasis ( rcnB family), osmotic stress defense ( ompW family), and stringent response regulator ( traR/dksA C4-type zinc finger) in tolerating the last-line antibiotic polymyxin B. Our findings may lead to potential targets for the development of novel therapeutics against the problematic pathogen A. baumannii .

Minocycline attenuates the bilirubin-induced developmental neurotoxicity through the regulation of innate immunity and oxidative stress in zebrafish embryos.

When liver or intestinal function is impaired, bilirubin accumulates in the body and leads to neonatal jaundice. However, the potential negative effects caused by excessive accumulation of bilirubin such as developmental immunotoxicity and neurotoxicity remain unclear. We used a zebrafish model to establish bilirubin-induced jaundice symptoms and evaluated the toxic effects of bilirubin in aquatic organisms. Firstly, our results suggested that bilirubin exposure markedly decreased the survival rate, induced the developmental toxicity and increased the yellow pigment deposited in the zebrafish tail. Meanwhile, the number of macrophages and neutrophils was substantially reduced in a concentration-dependent manner. Besides, the antioxidant enzyme activities were greatly elevated while the inflammatory genes were significantly decreased after bilirubin exposure. Secondly, transcriptome analysis identified 708 genes were differentially expressed after bilirubin exposure, which animal organ morphogenesis, chemical synaptic transmission, and MAPK / mTOR signaling pathways were significantly enriched. Thirdly, bilirubin exposure leads to a significant decrease in the motility of zebrafish, including a dose-dependent decrease in the travelled distance, movement time, and average velocity. Moreover, the innate immune genes and apoptosis-related genes such as TLR4, NF-κB p65, STAT3 and p53 were elevated at a concentration of 10 µg/mL of bilirubin. Finally, our results further revealed that the anti-inflammatory and neuroprotective minocycline could partially rescue the bilirubin-induced neurobehavioral disorders in zebrafish embryos. In conclusion, our study explored the bilirubin-induced immunotoxicity and neurotoxicity in aquatic organisms, which will provide a theoretical basis for the treatment of neonatal jaundice in clinical practice.

Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Characterization of outer membrane vesicles released by clinical isolates of Neisseria gonorrhoeae.

The sexually transmitted pathogen Neisseria gonorrhoeae releases membrane vesicles including outer membrane vesicles (OMVs) during infections. OMVs traffic outer membrane molecules, such as the porin PorB and lipo-oligosaccharide (LOS), into host innate immune cells, eliciting programmed cell death pathways, and inflammation. Little is known, however, about the proteome and LOS content of OMVs released by clinical strains isolated from different infection sites, and whether these vesicles similarly activate immune responses. Here, we characterized OMVs from four N. gonorrhoeae isolates and determined their size, abundance, proteome, LOS content, and activation of inflammatory responses in macrophages. The overall proteome of the OMVs was conserved between the four different isolates, which included major outer membrane and periplasm proteins. Despite this, we observed differences in the rate of OMV biogenesis and the relative abundance of membrane proteins and LOS. Consequently, OMVs from clinical isolates induced varying rates of macrophage cell death and the secretion of interleukin-1 family members, such as IL-1α and IL-1ß. Overall, these findings demonstrate that clinical isolates of N. gonorrhoeae utilize membrane vesicles to release proteins and lipids, which affects innate immune responses.

Natural variants of molybdate transporters contribute to yield traits of soybean by affecting auxin synthesis.

Soybean (Glycine max) is a crop with high demand for molybdenum (Mo) and typically requires Mo fertilization to achieve maximum yield potential. However, the genetic basis underlying the natural variation of Mo concentration in soybean and its impact on soybean agronomic performance is still poorly understood. Here, we performed a genome-wide association study (GWAS) to identify GmMOT1.1 and GmMOT1.2 that drive the natural variation of soybean Mo concentration and confer agronomic traits by affecting auxin synthesis. The soybean population exhibits five haplotypes of the two genes, with the haplotype 5 demonstrating the highest expression of GmMOT1.1 and GmMOT1.2, as well as the highest transport activities of their proteins. Further studies showed that GmMOT1.1 and GmMOT1.2 improve soybean yield, especially when cultivated in acidic or slightly acidic soil. Surprisingly, these two genes contribute to soybean growth by enhancing the activity of indole-3-acetaldehyde (IAAld) aldehyde oxidase (AO), leading to increased indole-3-acetic acid (IAA) synthesis, rather than being involved in symbiotic nitrogen fixation or nitrogen assimilation. Furthermore, the geographical distribution of five haplotypes in China and their correlation with soil pH suggest the potential significance of GmMOT1.1 and GmMOT1.2 in soybean breeding strategies.

