查找使用阿拉丁产品的科研文献

Impact of DnaK on protein expression and deamidation modification in Cronobacter sakazakii: insights from proteomic analysis

Ping Lu,Juan Xue,Xi Chen,Xuemeng Ji

An ultrasensitive sandwich immunoassay with a glucometer readout for portable and quantitative detection of Cronobacter sakazakii

Lingxian Ye,Guangying Zhao,Wenchao Dou

Developing portable and low-cost methods for quantitative detection of Cronobacter sakazakii (C. sakazakii) from powdered infant formula plays a significant role in controlling and preventing a range of serious neonatal infections. Here, we reported a novel methodology for C. sakazakii detection using a low-cost, simple, and widely accessible personal glucose meter (PGM) as the readout. The electrochemical immunoassay strategy was based on antibody and glucose oxidase coated silica nanoparticles (SiNP–GOX–IgG) and antibody functionalized silica-coated magnetic nanoparticles (MNP–IgG). In the process of detecting a positive sample, a sandwich immunocomplex was formed between the MNP–IgG, C. sakazakii and SiNP–GOX–IgG. Then, the above sandwich composites were dispersed in glucose solution, and the concentration of glucose was measured using the PGM. Under optimal conditions, a linear relationship between the decrease of glucose concentration and the logarithm of C. sakazakii concentration was obtained in the concentration range from 9.0 × 102 to 9.0 × 107 CFU mL−1 with a detection limit of 4.2 × 101 CFU mL−1 (S/N = 3). This simple immunoassay holds great potential as an efficient, sensitive and selective point-of-care testing (POCT) tool for C. sakazakii detection.

Hydrophilic Guardians: Putative Core Proteins Unaltered by 22 Gene Deletions Mediate Desiccation Resistance in Cronobacter sakazakii

Ping Lu,Juan Xue,Aiying Shi,Xuemeng Ji

Cronobacter sakazakii is a notorious foodborne pathogen linked to severe infections in neonates, with powdered infant formula serving as a common transmission route due to its ability to survive under desiccated conditions. Understanding the molecular mechanisms underlying its desiccation resistance is crucial for developing effective control strategies. In this study, we employed comparative proteomics and hydropathy profiling to investigate putative core proteins potentially involved in desiccation resilience. Proteomic analysis of wild-type and 22 knockout strains identified 1,354 proteins, of which 156 were defined as putative core proteins due to stable abundance profiles. Among these, two uncharacterized proteins, ESA_03998 and ESA_01764 , exhibited strong hydrophilic properties and were predicted to be surface-associated based on signal peptide analysis. Gene knockout of ESA_03998 or ESA_01764 significantly reduced surface hydrophilicity and survival under prolonged desiccation in both PBS and milk powder matrices. Additionally, Laurdan-based membrane fluidity assays revealed increased membrane rigidification (ΔGP) in mutant strains, indicating that these proteins help preserve membrane hydration and dynamics under low-moisture conditions. These findings establish a novel link between hydrophilic putative core proteins and membrane-associated desiccation resistance in C. sakazakii , offering insights into its persistence in dry food environments and highlighting potential targets for mitigating contamination in powdered infant formula.

Antibacterial activity and its mechanisms of a recombinant Funme peptide against Cronobacter sakazakii in powdered infant formula

Yong Chen,Yifan Zhang,Xiaohou Wang,Jianqun Ling,Guanghua He,Lirong Shen

Cronobacter sakazakii ( Cs ) is a typical foodborne bacterium that infect powdered infant formula (PIF) worldwide. In this study, a recombinant antimicrobial peptide, branded as Funme peptide (FP)was applied to protect PIF from Cs contamination. The result from the antimicrobial activity assay showed that the minimum inhibitory concentration (MIC) of BMAP-27 peptide, FP and Ampicillin against Cs were 250.0, 125.0 and 15.6 μg/mL, respectively, indicating FP possessed higher MIC than that of Ampicillin, and lower MIC than that of BMAP-27. The minimum biofilm eradication concentration (MBEC) assay showed that FP at 2 × MIC (250.0 μg/mL) could completely eradicated Cs biofilms. The antibacterial activity of FP might be due to the increasing permeability and the release of cytoplasmic β-galactosidase of Cs . The results acquired from transmission electron microscopy and scanning electron microscopy indicated that FP induced the disruption and dysfunction of cell walls and membranes. Moreover, safety assay showed that FP had low cytotoxicity to human erythrocytes. The present study investigated the antibacterial effects and mechanisms of FP against Cs , providing promising evidence to apply this novel antimicrobial agent against Cs contamination in foods and food processing facilities.

