Thanatin, a potent cationic antimicrobial peptide, has demonstrated remarkable efficacy against new NDM-1 producing bacteria. However, its clinical application is hampered by suboptimal stability in circulation and limited bioavailability in the human body. To overcome these challenges, a novel thanatin nanomedicine has been developed, which encapsulated thanatin in nanoparticles formed by electrostatic interactions between negatively charged HA and PLGA. The obtained ThaNPs demonstrated good stability, low cytotoxicity, and good metabolic ratio. ThaNPs significantly improve the stability of thanatin in the circulation, increasing its half-life in 50 % serum from 0.6 h to 3.2 h. Notably, the protective effect of ThaNPs against sepsis induced by NDM1-producing Escherichia coli. was 10-fold higher than that of unencapsulated thanatin. These findings suggest that hyaluronic acid-based nanoparticles have the potentiality to overcome the clinical limitations associated with cationic antimicrobial peptides, thereby providing a novel and effective strategy for treating severe infections caused by antibiotic-resistant bacteria.

Trans-cinnamaldehyde (CA), a natural compound from cinnamon oil, is known for its broad-spectrum but limited antibacterial activity. In this study, we synthesized and evaluated a series of CA derivatives, identifying α-Bromocinnamaldehyde (BCA) as the most potent antibacterial agent against ESKAPE pathogens. Through minimum inhibitory concentration (MIC) assays and a detailed structure-activity analysis, we determined that BCA’s enhanced antibacterial potency is due to the α-bromine substitution, the aldehyde group, and the conjugated double bond. In an in vivo septic mouse model infected with NDM-1-producing E. coli, BCA treatment significantly improved survival rates, and no acute renal or liver toxicity was observed at therapeutic doses. Mechanistic studies, including scanning electron microscopy, fluorescence microscopy, Gram staining, light-scattering assays for FtsZ polymerization, and GTP hydrolysis assays, have elucidated that BCA exerts its bactericidal effects through a dual-mechanism approach. Firstly, BCA increases membrane permeability, disrupting the integrity of the bacterial cell membrane. Secondly, it inhibits bacterial cell division, likely by interfering with the polymerization of FtsZ, a key protein involved in cell division, and affecting GTP hydrolysis processes essential for cell division progression. These findings highlight BCA’s dual bactericidal mechanisms and support its potential as a promising therapeutic agent for treating multidrug-resistant bacterial infections. This study explores chemical modifications of cinnamaldehyde (CA) to enhance its antibacterial activity. α-Bromocinnamaldehyde (BCA), a key derivative, exhibits potent, broad-spectrum activity against multidrug-resistant ESKAPE pathogens. Mechanistic insights reveal that BCA disrupts bacterial membranes and inhibits cell division, highlighting its potential as a novel antibacterial agent in the fight against antibiotic-resistant infections

New Delhi metallo-β-lactamase-1 (NDM-1)-producing bacteria are resistant to nearly all available β-lactam antibiotics and have become a public health threat. Antibiotic resistance often carries fitness costs, which typically manifest as a reduced bacterial growth rate. Here, we investigated the mechanism of fitness cost in NDM-1-producing bacteria. Our findings revealed that strains expressing blaNDM-1 exhibited a significant growth defect under high osmotic stress. This fitness cost was attributed to the anchoring of NDM-1 to the bacterial outer membrane via its leader peptide, which destabilized the outer membrane. Replacing the membrane-anchoring residue Cys26 in the leader peptide with alanine not only restored outer membrane stability but also ameliorated the bacterial fitness cost. Furthermore, the anchoring of NDM-1 to the membrane increased bacterial susceptibility to the membrane-disrupting antibiotic colistin, both in vitro and in vivo, as confirmed in engineered and clinically isolated strains. In conclusion, membrane anchoring of NDM-1 increased the permeability of the bacterial outer membrane, thereby reducing the fitness of NDM-1-producing bacteria and enhancing their susceptibility to colistin. These results not only elucidate the mechanism of fitness cost associated with NDM-1 but also provide new insights into the rational use of colistin to combat infections caused by NDM-1-producing bacteria.