The evolutionary innovation of root suberin lamellae contributed to the rise of seed plants.

Seed plants overtook ferns to become the dominant plant group during the late Carboniferous, a period in which the climate became colder and dryer1,2. However, the specific innovations driving the success of seed plants are not clear. Here we report that the appearance of suberin lamellae (SL) contributed to the rise of seed plants. We show that the Casparian strip and SL vascular barriers evolved at different times, with the former originating in the most recent common ancestor (MRCA) of vascular plants and the latter in the MRCA of seed plants. Our results further suggest that most of the genes required for suberin formation arose through gene duplication in the MRCA of seed plants. We show that the appearance of the SL in the MRCA of seed plants enhanced drought tolerance through preventing water loss from the stele. We hypothesize that SL provide a decisive selective advantage over ferns in arid environments, resulting in the decline of ferns and the rise of gymnosperms. This study provides insights into the evolutionary success of seed plants and has implications for engineering drought-tolerant crops or fern varieties.

A new family of proteins is required for tethering of Casparian strip membrane domain and nutrient homoeostasis in rice.

Cell-cell junctions are essential for multicellular organisms to maintain nutrient homoeostasis. A plant-type tight junction, the Casparian strip (CS)-Casparian strip membrane domain (CSD) that seals the paracellular space between adjacent endodermal cells, has been known for more than one hundred years. However, the molecular basis of this structure remains unknown. Here we report that a new family of proteins containing a glycine/alanine/proline-rich domain, a lectin domain and a secretory signal peptide (GAPLESS) mediates tethering of the plasma membrane to the CS in rice. The GAPLESS proteins are specifically localized in the CS of root endodermal cells, and loss of their functions results in a disabled cell-cell junction and disrupted nutrient homoeostasis. The GAPLESS protein forms a tight complex with OsCASP1 in the plasma membrane, thereby mediating the CS-CSD junction. This study provides valuable insights into the junctional complex of plant endodermal cells, shedding light on our understanding of nutrient homoeostasis in crops and the cell junctions of eukaryotes.

Metabolic perturbations and key pathways associated with the bacteriostatic activity of Clitoria ternatea flower anthocyanin fraction against Escherichia coli.

Clitoria ternatea flowers are rich in anthocyanins and possess various biological activities. Specifically, the antibacterial mechanism of action of C. ternatea anthocyanins remains unknown and was investigated in Escherichia coli . A time-kill assay was used to assess the antibacterial activity and the metabolic perturbations in E. coli were investigated utilizing liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. Pathway analyses were carried out for metabolites showing ≥2-fold changes. The anthocyanin fraction remarkably reduced the growth of E. coli at 4 h by 95.8 and 99.9 % at minimum inhibitory concentration (MIC) and 2× MIC, respectively. The anthocyanin fraction (MIC) had a bacteriostatic effect and was shown to have perturbed glycerophospholipids (1-acyl-sn-glycero-3-phosphoethanolamine, phosphatidylglycerol, diacylglycerol and cardiolipin), amino acids (valine, tyrosine and isoleucine) and energy (ubiquinone and NAD) metabolites at 1 and 4 h. This study demonstrated significant metabolic perturbations of the glycerophospholipid, amino acid and energy metabolism, with these being the key pathways involved in the bacteriostatic activity of anthocyanins from C. ternatea, which may have promise as bacteriostatic agents for E. coli -related infections.

Lipid A Modification and Metabolic Adaptation in Polymyxin-Resistant, New Delhi Metallo-ß-Lactamase-Producing Klebsiella pneumoniae.