Proteomic Analysis Revealed Metabolic Inhibition and Elongation Factor Tu Deamidation by p-Coumaric Acid in Cronobacter sakazakii

Lu Ping,Ji Xuemeng,Xue Juan,Dong Yinping,Chen Xi

Screening drugs and compounds to fight against Cronobacter sakazakii (C. sakazakii), one of the most common pathogens that can cause fatal necrotizing enterocolitis, septicema and meningitis, is still needed. We found that p-coumaric acid (pCA) has an inhibitory effect on C. sakazakii in vitro and in vivo. Proteomic changes of C. sakazakii BAA-894 exposed to pCA were studied to reveal the antibacterial mechanisms involved. A total of 1,553 proteins were identified in C. sakazakii BAA-894 by label-free proteomics analysis. Fuzzy cluster analysis showed that 33 were up-regulated, and 110 were down-regulated with pCA treatment. Gene Ontology (GO) analysis concluded that pCA caused the change of metabolic state of bacteria and generally in the state of metabolic inhibition. KEGG Enrichment Analysis (KEGG) analysis showed that pCA inhibited energy metabolism and distorted the balance of amino acid metabolism. Posttranslational modification analysis showed that pCA affected the deamidation of three proteins, including Elongation factor Tu, one of the vital proteins in bacteria. Molecular docking suggested the hydrogen bond between the pCA carboxyl group and Elongation factor Tu Asn-64 might contribute to deamidation. Overall, we found that pCA interfered with cellular energy and amino acid metabolism and promoted elongation factor Tu deamidation, suggesting that pCA can be an effective natural substitute to control C. sakazakii.

Disruption of the rpfB Gene Impairs Growth and Virulence in Cronobacter sakazakii through Downregulation of the Shikimate Pathway

Aiying Shi,Wei Wang,Chunfei Wang,Xiaoyu Weng,Qiang Tian,Shanming Sun

The rpfB gene, encoding long-chain acyl-CoA synthetase, plays an essential role in the fatty acid metabolism of Cronobacter sakazakii, a pathogenic bacterium known to cause neonatal infections. Here, we investigate the functional consequences of rpfB gene knockout and its effect on bacterial growth and virulence. Our findings demonstrate that rpfB deletion leads to significantly reduced bacterial growth and virulence in a murine infection model. Proteomic analysis revealed that the disruption of rpfB markedly downregulates key enzymes in the shikimate pathway, including aroA and aroK, which are essential for the biosynthesis of aromatic amino acids. Supplementation with aromatic amino acids restored the growth defect, highlighting the critical link between fatty acid metabolism and the shikimate pathway. These results uncover a novel regulatory role for rpfB in coordinating metabolic networks that control bacterial growth and virulence. This study provides a new understanding of the metabolic mechanisms underlying C. sakazakii pathogenicity and identifies possible metabolic targets for antimicrobial development.

Cronobacter sakazakii Pyridoxal Kinase PdxY Mediated by TreR and pESA3 Is Essential for Vitamin B6 (PLP) Maintenance and Virulence

Ping Lu,Xiaoli Dong,Xuemeng Ji

Cronobacter sakazakii is an opportunistic pathogen capable of causing severe infections, particularly in neonates. Despite the bacterium’s strong pathogenicity, the pathogenicity of C. sakazakii is not yet well understood. Using a comparative proteomic profiling approach, we successfully identified pdxY, encoding a pyridoxal kinase involved in the recycling of pyridoxal 5′-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in slower growth and reduced virulence. Our study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii. The identification of pdxY as gene essential for successful pathogenesis provides a potential target for the development of new antibiotic treatments.IMPORTANCE The opportunistic pathogen Cronobacter sakazakii is known to cause severe infections, particularly in neonates, and can result in high mortality rates. In this study, we used a comparative proteomic profiling approach to identify genes essential for the successful pathogenesis of C. sakazakii. We successfully identified pdxY, encoding a pyridoxal kinase involved in the salvage pathway of pyridoxal 5′-phosphate (PLP), as a gene essential for the successful pathogenesis of C. sakazakii. Knocking out the pdxY gene resulted in impaired growth and reduced virulence. This study sheds light on the fundamental importance of pyridoxal kinase for the survival and virulence of C. sakazakii, which can be a potential target for the development of new antibiotic treatments. This study highlights the importance of comparative proteomic profiling in identifying virulence factors that can be targeted for the development of new antibiotics.