The emergence of antibiotic-resistant-bacteria is a serious public health threat, which prompts us to speed up the discovery of novel antibacterial agents. Phage display technology has great potential to screen peptides or antibodies with high binding capacities for a wide range of targets. This property is significant in the rapid search for new antibacterial agents for the control of bacterial resistance. In this paper, we not only summarized the recent progress of phage display for the discovery of novel therapeutic agents, identification of action sites of bacterial target proteins, and rapid detection of different pathogens, but also discussed several problems of this technology that must be solved. Breakthrough in these problems may further promote the development and application of phage display technology in the biomedical field in the future.

Sepsis is a leading cause of in-hospital mortality resulting from a dysregulated response to infection. Novel immunomodulatory therapies targeting macrophage metabolism have emerged as an important focus for current sepsis research. However, understanding the mechanisms underlying macrophage metabolic reprogramming and how they impact immune response requires further investigation. Here, we identify macrophage-expressed Spinster homolog 2 (Spns2), a major transporter of sphingosine-1-phosphate (S1P), as a crucial metabolic mediator that regulates inflammation through the lactate-reactive oxygen species (ROS) axis. Spns2 deficiency in macrophages significantly enhances glycolysis, thereby increasing intracellular lactate production. As a key effector, intracellular lactate promotes pro-inflammatory response by increasing ROS generation. The overactivity of the lactate-ROS axis drives lethal hyperinflammation during the early phase of sepsis. Furthermore, diminished Spns2/S1P signaling impairs the ability of macrophages to sustain an antibacterial response, leading to significant innate immunosuppression in the late stage of infection. Notably, reinforcing Spns2/S1P signaling contributes to balancing the immune response during sepsis, preventing both early hyperinflammation and later immunosuppression, making it a promising therapeutic target for sepsis.

In recent years, infectious diseases have again become a critical threat to global public health largely due to the challengesposed by antimicrobial resistance. Conventional antibiotics haveplayed a crucial role in combating bacterial infections; however, theirefficacy is significantly impaired by widespread drug resistance.Natural antimicrobial peptides (AMPs) and their polymeric mimicsdemonstrate great potential for killing bacteria with low propensity ofresistance as they target the microbial membrane rather than aspecific molecular target, but they are also toxic to the host eukaryoticcells. To minimize antibiotics systemic spread and the required dosethat promote resistance and to advocate practical realization of thepromising activity of AMPs and polymers, smart systems to targetbacteria are highly sought after. This review presents bacterialrecognition by various specific targeting molecules and the delivery systems of active components in supramolecules. Bacteria-induced activations of antimicrobial-based nanoformulations are also included. Recent advances in the bacteria targeting and delivery of synthetic antimicrobial agents may assist in developing new classes of highly selective antimicrobial systems which can improve bactericidal efficacy and greatly minimize the spread of bacterial resistance

细菌囊泡(membrane vesicles,MVs)是一类由细菌分泌产生的膜性小体,通过携带运输蛋白质、核酸等物质,参与群体交流、宿主感染、生存防御等过程.MVs常见于革兰氏阴性菌,由细菌外膜向外出芽产生.最新研究表明,MVs也是革兰氏阳性菌的重要分泌方式;此外,噬菌体裂解细菌亦可诱发MVs的产生.理解MVs的多样性可为深入探究细菌群体行为提供理论基础,并为推进生物医药及纳米技术的研发提供新思路.本文将对MVs的分类、发生机制及其功能的最新研究进展进行综述.

