阪崎肠杆菌(Cronobacter sakazakii)是重要的乳品致病菌,在世界范围污染频发,由于其具有较强的环境耐受性,挖掘针对C.sakazakii的新型抗菌剂具有重要意义。昆虫死亡素(thanatin,Tn)是一种极具潜力的靶向细胞外膜的抗菌肽,含有21个氨基酸并具有β-发夹结构。Tn对大肠杆菌具有较好杀菌作用,而对C.sakazakii作用尚无报道。基于此,该文对Tn进行异源表达,并首次研究了Tn对C.sakazakii的抗菌作用。利用毕赤酵母(Pichia pastoris),分别借助PAOX和PGAP对密码子优化后的Tn基因进行异源表达,分析Tn对C.sakazakii BAA894的抑菌效果,并通过比较BAA894及其脂多糖缺失株BAA894ΔwaaC的敏感性差异,探究其可能作用机制。结果显示,组成型P.pastoris GS115/pGAPZα A-than-1和诱导型GS115/pPICZα A-than-1均能有效表达活性Tn,合成量分别为24.34 mg/L和28.03 mg/L。Tn经His-tag亲和层析后,80 mg/L Tn对C.sakazakii BAA894生长抑制率达到98.49%,显示Tn对C.sakazakii具有较强杀菌作用,而对BAA894ΔwaaC生长抑制率为91.01%,较BAA894降低了7.48%,显示Tn的作用靶标与脂多糖的核心多糖和O-抗原有关。该研究为挖掘并改进C.sakazakii的新型抗菌剂奠定了重要基础。
Cronobacter sakazakii, a globally prevalent dairy-borne pathogen with frequent pollution accidents worldwide, exhibited strong resistance to environmental stress, thus it was of great significance to develop novel antimicrobial agents against C.sakazakii. Thanatin (Tn), a promising antimicrobial peptide targeting the cell outer membrane, consists of 21 amino acids with a β-hairpin structure.Tn has been shown to exhibit bactericidal activity against Escherichia coli, yet there was a lack of reports regarding its antimicrobial effects against C.sakazakii.On the basis of this point, Tn was overexpressed, and its antimicrobial effect against C.sakazakii was revealed for the first time.The recombinant strains of Pichia pastoris were constructed to produce codon-optimized Tn variants via heterologous expression systems driven by PAOX and PGAP promoters respectively.The bactericidal activity against C.sakazakii BAA894 of purified Tn was assessed.To elucidate the underlying mechanisms, the sensitivities to Tn of the wild-type of C.sakazakii BAA894 and its lipopolysaccharide-deficient strain BAA894ΔwaaC were compared.Results demonstrated that both the constitutive GS115/pGAPZα A-than-1 and the inducible GS115/pPICZα A-than-1 produced the active Tn, with levels at 24.34 mg/L and 28.03 mg/L, respectively.After purified by His-tag affinity chromatography, Tn at 80 mg/L showed a bacteriostatic efficacy of 98.49% against C.sakazakii BAA894, demonstrating potent antibacterial activity.In contrast, the inhibitory effect on the BAA894ΔwaaC was decreased to 91.01%, displaying a 7.48% reduction compared to BAA894.This differential efficacy strongly suggested that the target of Tn was related to the core polysaccharide and O-antigen components of lipopolysaccharide.The present study lay a foundation for development and optimization of innovative antimicrobial agents against C.sakazakii.
[1] MULLANE N R, MURRAY J, DRUDY D, et al. Detection of Enterobacter sakazakii in dried infant milk formula by cationic-magnetic-bead capture[J]. Applied and Environmental Microbiology, 2006, 72(9):6325-6330.
[2] NAZAROWEC-WHITE M, FARBER J M. Enterobacter sakazakii: A review[J]. International Journal of Food Microbiology, 1997, 34(2):103-113.
[3] CHEN S S, CHEN S, WANG J L, et al. Characterization of a gene cluster containing four genes relevant to biosynthesis of inner core of lipopolysaccharide in Cronobacter sakazakii[J]. Biotechnology and Applied Biochemistry, 2022, 69(3):1080-1093.