Preparation of silver nanoparticles/polymethylmethacrylate/cellulose acetate film and its inhibitory effect on Cronobacter sakazakii in infant formula milk

Hui Li,Xiaoyan Feng,Xianxiang Zeng,Qixiu You,Wen Li,Hengyi Xu

ABSTRACT Cronobacter sakazakii is a harmful foodborne pathogen, and its contaminated food will pose a huge threat to human health. Prevention of C. sakazakii contamination of food is valuable for food safety as well as for human health. In this study, silver nanoparticles (AgNP) were successfully immobilized on the surface of cellulose acetate (CA) and polymethylmethacrylate (PMMA) composite to obtain AgNP/PMMA/CA film. Through the inhibition zone and growth curve experiments, we found that AgNP/PMMA/CA films has excellent antibacterial activity on C. sakazakii . The AgNP/PMMA/CA film can prolong the lag phase of the growth curve of C. sakazakii from 2 to 8 h. The antibacterial films were found to reduce the survival of C. sakazakii in Luria-Bertani and infant formula by combining it with a mild heat treatment (45°C, 50°C, and 55°C). The AgNP/PMMA/CA film combined with 55°C water bath can completely inactivate C. sakazakii in infant formula within 120 min. Finally, the potential mechanism by which AgNP/PMMA/CA films reduce the heat tolerance of C. sakazakii was investigated by quantitative real-time PCR. The results showed that AgNP/PMMA/CA films could reduce the expression of environmental tolerance-related genes in C. sakazakii . The current research shows that AgNP/PMMA/CA film has strong antibacterial activity, and the antibacterial film combined with mild heat treatment can accelerate the inactivation of C. sakazakii and effectively reduce the harm of foodborne pathogens. The AgNP/PMMA/CA film can be used as a potential packaging material or antibacterial surface coating.

Directional immobilization of phage on the palladium-based nanozyme for colorimetric detection of Cronobacter sakazakii in powdered infant formula

Yuan-song Zhang,Xuechao Xu,Juanli Yang,Mengyuan Tan,Wenyuan Zhou,Lu Gao,Zhen-quan Yang

Cronobacter sakazakii in powdered infant formula (PIF) can cause infections in newborns that are life-threatening. Establishing a High-performance evaluation method for C. sakazakii in the PIF is urgently needed. In this study, a chromogenic system based on phage-directed immobilized nanozyme for colorimetric measurement of C. sakazakii in PIF was constructed . Initially, our acquired phage EspYZU15 was electrostatically immobilized on the surface of a palladium-based nanozyme to produce EspYZU15 directionally immobilized Pd (EspYZU15@Pd). EspYZU15@Pd possesses outstanding peroxidase-like properties, catalyzing the chromogenic reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) in the presence of H 2 O 2 . The ability of EspYZU15@Pd to recognize and trap host bacteria allows C. sakazakii to specifically inhibit the TMB chromogenic process catalyzed by EspYZU15@Pd. Based on this detection strategy, a visual quantification method for C. sakazakii ranging from 3.2 × 10 2 to 3.2 × 10 7  CFU mL −1 was developed, yielding a low limit of detection (LOD) of 3.6 × 10 1  CFU mL −1 . This method was able to successfully detect C. sakazakii in PIF, with outstanding recoveries ranging from 95.8% to 104.9%. These characteristics indicate that the EspYZU15@Pd-based chromogenic system has considerable promise for the colorimetric determination of C. sakazakii in PIF.