Abstract Background Outbreaks of infection due to multidrug-resistant (MDR) bacteria, especially Gram-negative bacteria, have become a global health issue in both hospitals and communities. Antisense oligonucleotides (ASOs) based therapeutics hold a great promise for treating infections caused by MDR bacteria. However, ASOs therapeutics are strangled because of its low cell penetration efficiency caused by the high molecular weight and hydrophilicity. Results Here, we designed a series of dendritic poly-peptides (DPP1 to DPP12) to encapsulate ASOs to form DSPE-mPEG2000 decorated ASOs/DPP nanoparticles (DP-AD1 to DP-AD12) and observed that amphipathic DP-AD2, 3, 7 or 8 with a positive charge ≥ 8 showed great efficiency to deliver ASOs into bacteria, but only the two histidine residues contained DP-AD7 and DP-AD8 significantly inhibited the bacterial growth and the targeted gene expression of tested bacteria in vitro. DP-AD7anti-acpP remarkably increased the survival rate of septic mice infected by ESBLs-E. coli, exhibiting strong antibacterial effects in vivo. Conclusions For the first time, we designed DPP as a potent carrier to deliver ASOs for combating MDR bacteria and demonstrated the essential features, namely, amphipathicity, 8–10 positive charges, and 2 histidine residues, that are required for efficient DPP based delivery, and provide a novel approach for the development and research of the antisense antibacterial strategy. Graphical Abstract

DMB (6,7-dichloro-2-methylsulfonyl-3-Ntert-butylaminoquinoxaline) is a quinoxaline-based compound that has been investigated as a glucagon-like peptide-1 receptor (GLP-1R) agonist. To clarify anti-osteoporosis effect of DMB, an osteoporotic mice model was established by ovariectomy (OVX) operation. The OVX mice were given intraperitoneally DMB, exendin-4 (EX-4), or 17β-estradiol (E2) for two months. Then bone mass and structure, and bone morphometric parameters were examined by micro-CT. Weight gain and food consumption, bone turnover markers, and biomechanical strength of the femur were tested, and bone histomorphometry was analyzed. The food intake and weight gain was obviously reduced by E2 or EX-4, but not DMB. However, DMB or EX-4 treatment obviously inhibited skeletal deterioration and enhanced bone strength. The improvement involved in the increased osteoblast number and level of bone formation markers, and reduced osteoclasts number and level of bone resorption markers. In addition, DMB was found to stimulate osteoblastogenesis-related marker gene expression. These results demonstrated that DMB ameliorated bone loss mainly via induction of bone formation, which suggests that the small molecule compound might be applied to the management of postmenopausal osteoporosis.

Bacterial colonization on biomedical devices often leads to biofilms that are recalcitrant to antibiotic treatment and the leading cause of hospital-acquired infections. We have invented a novel pretreatment chemistry for device surfaces to produce a high-density three-dimensional (3-D) network of covalently linked S-nitrosothiol (RSNO), which is a nitric oxide (NO) donor. Poly(polyethylene glycol-hydroxyl-terminated) (i.e., PPEG-OH) brushes were grafted from an ozone-pretreated polyurethane (PU) surface. The high-density hydroxyl groups on the dangling PPEG-OH brushes then underwent condensation with a mercapto-silane (i.e., MPS, mercaptopropyl trimethoxysilane) followed by S-nitrosylation to produce a 3-D network of NO-releasing RSNO to form the PU/PPEG-OH-MPS-NO coating. This 3-D coating produces NO flux of up to 7 nmol/(cm2 min), which is nearly 3 orders of magnitude higher than the picomole/(cm2 min) levels of other NO-releasing biomedical implants previously reported. The covalent immobilization of RSNO avoids donor leaching and reduces the risks of cytotoxicity arising from leachable RSNO. Our coated PU surfaces display good biocompatibility and exhibit excellent antibiofilm formation activity in vitro (up to 99.99%) against a broad spectrum of Gram-positive and Gram-negative bacteria. Further, the high-density RSNO achieves nearly 99% and 99.9% in vivo reduction of Pseudomonas aeruginosa (P. aeruginosa) and methicillin-resistant Staphylococcus aureus (MRSA) in a murine subcutaneous implantation infection model. Our surface chemistry to create high NO payload without NO-donor leaching can be applied to many biomedical devices.

