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Abstract：
Phages and bacteria have acquired resistance mechanisms for protection. In this context, the aims of the present study were to analyze the proteins isolated from 21 novel lytic phages of Klebsiella pneumoniae in search of defense mechanisms against bacteria and also to determine the infective capacity of the phages. A proteomic study was also conducted to investigate the defense mechanisms of two clinical isolates of K. pneumoniae infected by phages. For this purpose, the 21 lytic phages were sequenced and de novo assembled. The host range was determined in a collection of 47 clinical isolates of K. pneumoniae, revealing the variable infective capacity of the phages. Genome sequencing showed that all of the phages were lytic phages belonging to the order Caudovirales. Phage sequence analysis revealed that the proteins were organized in functional modules within the genome. Although most of the proteins have unknown functions, multiple proteins were associated with defense mechanisms against bacteria, including the restriction-modification system, the toxin-antitoxin system, evasion of DNA degradation, blocking of host restriction and modification, the orphan CRISPR-Cas system, and the anti-CRISPR system. Proteomic study of the phage-host interactions (i.e., between isolates K3574 and K3320, which have intact CRISPR-Cas systems, and phages vB_KpnS-VAC35 and vB_KpnM-VAC36, respectively) revealed the presence of several defense mechanisms against phage infection (prophage, defense/virulence/resistance, oxidative stress and plasmid proteins) in the bacteria, and of the Acr candidate (anti-CRISPR protein) in the phages. IMPORTANCE Researchers, including microbiologists and infectious disease specialists, require more knowledge about the interactions between phages and their bacterial hosts and about their defense mechanisms. In this study, we analyzed the molecular mechanisms of viral and bacterial defense in phages infecting clinical isolates of K. pneumoniae. Viral defense mechanisms included restriction-modification system evasion, the toxin-antitoxin (TA) system, DNA degradation evasion, blocking of host restriction and modification, and resistance to the abortive infection system, anti-CRISPR and CRISPR-Cas systems. Regarding bacterial defense mechanisms, proteomic analysis revealed expression of proteins involved in the prophage (FtsH protease modulator), plasmid (cupin phosphomannose isomerase protein), defense/virulence/resistance (porins, efflux pumps, lipopolysaccharide, pilus elements, quorum network proteins, TA systems, and methyltransferases), oxidative stress mechanisms, and Acr candidates (anti-CRISPR protein). The findings reveal some important molecular mechanisms involved in the phage-host bacterial interactions; however, further study in this field is required to improve the efficacy of phage therapy.
摘要：
噬菌体和细菌已经获得了保护的抗性机制。在这种情况下，本研究的目的是分析从21种新型肺炎克雷伯菌裂解噬菌体中分离出的蛋白质，以寻找针对细菌的防御机制，并确定噬菌体的感染能力。还进行了蛋白质组学研究以研究被噬菌体感染的两种肺炎克雷伯菌临床分离株的防御机制。为此,对21个裂解噬菌体进行测序和从头组装。宿主范围是在47个肺炎克雷伯菌临床分离株的集合中确定的，揭示噬菌体的可变感染能力。基因组测序表明，所有噬菌体都是属于Caudovirales目的裂解噬菌体。噬菌体序列分析显示，蛋白质在基因组中被组织在功能模块中。虽然大多数蛋白质具有未知的功能，多种蛋白质与针对细菌的防御机制有关，包括限制-修改系统，毒素-抗毒素系统,逃避DNA降解，阻止主机限制和修改，孤儿CRISPR-Cas系统，和反CRISPR系统。噬菌体-宿主相互作用的蛋白质组学研究(即，在分离株K3574和K3320之间，它们具有完整的CRISPR-Cas系统，和噬菌体vB_KpnS-VAC35和vB_KpnM-VAC36分别）揭示了针对噬菌体感染的几种防御机制（prophage，防御/毒力/抗性，细菌中的氧化应激和质粒蛋白），和噬菌体中的Acr候选物(抗CRISPR蛋白)。重要性研究人员，包括微生物学家和传染病专家，需要更多有关噬菌体与其细菌宿主之间的相互作用及其防御机制的知识。在这项研究中,我们分析了感染肺炎克雷伯菌临床分离株的噬菌体中病毒和细菌防御的分子机制.病毒防御机制包括限制性修饰系统规避，毒素-抗毒素（TA）系统，DNA降解逃避，阻止主机限制和修改，和对流产感染系统的抵抗力，抗CRISPR和CRISPR-Cas系统。关于细菌防御机制，蛋白质组学分析揭示了与原蛋白（FtsH蛋白酶调节剂）有关的蛋白质表达，质粒（cupin磷酸甘露糖异构酶蛋白），防御/毒力/抗性(孔，外排泵,脂多糖，菌毛元素,群体网络蛋白，TA系统,和甲基转移酶)，氧化应激机制，和Acr候选物(抗CRISPR蛋白)。研究结果揭示了噬菌体-宿主细菌相互作用的一些重要分子机制；然而，需要在这一领域进行进一步的研究，以提高噬菌体疗法的疗效。