[4] FOLCH J, LEES M, SLOANE STANLEY G H. A simple method for the isolation and purification of total lipides from animal tissues[J]. Journal of Biological Chemistry, 1957, 226(1):497-509.
[5] MAHLAPUU M, HÅKANSSON J, RINGSTAD L, et al. Antimicrobial peptides: An emerging category of therapeutic agents[J]. Frontiers in Cellular and Infection Microbiology, 2016, 6:194.
[6] HANEY E F, STRAUS S K, HANCOCK R E W. Reassessing the host defense peptide landscape[J]. Frontiers in Chemistry, 2019, 7:43.
[7] FEHLBAUM P, BULET P, CHERNYSH S, et al. Structure-activity analysis of thanatin, a 21-residue inducible insect defense peptide with sequence homology to frog skin antimicrobial peptides[J]. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(3):1221-1225.
[8] WU Z Y, LI Y R, ZHANG L, et al. Microbial production of small peptide: Pathway engineering and synthetic biology[J]. Microbial Biotechnology, 2021, 14(6):2257-2278.
[9] TIAN L, ZHANG D, SU P, et al. Design, recombinant expression, and antibacterial activity of a novel hybrid Magainin-thanatin antimicrobial peptide[J]. Preparative Biochemistry & Biotechnology, 2019, 49(5):427-434.
[10] UNVER Y, DAGCI I. Komagataella phaffii (Pichia pastoris) as a powerful yeast expression system for biologics production[J]. Frontiers in Bioscience, 2024, 16(2):19.
[11] PIPIYA S O, KUDZHAEV A M, MIRZOEVA N Z, et al. Bioengineering the antimicrobial activity of yeast by recombinant thanatin production[J]. Antibiotics, 2023, 12(12):1719.
[12] WU G Q, LI X F, FAN X B, et al. The activity of antimicrobial peptide S-thanatin is independent on multidrug-resistant spectrum of bacteria[J]. Peptides, 2011, 32(6):1139-1145.
[13] LIU S, WANG W J, DENG L L, et al. Control of sour rot in citrus fruit by three insect antimicrobial peptides[J]. Postharvest Biology and Technology, 2019, 149:200-208.
[14] CHEN L, COLEMAN W G Jr. Cloning and characterization of the Escherichia coli K-12 rfa-2 (rfaC) gene, a gene required for lipopolysaccharide inner core synthesis[J]. Journal of Bacteriology, 1993, 175(9):2534-2540.
[15] RAETZ C R H, WHITFIELD C. Lipopolysaccharide endotoxins[J]. Annual Review of Biochemistry, 2002, 71:635-700.
[16] WANG L, HU X, TAO G, et al. Outer membrane defect and stronger biofilm formation caused by inactivation of a gene encoding for heptosyltransferase Ⅰ in Cronobacter sakazakii ATCC BAA-894[J]. Journal of Applied Microbiology, 2012, 112(5):985-997.
[17] KADRMAS J L, RAETZ C R. Enzymatic synthesis of lipopolysaccharide in Escherichia coli.: Purification and properties of heptosyltransferase Ⅰ[J]. The Journal of Biological Chemistry, 1998, 273(5):2799-2807.
[18] SINHA S, BHATTACHARJYA S. NMR structure and localization of the host defense peptide ThanatinM21F in zwitterionic dodecylphosphocholine micelle: Implications in antimicrobial and hemolytic activity[J]. The Journal of Membrane Biology, 2022, 255(2-3):151-160.
[19] SINHA S, ZHENG L Z, MU Y G, et al. Structure and interactions of a host defense antimicrobial peptide thanatin in lipopolysaccharide micelles reveal mechanism of bacterial cell agglutination[J]. Scientific Reports, 2017, 7:17795.
[20] LEE S Y, JIN H H. Inhibitory activity of natural antimicrobial compounds alone or in combination with nisin against Enterobacter sakazakii[J]. Letters in Applied Microbiology, 2008, 47(4):315-321.