Cationic polymers are promising antibacterial agents because bacteria have a low propensity to develop resistance against them, but they usually have low biocompatibility because of their hydrophobic moieties. Herein, we report a new biodegradable and biocompatible chitosan-derived cationic antibacterial polymer, 2,6-diamino chitosan (2,6-DAC). 2,6-DAC shows excellent broad-spectrum antimicrobial activity with minimum inhibitory concentrations (MICs) of 8-32 μg/mL against clinically relevant and multidrug-resistant (MDR) bacteria including Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii. Furthermore, 2,6-DAC shows an excellent synergistic effect with various clinically relevant antibiotics proved by decreasing the MICs of the antibiotics against MDR A. baumannii and methicillin-resistant Staphylococcus aureus to <1 μg/mL. In vivo biocompatibility of 2,6-DAC is proved by a dosage of 100 mg/kg compound via oral administration and 25 mg/kg compound via intraperitoneal injection to mice; 2,6-DAC does not cause any weight loss and any significant change in liver and kidney biomarkers or the important blood electrolytes. The combinations of 2,6-DAC together with novobiocin and rifampicin show >2.4 log10 reduction of A. baumannii in murine intraperitoneal and lung infection models. The novel chitosan derivative, 2,6-DAC, can be utilized as a biocompatible broad-spectrum cationic antimicrobial agent alone or in synergistic combination with various antibiotics.

Zwitterionic polymers are classical antifouling polymers but they require specialized monomers that have cationic and anionic charges integrated into a single monomer. Herein, we show that pseudo-zwitterionic copolymers synthesized from a mixture of 2 monomers each having a single opposite polarity has excellent antibiofilm efficacy. We have discovered a new mixed-charge copolymer brush (#1-A) synthesized from 2 oppositely charged monomers, the anionic SPM (3-Sulfopropyl methacrylate) and the cationic AMPTMA ((3-Acrylamidopropyl) trimethylammonium chloride), that achieves broad spectrum in vitro antibiofilm effect of greater than 99% reductions against all six Gram-positive and Gram-negative bacteria tested. In the murine subcutaneous wound catheter infection models, the #1-A has good long-term anti-biofilm efficacy against MRSA and Pseudomonas aeruginosa of 3.41 and 3.19 orders respectively, outperforming previous mixed-charge copolymer coatings. We discovered a new method to choose the cationic/anionic pair combination to form the best antibiofilm copolymer brush coating by exploiting the solution polymerization kinetics disparity between the cationic and anionic monomers. We also showed that #1-A is softer and has higher hydration than the classical zwitterionic polymer. This study shows the possibility of achieving potent antibiofilm efficacy by combining readily available opposite singly charged monomers.

Bacterial cell division is a highly controlled process regulated accurately by a diverse array of proteins spatially and temporally working together. Among these proteins, FtsZ is recognized as a cytoskeleton protein because it can assemble into a ring-like structure called Z-ring at midcell. Z-ring recruits downstream proteins, thus forming a multiprotein complex termed the divisome. When the Z-ring scaffold is established and the divisome matures, peptidoglycan (PG) biosynthesis and chromosome segregation are triggered. In this review, we focus on multiple interactions between FtsZ and its accessory proteins in bacterial cell cytokinesis, including FtsZ localization, Z-ring formation and stabilization, PG biosynthesis, and chromosome segregation. Understanding the interactions among these proteins may help discover superior targets on treating bacterial infectious diseases.