Polymyxins are last-line antibiotics employed against multidrug-resistant (MDR) Klebsiella pneumoniae. Worryingly, polymyxin resistance is rapidly on the rise globally. Polymyxins initially target lipid A of lipopolysaccharides (LPSs) in the cell outer membrane (OM), causing disorganization and cell lysis. While most studies focus on how genetic variations confer polymyxin resistance, the mechanisms of membrane remodeling and metabolic changes in polymyxin-resistant strains remain unclear, thus hampering the development of effective therapies to treat severe K. pneumoniae infections. In the present study, lipid A profiling, OM lipidomics, genomics, and metabolomics were integrated to elucidate the global mechanisms of polymyxin resistance and metabolic adaptation in a polymyxin-resistant strain (strain S01R; MIC of >128 mg/L) obtained from K. pneumoniae strain S01, a polymyxin-susceptible (MIC of 2 mg/L), New Delhi metallo-ß-lactamase (NDM)-producing MDR clinical isolate. Genomic analysis revealed a novel in-frame deletion at position V258 of PhoQ in S01R, potentially leading to lipid A modification with 4-amino-4-deoxy-l-arabinose (L-Ara4N) despite the absence of polymyxin B. Comparative metabolomic analysis revealed slightly elevated levels of energy production and amino acid metabolism in S01R compared to their levels in S01. Exposure to polymyxin B (4 mg/L for S01 and 512 mg/L for S01R) substantially altered energy, nucleotide, and amino acid metabolism and resulted in greater accumulation of lipids in both strains. Furthermore, the change induced by polymyxin B treatment was dramatic at both 1 and 4 h in S01 but only significant at 4 h in S01R. Overall, profound metabolic adaptation was observed in S01R following polymyxin B treatment. These findings contribute to our understanding of polymyxin resistance mechanisms in problematic NDM-producing K. pneumoniae strains and may facilitate the discovery of novel therapeutic targets. IMPORTANCE Antimicrobial resistance (AMR) is a major threat to global health. The emergence of resistance to the polymyxins that are the last line of defense in so-called Gram-negative "superbugs" has further increased the urgency to develop novel therapies. There are frequent outbreaks of K. pneumoniae infections in hospitals being reported, and polymyxin usage is increasing remarkably. Importantly, the polymyxin-resistant K. pneumoniae strains are imposing more severe consequences to health systems. Using metabolomics, lipid A profiling, and outer membrane lipidomics, our findings reveal (i) changes in the pentose phosphate pathway and amino acid and nucleotide metabolism in a susceptible strain following polymyxin treatment and (ii) how cellular metabolism, lipid A modification, and outer membrane remodeling were altered in K. pneumoniae following the acquisition of polymyxin resistance. Our study provides, for the first time, mechanistic insights into metabolic responses to polymyxin treatment in a multidrug-resistant, NDM-producing K. pneumoniae clinical isolate with acquired polymyxin resistance. Overall, these results will assist in identifying new therapeutic targets to combat and prevent polymyxin resistance.

Spinal CBX2 contributes to neuropathic pain by activating ERK signaling pathway in male mice.

The deregulated spinal cord proteins induced by nerve injury are the key to neuropathic pain. Integrated transcriptome and translatome analyses can screen out deregulated proteins controlled by only post-transcriptional regulation. By comparing RNA sequencing (RNA-seq) and ribosome profiling sequencing (Ribo-seq) data, we identified an upregulated protein, chromobox 2 (CBX2), with its mRNA level unchanged in the spinal cord after peripheral nerve injury. CBX2 was mainly distributed in the spinal cord neurons. Blocking the SNL-induced increase of spinal CBX2 attenuated the neuronal and astrocytes hyperactivities and pain hypersensitivities in both the development and maintenance phases. Conversely, mimicking the upregulation of CBX2 in the spinal cord facilitated the activities of neurons and astrocytes and produced evoked nociceptive hypersensitivity and spontaneous pain. Our results also revealed that activating the ERK pathway, upregulating CXCL13 in neurons, and CXCL13 further inducing astrocyte activation were possible downstream signaling mechanisms of CBX2 in pain processing. In conclusion, upregulation of CBX2 after nerve injury leads to nociceptive hyperalgesia by promoting neuronal and astrocyte hyperactivities through the ERK pathway. Inhibiting CBX2 upregulation may be therapeutically beneficial.

