Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.
Cationic Peptides Facilitate Iron-induced Mutagenesis in Bacteria.
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DOI：
10.1371/journal.pgen.1005546
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发表时间：
2015-10
期刊：
影响因子：
4.5
通讯作者：
Rolff J
中科院分区：
文献类型：
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作者：
Rodríguez-Rojas A;Makarova O;Müller U;Rolff J
铜绿假单胞菌是慢性呼吸道感染的病原体，也是囊性纤维化患者的重要病原体。适应性突变对抗菌素耐药性和持久性起着至关重要的作用。在这种环境中导致细菌诱变的因素尚不清楚。最近有人提出，阳离子抗菌肽如LL-37可作为铜绿假单胞菌的诱变剂。在这里，我们提供的实验证据表明，诱变是产品的联合行动的LL-37和游离铁。通过估计用LL-37、铁或两者的组合处理的铜绿假单胞菌中的突变率、突变频率和评估突变谱，我们证明了仅当游离铁和LL-37同时存在时突变率和突变频率增加。粘菌素也有同样的效果。铁螯合剂的加入完全消除了这种致突变作用，表明LL-37使铁进入细胞，通过芬顿反应导致DNA损伤。这也得到了以下观察结果的支持，即在LL-37-铁方案下细菌的突变谱显示出特征性芬顿反应指纹之一：C至T转换。自然界和宿主体内游离铁浓度保持在很低的水平，但囊性纤维化患者感染肺中的情况则不同。间歇性出血和肺部上皮细胞的损伤可能导致游离铁的释放，从而导致活性氧的产生和呼吸道的恶化，使其更容易受到感染。阳离子抗菌肽（cAMPs）是由免疫系统天然产生的小蛋白，主要通过膜中的孔形成来限制细菌生长。最近有人提出，与抗生素类似，cAMP的亚抑制浓度促进细菌诱变。然而，我们以前报道过cAMPs不会增加突变率，也不会激活细菌应激反应。我们在这里解决这个矛盾。我们报告说，游离铁在培养基中增加突变的cAMP的存在下。我们发现，亚抑制浓度的cAMP促进游离铁进入细菌细胞，在那里它与过氧化氢相互作用，从而导致DNA损伤活性氧的产生和增加诱变。此外，这些结果可能具有临床相关意义：虽然健康个体中通常存在很少的游离铁，但囊性纤维化患者的情况并非如此，其中细菌诱变的增加促进了抗生素耐药性，并导致感染的持续性和严重性。因此，旨在减少肺中游离铁的干预可以减少铁介导的诱变的cAMP促进作用;因此抗生素抗性和病理适应。
Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. Cationic antimicrobial peptides (cAMPs) are small proteins naturally produced by the immune system to limit bacterial growth mainly through pore formation in the membrane. It has recently been suggested that sub-inhibitory concentrations of cAMPs promote bacterial mutagenesis, similarly to antibiotics. However, we previously reported that cAMPs do not increase mutation rate and do not activate bacterial stress responses. Here we resolve this contradiction. We report that free iron in the culture medium increases mutagenesis in the presence of cAMPs. We show that sub-inhibitory concentrations of cAMPs facilitate entry of free iron into bacterial cells, where it interacts with hydrogen peroxide, thereby resulting in production of DNA-damaging reactive oxygen species and increased mutagenesis. Moreover, these results may have clinically-relevant implications: while very little free iron is normally present in healthy individuals, this is not the case in patients suffering from cystic fibrosis, where elevated bacterial mutagenesis promotes antibiotic resistance and contributes to persistence and severity of infection. Thus, an intervention aimed at reduction of free iron in the lungs could reduce the cAMPs-facilitation of iron-mediated mutagenesis; hence antibiotic resistance and pathoadaptation.