BACKGROUND:Antisense oligonucleotides (ASOs) based technology is considered a potential strategy against antibiotic-resistant bacteria; however, a major obstacle to the application of ASOs is how to deliver them into bacteria effectively. DNA tetrahedra (Td) is an emerging carrier for delivering ASOs into eukaryotes, but there is limited information about Td used for bacteria. In this research, we investigated the uptake features of Td and the impact of linkage modes between ASOs and Td on gene-inhibition efficiency in bacteria.RESULTS:Td was more likely to adhere to bacterial membranes, with moderate ability to penetrate into the bacteria. Strikingly, Td could penetrate into bacteria more effectively with the help of Lipofectamine 2000 (LP2000) at a 0.125 μL/μg ratio to Td, but the same concentration of LP2000 had no apparent effect on linear DNA. Furthermore, linkage modes between ASOs and Td influenced gene-knockdown efficiency. Looped structure of ASOs linked to one side of the Td exhibited better gene-knockdown efficiency than the overhung structure.CONCLUSIONS:This study established an effective antisense delivery system based on loop-armed Td, which opens opportunities for developing antisense antibiotics.

目的 探究RNAⅢ抑制肽(RIP)在细菌的不同生长时相对葡萄球菌溶素和黏附相关蛋白表达水平的调节作用.方法 按比例接种耐甲氧西林金黄色葡萄球菌至培养基中,将过夜培养的细菌分别转接至TSB培养基(含0.5％的葡萄糖),将其浓度调整至1×108 CFU/mL.RIP组加入课题组设计合成的RIP衍生物(RIP1183),对照组加入无菌磷酸盐缓冲液.检测其对细菌生长曲线和生物膜形成的影响,应用实时荧光定量聚合酶链式反应的方法 ,在细菌迟缓期(2 h)、对数期(6 h)和稳定期(10 h)检测RIP1183对RNAⅢ、人类白细胞抗原(HLA)、icaA和fnbA基因的转录水平的影响,并与对照组进行比较.结果 与对照组比较,RIP1183不影响细菌生长,差异无统计学意义(P>0.05).与对照组比较,RIP组细菌生物膜形成显著减少(P<0.01).迟缓期(2 h),与对照组比较,RIP组RNAⅢ表达明显降低(P<0.05).对数期(6 h),与对照组比较,RIP组RNAⅢ、HLA表达显著降低(P<0.01);稳定期(10 h),与对照组比较,RIP组icaA、fnbA和RNAⅢ表达显著降低(P<0.05或P<0.01).结论 RIP1183通过抑制附属基因调节系统的效应分子RNAⅢ的表达,显著降低不同时相不同毒力因子的表达,从而破坏细菌对环境适应机制,可能是其抗菌活性和抗生物膜形成的机制之一.

Multidrug resistance poses a severe threat to public health and urgently requires new solutions. The natural product chelerythrine (CHE) is a benzophenanthridine alkaloid with antimicrobial potential. In this study, CHE was effective against seven gram-positive bacterial strains, and the minimum inhibitory concentrations (MICs) ranged from 2 to 4 μg/mL. By contrast, CHE showed inferior antibacterial activities against 11 gram-negative strains, and the MICs varied from 16 to 256 μg/mL. We also determined the synergistic/additive effects of combining CHE with nine currently used antibiotics. CHE restored the antibacterial efficacy of the antibiotics against methicillin-resistant Staphylococcus aureus and extended-spectrum β-lactamases producing Escherichia coli. This study suggests that the combination of CHE with conventional antibiotics may be a promising strategy to combat infections caused by multidrug-resistant organisms.

The emergence and rapid spread of multidrug resistance in bacteria have led to the urgent need for novel antibacterial agents. Membrane permeabilization is the mechanism for many antibacterial molecules that are being developed against gram-negative bacteria. Thus, to determine the efficacy of a potential antibacterial molecule, it is important to assess the change in bacterial membrane permeability after treatment. This study describes the protocol for the assays of outer and inner membrane permeability using the fluorescent probes N-phenyl-1-naphthylamine and propidium iodide. Compared with other experiments, such as electron microscopy and the assay of minimal bactericidal concentration, this methodology provides a simpler, faster, and cost-effective way of estimating the membrane-permeabilizing effect and bactericidal efficacy of antibacterial molecules. This study presents an optimized protocol with respect to the classical protocols by incubating bacteria with antibacterial molecules in the culture condition identical to that of antibacterial assays and then detecting the signal of the fluorescent probe in the buffer without broth and antibacterial molecules. This protocol avoids the effect of nutrient deficiency on the physiological status of bacteria and the interference of antibacterial molecules towards the fluorescent probe. Thus, this method can effectively and precisely evaluate the membrane permeability and match the results obtained from other antibacterial assays, such as minimum inhibitory concentration and time-kill curve assays.