Model-informed dose optimisation of polymyxin-rifampicin combination therapy against multidrug-resistant Acinetobacter baumannii.

OBJECTIVES: Antimicrobial resistance is a major global threat. Because of the stagnant antibiotic pipeline, synergistic antibiotic combination therapy has been proposed to treat rapidly emerging multidrug-resistant (MDR) pathogens. We investigated antimicrobial synergy of polymyxin/rifampicin combination against MDR Acinetobacter baumannii. METHODS: In vitro static time-kill studies were performed over 48 h at an initial inoculum of ∼107 CFU/mL against three polymyxin-susceptible but MDR A. baumannii isolates. Membrane integrity was examined at 1 and 4 h post-treatment to elucidate the mechanism of synergy. Finally, a semi-mechanistic PK/PD model was developed to simultaneously describe the time course of bacterial killing and prevention of regrowth by mono- and combination therapies. RESULTS: Polymyxin B and rifampicin alone produced initial killing against MDR A. baumannii but were associated with extensive regrowth. Notably, the combination showed synergistic killing across all three A. baumannii isolates with bacterial loads below the limit of quantification for up to 48 h. Membrane integrity assays confirmed the role of polymyxin-driven outer membrane remodelling in the observed synergy. Subsequently, the mechanism of synergy was incorporated into a PK/PD model to describe the enhanced uptake of rifampicin due to polymyxin-induced membrane permeabilisation. Simulations with clinically utilised dosing regimens confirmed the therapeutic potential of this combination, particularly in the prevention of bacterial regrowth. Finally, results from a neutropenic mouse thigh infection model confirmed the in vivo synergistic killing of the combination against A. baumannii AB5075. CONCLUSION: Our results showed that polymyxin B combined with rifampicin is a promising option to treat bloodstream and tissue infection caused by MDR A. baumannii and warrants clinical evaluations.

The effect of different representations of pictures on the activation of gender stereotypes.

The purpose of this study is to explore whether there is N400 effect on the representation of gender stereotype in the different picture priming condition from the behavioral and ERP levels, and further explore whether there is hierarchical structure of upper category, secondary category, typical example and counter-example based on this. The results showed: (1) under the condition of picture priming, N400 effect would be induced when representing the conflict of gender stereotypes. (2) Category representation and example representation can activate different regions of the brain. When the priming stimulus was upper category (gender picture) and secondary category (occupational gender picture), N400 effect mainly appeared on the electrode of frontal region in left hemisphere.When the priming stimuli were typical example (typical example picture) and counter-example, the N400 effect mainly appeared on the electrodes in the frontal region of the right hemisphere.(3) the gender stereotype representation of picture activation has hierarchical structure, that is, N400 amplitude induced by upper category activation < secondary category activation < typical sample activation < counter-example activation. These findings suggest that the representation of gender stereotypes has a hierarchical structure at the picture level.

The eTM-miR858-MYB62-like module regulates anthocyanin biosynthesis under low-nitrogen conditions in Malus spectabilis.

The anthocyanin content increases in Malus spectabilis leaves under low-nitrogen conditions. Noncoding RNAs are indicated to play key regulatory roles in anthocyanin biosynthesis. However, the functional roles of noncoding RNAs in anthocyanin biosynthesis under low-nitrogen conditions remain elusive. In this study, miR858 was screened as a key regulator of anthocyanin biosynthesis under low-nitrogen conditions through whole-transcriptome sequencing. Then, we used miR858 as an entry point to explore the regulatory network of lncRNA-miRNA-mRNA by dual-luciferase reporter assays and GUS histochemical staining assays, as well as to identify the mechanism of this regulatory network in anthocyanin biosynthesis by both transient and stable transformation experiments in Malus. MiR858 overexpression increased total anthocyanin content. MiR858 acted by negatively regulating its target gene, MsMYB62-like, under the low-nitrogen condition. MsMYB62-like inhibited the expression of MsF3'H, thereby negatively regulating anthocyanin biosynthesis. In addition, eTM858-1 and eTM858-2 were identified as endogenous target mimics of miR858 that bind to miR858 to prevent cleavage of MsMYB62-like and thereby negatively regulate anthocyanin biosynthesis. The results clarify the mechanism through which the eTM-miR858-MYB62-like module regulates anthocyanin biosynthesis in Malus under low-nitrogen conditions.

