Mechanisms and physiological effects of protamine resistance in Salmonella enterica serovar Typhimurium LT2
Mechanisms and physiological effects of protamine resistance in Salmonella enterica serovar Typhimurium LT2
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DOI：
10.1093/jac/dkq059
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发表时间：
2010-05-01
影响因子：
5.2
通讯作者：
Andersson, Dan I.
中科院分区：
文献类型：
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作者：
Pranting, Maria;Andersson, Dan I.
鱼精蛋白是具有抗菌活性的阳离子肽。我们已经研究了细菌对硫酸鱼精蛋白的耐药性的演变以及由此产生的对健身和生理的影响，目的是增加对抗菌肽的细菌耐药性机制的了解。自发的，抗鱼精蛋白的肠道沙门氏菌血清型鼠伤寒沙门氏菌（即鼠伤寒沙门氏菌）LT 2突变体在含有硫酸鱼精蛋白的琼脂糖平板上分离。使用转座子插入和DNA测序鉴定抗性突变。通过肉汤稀释试验测定肽敏感性，并使用Etests测定抗生素敏感性。适合度测定为对数生长期生长速率。通过连续传代通过群体瓶颈，然后目视筛选大菌落，分离出生长补偿菌株，抗鱼精蛋白突变体的出现率为2.3 × 10 ~（-7）/细胞/代。这些突变体对鱼精蛋白的抗性是亲本菌株的2-20倍，并且对几种其他抗微生物剂（包括粘菌素、庆大霉素、乳铁蛋白和人防御素HNP-1）的敏感性较低。抗性突变被定位到涉及血红素生物合成和呼吸的基因上，并且与细菌适应性的降低有关。一些突变体，在鱼精蛋白的情况下，可以通过获得第二个位点的补偿突变进化到更高的fitness。自发突变体抗鱼精蛋白硫酸盐很容易在鼠伤寒沙门氏菌LT 2中选择。这些突变体对其他几种肽和抗生素的敏感性较低，并且具有小菌落变体的特征，这是一种通常与难以治疗的持续性和复发性感染相关的表型，并且可能是细菌逃避抗菌肽杀伤作用的策略。
Protamines are cationic peptides that exert antimicrobial activity. We have examined the evolution of bacterial resistance to protamine sulphate and the resulting effects on fitness and physiology, with the objective of increasing knowledge about mechanisms of bacterial resistance to antimicrobial peptides.Spontaneous, protamine-resistant Salmonella enterica serovar Typhimurium (i.e. Salmonella Typhimurium) LT2 mutants were isolated on agarose plates containing protamine sulphate. Resistance mutations were identified using transposon insertions and DNA sequencing. Peptide susceptibility was determined by broth dilution tests and antibiotic susceptibility using Etests. Fitness was determined as log-phase growth rates. Growth-compensated strains were isolated by serial passage through population bottlenecks followed by visual screening for large colonies.Protamine-resistant mutants appeared at a rate of 2.3 x 10(-7)/cell/generation. These mutants were 2-20 times more resistant to protamine than the parental strain and less susceptible to several other antimicrobials, including colistin, gentamicin, lactoferricin and human defensin HNP-1. The resistance mutations were mapped to genes involved in haem biosynthesis and respiration, and were associated with a reduction in bacterial fitness. Some mutants could, in the absence of protamine, be evolved to higher fitness by acquiring second-site compensatory mutations.Spontaneous mutants resistant to protamine sulphate were readily selected in Salmonella Typhimurium LT2. These mutants were less susceptible to several other peptides and antibiotics, and had the characteristics of small colony variants, a phenotype often associated with persistent and recurrent infections that are difficult to treat and which could be a strategy for bacteria to escape the killing effects of antimicrobial peptides.