Fatty acid metabolism governed by FadR is essential for Cronobacter sakazakii survival in dry food matrices and neonatal infection

Ping Lu,Juan Xue,Aiying Shi,Xuemeng Ji

Cronobacter sakazakii poses significant neonatal health risks due to its exceptional desiccation tolerance and virulence, yet the molecular mechanisms linking membrane lipid regulation to these traits remain poorly defined. This study characterizes the global regulator FadR (ESA_01467) as a key modulator of fatty acid metabolism, membrane fluidity, and pathogenicity. Deletion of fadR resulted in a 52 % decrease in adhesion and 61 % reduction in invasion of intestinal epithelial cells, alongside attenuated virulence and reduced bacterial loads in a neonatal rat model. Proteomic and lipidomic analyses revealed downregulation of FabA and FabB, upregulation of beta-oxidation enzymes, and a 25 % increase in membrane rigidity associated with decreased levels of palmitoleic and oleic acids. The Δ fadR mutant showed a 3.2-log reduction in viability following 14 days of desiccation in phosphate-buffered saline, while survival in milk powder remained unaffected. Supplementation with milk-derived long-chain fatty acids restored both membrane fluidity and desiccation survival to wild-type levels. These findings establish FadR as a central regulator linking lipid homeostasis to environmental adaptation and virulence in C. sakazakii , providing mechanistic insight into its persistence in dry food environments and infection risk.

Cronobacter sakazakii lysozyme inhibitor LprI mediated by HmsP and c-di-GMP is essential for biofilm formation and virulence

Xuemeng Ji,Dancai Fan,Jin Wang,Bowei Zhang,Yaozhong Hu,Huan Lv,Jing Wu,Yi Sun,Jingmin Liu,Yan Zhang,Shuo Wang

Cronobacter sakazakii poses a significant threat, particularly to neonates and infants. Despite its strong pathogenicity, understanding of C. sakazakii biofilms and their role in infections remains limited. This study investigates the roles of HmsP and c-di-GMP in biofilm formation and identifies key genetic and proteomic elements involved. Gene knockout experiments reveal that HmsP and c-di-GMP are linked to biofilm formation in C. sakazakii. Comparative proteomic profiling identifies the lysozyme inhibitor protein LprI, which is downregulated in hmsP knockouts and upregulated in c-di-GMP knockouts, as a potential biofilm formation factor. Further investigation of the lprI knockout strain shows significantly reduced biofilm formation and decreased virulence in a rat infection model. Additionally, LprI is demonstrated to bind extracellular DNA, suggesting a role in anchoring C. sakazakii within the biofilm matrix. These findings enhance our understanding of the molecular mechanisms underlying biofilm formation and virulence in C. sakazakii, offering potential targets for therapeutic intervention and food production settings.

The lipoprotein NlpD in Cronobacter sakazakii responds to acid stress and regulates macrophage resistance and virulence by maintaining membrane integrity

Xuemeng Ji,Ping Lu,Juan Xue,Ning Zhao,Yan Zhang,Lu Dong,Xuejiao Zhang,Ping Li,Yaozhong Hu,Jin Wang,Bowei Zhang,Jingmin Liu,Huan lv,Shuo Wang

Cronobacter sakazakii, an emerging opportunistic pathogen, is implicated in severe foodborne outbreak infections in premature and full-term infants. Generally, acid tolerance is vital for the pathogenesis of foodborne pathogens; however, its role in C. sakazakii virulence remains largely unknown. To screen out acid-tolerance determinants from transposon mutants, anovel counterselection method using gentamicin and acid was developed. Using the counterselection method and growth assay, we screened several acid-sensitive mutants and found that nlpD encodes an acid-resistance factor in C. sakazakii. Compared to the wild-type strain, the nlpD mutant exhibited attenuated virulence in a rat model. Using macrophage THP-1 cells and a pH probe, we verified that nlpD enables bacteria to resist macrophages by resisting acidification. Finally, we confirmed that nlpD maintains C. sakazakii membrane integrity in acid using propidium iodide permeabilization assays via flow cytometry. Our results confirm that nlpD is a novel virulence factor that permits C. sakazakii to survive under acid stress conditions. Considering that NlpD is a conserved lipoprotein located in the bacterial outer membrane, NlpD could be used as a target for drug development.