Swelling-Mediated Mechanical Stimulation Regulates Differentiation of Adipose-Derived Mesenchymal Stem Cells for Intervertebral Disc Repair Using Injectable UCST Microgels.

Mechanical stimulation is an effective approach for controlling stem cell differentiation in tissue engineering. However, its realization in in vivo tissue repair remains challenging since this type of stimulation can hardly be applied to injectable seeding systems. Here, it is presented that swelling of injectable microgels can be transformed to in situ mechanical stimulation via stretching the cells adhered on their surface. Poly(acrylamide-co-acrylic acid) microgels with the upper critical solution temperature property are fabricated using inverse emulsion polymerization and further coated with polydopamine to increase cell adhesion. Adipose-derived mesenchymal stem cells (ADSCs) adhered on the microgels can be omnidirectionally stretched along with the responsive swelling of the microgels, which upregulate TRPV4 and Piezo1 channel proteins and enhance nucleus pulposus (NP)-like differentiation of ADSCs. In vivo experiments reveal that the disc height and extracellular matrix content of NP are promoted after the implantation with the microgels. The findings indicate that swelling-induced mechanical stimulation has great potential for regulating stem cell differentiation during intervertebral disc repair.

A genome-wide association study identifies a transporter for zinc uploading to maize kernels.

The Zn content in cereal seeds is an important trait for crop production as well as for human health. However, little is known about how Zn is loaded to plant seeds. Here, through a genome-wide association study (GWAS), we identify the Zn-NA (nicotianamine) transporter gene ZmYSL2 that is responsible for loading Zn to maize kernels. High promoter sequence variation in ZmYSL2 most likely drives the natural variation in Zn concentrations in maize kernels. ZmYSL2 is specifically localized on the plasma membrane facing the maternal tissue of the basal endosperm transfer cell layer (BETL) and functions in loading Zn-NA into the BETL. Overexpression of ZmYSL2 increases the Zn concentration in the kernels by 31.6%, which achieves the goal of Zn biofortification of maize. These findings resolve the mystery underlying the loading of Zn into plant seeds, providing an efficient strategy for breeding or engineering maize varieties with enriched Zn nutrition.

Cognitive processing differences between stereotype activation and semantic activation.

Previous studies have demonstrated that the activation of stereotype conflict is similar to the N400 congruency effect shown by the activation of semantic violation. In order to distinguish the differences between the two, the first experiment used gender stereotype trait words as target stimuli, and used "male/female" and "synonym of trait words/antonym of trait words" as priming stimuli respectively, so that the subjects completed the consistency determination task. In experiment 2, gender stereotyped behavior pictures were used as target stimuli, and "male/female" was used as priming stimuli, so that the subjects completed the task of consistency determination. The results showed that both gender stereotype conflict and semantic violation could induce N400 a congruency effect. Importantly, the N400 amplitude and response latency induced by gender stereotype activation are both smaller than those induced by semantic activation. These results show that stereotype activation is distinct from semantic activation, further demonstrating that the brain preferentially processes information related to gender stereotypes, and gender stereotype cognitive processing is more likely to happen than semantic knowledge processing.

Cognitive processing differences between stereotype activation and semantic activation.

Previous studies have demonstrated that the activation of stereotype conflict is similar to the N400 congruency effect shown by the activation of semantic violation. In order to distinguish the differences between the two, the first experiment used gender stereotype trait words as target stimuli, and used "male/female" and "synonym of trait words/antonym of trait words" as priming stimuli respectively, so that the subjects completed the consistency determination task. In experiment 2, gender stereotyped behavior pictures were used as target stimuli, and "male/female" was used as priming stimuli, so that the subjects completed the task of consistency determination. The results showed that both gender stereotype conflict and semantic violation could induce N400 a congruency effect. Importantly, the N400 amplitude and response latency induced by gender stereotype activation are both smaller than those induced by semantic activation. These results show that stereotype activation is distinct from semantic activation, further demonstrating that the brain preferentially processes information related to gender stereotypes, and gender stereotype cognitive processing is more likely to happen than semantic knowledge processing.