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Genomic and Biochemical Characterization of Acinetobacter Podophage Petty Reveals a Novel Lysis Mechanism and Tail-Associated Depolymerase Activity
Journal of Virology ( IF 4.0 ) Pub Date : 2018-03-15 , DOI: 10.1128/jvi.01064-17
A C Hernandez-Morales 1, 2 , L L Lessor 2 , T L Wood 2 , D Migl 1, 2 , E M Mijalis 2 , J Cahill 2 , W K Russell 3 , R F Young 1, 2 , J J Gill 4, 5
Affiliation  

The increased prevalence of drug-resistant, nosocomial Acinetobacter infections, particularly from pathogenic members of the Acinetobacter calcoaceticus-baumannii complex, necessitates the exploration of novel treatments such as phage therapy. In the present study, we characterized phage Petty, a novel podophage that infects multidrug-resistant Acinetobacter nosocomialis and Acinetobacter baumannii. Genome analysis reveals that phage Petty is a 40,431-bp ϕKMV-like phage, with a coding density of 92.2% and a G+C content of 42.3%. Interestingly, the lysis cassette encodes a class I holin and a single-subunit endolysin, but it lacks canonical spanins to disrupt the outer membrane. Analysis of other ϕKMV-like genomes revealed that spaninless lysis cassettes are a feature of phages infecting Acinetobacter within this subfamily of bacteriophages. The observed halo surrounding Petty's large clear plaques indicated the presence of a phage-encoded depolymerase capable of degrading capsular exopolysaccharides (EPS). The product of gene 39, a putative tail fiber, was hypothesized to possess depolymerase activity based on weak homology to previously reported phage tail fibers. The 101.4-kDa protein gene product 39 (gp39) was cloned and expressed, and its activity against Acinetobacter EPS in solution was determined. The enzyme degraded purified EPS from its host strain A. nosocomialis AU0783, reducing its viscosity, and generated reducing ends in solution, indicative of hydrolase activity. Given that the accessibility to cells within a biofilm is enhanced by degradation of EPS, phages with depolymerases may have enhanced diagnostic and therapeutic potential against drug-resistant Acinetobacter strains.

IMPORTANCE Bacteriophage therapy is being revisited as a treatment for difficult-to-treat infections. This is especially true for Acinetobacter infections, which are notorious for being resistant to antimicrobials. Thus, sufficient data need to be generated with regard to phages with therapeutic potential, if they are to be successfully employed clinically. In this report, we describe the isolation and characterization of phage Petty, a novel lytic podophage, and its depolymerase. To our knowledge, it is the first phage reported to be able to infect both A. baumannii and A. nosocomialis. The lytic phage has potential as an alternative therapeutic agent, and the depolymerase could be used for modulating EPS both during infections and in biofilms on medical equipment, as well as for capsular typing. We also highlight the lack of predicted canonical spanins in the phage genome and confirm that, unlike the rounding of lambda lysogens lacking functional spanin genes, A. nosocomialis cells infected with phage Petty lyse by bursting. This suggests that phages like Petty employ a different mechanism to disrupt the outer membrane of Acinetobacter hosts during lysis.



中文翻译:


噬足不动杆菌的基因组和生化特征揭示了一种新的裂解机制和尾部相关的解聚酶活性



耐药性医院内不动杆菌感染(尤其是乙酸钙-鲍曼不动杆菌复合体的致病成员)的患病率不断增加,因此需要探索噬菌体疗法等新疗法。在本研究中,我们对噬菌体 Petty 进行了表征,这是一种新型的足噬菌体,可感染多重耐药的院内不动杆菌鲍曼不动杆菌。基因组分析表明,噬菌体 Petty 是一种 40,431 bp 的 phiKMV 样噬菌体,编码密度为 92.2%,G+C 含量为 42.3%。有趣的是,裂解盒编码 I 类穴蛋白和单亚基内溶素,但它缺乏破坏外膜的经典跨蛋白。对其他 phiKMV 样基因组的分析表明,无跨度裂解盒是感染该噬菌体亚科不动杆菌噬菌体的一个特征。观察到佩蒂大的透明斑块周围的光环表明存在噬菌体编码的解聚酶,该酶能够降解荚膜外多糖(EPS)。基因 39 的产物(一种假定的尾纤维)被假设具有解聚酶活性,这是基于与先前报道的噬菌体尾纤维的弱同源性。克隆并表达了101.4-kDa蛋白基因产物39(gp39),并测定了其在溶液中对抗不动杆菌EPS的活性。该酶降解来自其宿主菌株A. nosocomialis AU0783 的纯化 EPS,降低其粘度,并在溶液中产生还原端,表明水解酶活性。 鉴于 EPS 的降解增强了生物膜内细胞的可及性,具有解聚酶的噬菌体可能具有增强的针对耐药不动杆菌菌株的诊断和治疗潜力。


重要性噬菌体疗法作为难治性感染的治疗方法正在被重新审视。对于不动杆菌感染尤其如此,不动杆菌感染因对抗菌药物产生耐药性而臭名昭著。因此,如果要在临床上成功应用噬菌体,就需要生成有关具有治疗潜力的噬菌体的足够数据。在本报告中,我们描述了噬菌体 Petty(一种新型裂解性足噬菌体)及其解聚酶的分离和表征。据我们所知,这是第一个被报道能够感染鲍曼不动杆菌院内不动杆菌的噬菌体。裂解噬菌体具有作为替代治疗剂的潜力,解聚酶可用于在感染期间和医疗设备上的生物膜中调节 EPS,以及用于荚膜分型。我们还强调了噬菌体基因组中预测的经典跨蛋白的缺乏,并证实,与缺乏功能性跨蛋白基因的 lambda 溶原菌的四舍五入不同,感染噬菌体 Petty 的A. nosocomialis细胞会通过破裂而裂解。这表明像佩蒂这样的噬菌体在裂解过程中采用不同的机制来破坏不动杆菌宿主的外膜。

更新日期:2018-02-27
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