Staphylococcus aureus infection is difficult to eradicate because of biofilm formation and antibiotic resistance. The increasing prevalence of methicillin-resistant Staphylococcus aureus (MRSA) infection necessitates the development of a new agent against bacterial biofilms. We report a new coumarin compound, termed DCH, that effectively combats MRSA in vitro and in vivo and exhibits potent antibiofilm activity without detectable resistance. Cellular proteome analysis suggests that the molecular mechanism of action of DCH involves the arginine catabolic pathway. Using molecular docking and binding affinity assays of DCH, and comparison of the properties of wild-type and ArgR-deficient MRSA strains, we demonstrate that the arginine repressor ArgR, an essential regulator of the arginine catabolic pathway, is the target of DCH. These findings indicate that DCH is a promising lead compound and validate bacterial ArgR as a potential target in the development of new drugs against MRSA biofilms.

Acinetobacter baumannii (A. baumannii) is an important opportunistic pathogen causing serious nosocomial infections, which is considered as the most threatening Gram-negative bacteria (GNB). Outer membrane protein A (OmpA), a major component of outer membrane proteins (OMPs) in GNB, is a key virulence factor which mediates bacterial biofilm formation, eukaryotic cell infection, antibiotic resistance and immunomodulation. The characteristics of OmpA in Escherichia coli (E. coli) have been extensively studied since 1974, but only in recent years researchers started to clarify the functions of OmpA in A. baumannii. In this review, we summarized the structure and functions of OmpA in A. baumannii (AbOmpA), collected novel therapeutic strategies against it for treating A. baumannii infection, and emphasized the feasibility of using AbOmpA as a potential therapeutic target.

目的:观察RNAⅢ抑制肽衍生物RIP1183对SD大鼠、Beagle犬的急性毒性反应和对豚鼠过敏反应.方法:SD大鼠和Beagle犬随机分为RIP1183给药组和对照组.SD大鼠尾静脉注射RIP1183,单次剂量为50 mg·kg-1,间隔4～5 h,连续2次,总剂量为100 mg·kg-1,对照组给予等体积的0.9％氯化钠注射液,给药后连续14 d观察RIP1183对大鼠的毒性作用.Beagle犬静脉注射RIP1183,最大给药剂量为50 mg·kg-1,对照组给予等体积的0.9％氯化钠注射液,给药后连续14 d观察RIP1183对Beagle犬的体质量、体温、摄食量、血清生化指标、血液学指标、眼科检查和心电图的影响.采用豚鼠全身主动过敏实验,Hartley豚鼠随机分为阴性对照组(0.9％氯化钠注射液)、阳性对照组(牛血清白蛋白V)、RIP1183低(9.3 mg·kg-1)、高(18.6 mg·kg-1)剂量组,每组6只.动物致敏腹腔注射给药3次,激发静脉注射给药2次,激发给药后观察动物是否出现过敏反应.结果:给予RIP1183后,SD大鼠外观、行为活动、饮食等均未见明显异常,体质量增长正常,结束时全部存活,大体解剖未见肉眼可见变化.Beagle犬给药后,其体质量、体温和摄食量与对照组比较,差异无统计学意义(P>0.05),其他各项指标均无异常.阳性对照组豚鼠在激发后出现过敏症状,RIP1183各剂量组豚鼠激发后未见明显异常和过敏反应症状.结论:SD大鼠和Beagle犬分别静脉给予100 mg·kg-1和50 mg·kg-1 RIP1183后均无异常反应,亦无死亡;豚鼠的RIP1183过敏反应为阴性.