Davies Courtney G, Reilly Kerri, Altermann Eric, Hendrickson Heather L
Massey Phage Whānau, School of Natural and Computational Sciences, Massey University, Auckland, New Zealand.
AgResearch Ltd., Palmerston North, New Zealand.
Front Microbiol. 2021 Apr 26;12:562748. doi: 10.3389/fmicb.2021.562748. eCollection 2021.
The Mycobacteria are a genus of Actinobacteria that include human pathogens such as (TB). Active TB disease can spread by airborne transmission to healthcare workers and to their community. The HHMI SEA-PHAGES program has contributed to discovering bacteriophages that are able to infect MC 155, a close relative of . This collection of diverse Mycobacteriophages is an excellent resource for trialling bacteriophage-sourced enzymes in novel applications. Herein we measured the ability Mycobacteriophage endolysins to lyse their host strain when functionally fused to biodegradable polyhydroxyalkanoate (PHA) nanobeads. PHA nanobeads facilitate both the expression and the application of enzymes to surfaces and have been demonstrated to stabilize a wide array of proteins for practical applications whilst eliminating the challenges of traditional protein purification. We selected two Lysin A and six Lysin B homologs to be functionally fused to the polyhydroxyalkanoate synthase C (PhaC). Expression of these constructs resulted in functional lysins displayed on the surface of PHA nanobeads. The lysins thus directionally displayed on nanobeads lysed up to 79% of the MC 155 population using 80 mg/mL of nanobeads in pure culture. In order to determine whether the nanobeads would be effective as a protective layer in PPE we adapted a fabric-based test and observed a maximum of 1 log loss of the cell population after 5 h of exposure on a textile (91% cell lysis). Lysin B enzymes performed better than the Lysin A enzymes as a protective barrier on textiles surface assays. These results suggest that bacterial endolysins are efficient in their action when displayed on PHA nanobeads and can cause significant population mortality in as little as 45 min. Our results provide the proof-of-principle that Mycobacteriophage endolysins can be used on functionalized nanobeads where they can protect surfaces such as personal protective equipment (PPE) that routinely come into contact with aerosolised bacteria.
分枝杆菌是放线菌属的一个属，其中包括人类病原体，如结核分枝杆菌（TB）。活动性结核病可通过空气传播给医护人员及其社区。霍华德·休斯医学研究所（HHMI）的SEA-PHAGES项目有助于发现能够感染结核分枝杆菌近亲MC 155的噬菌体。这种多样的分枝杆菌噬菌体集合是在新应用中试验噬菌体来源酶的极佳资源。在此，我们测量了分枝杆菌噬菌体溶菌酶在与可生物降解的聚羟基脂肪酸酯（PHA）纳米珠功能融合时裂解其宿主菌株的能力。PHA纳米珠有助于酶在表面的表达和应用，并且已被证明可稳定多种蛋白质以用于实际应用，同时消除了传统蛋白质纯化的挑战。我们选择了两个溶菌酶A和六个溶菌酶B同源物与聚羟基脂肪酸酯合酶C（PhaC）进行功能融合。这些构建体的表达导致功能性溶菌酶展示在PHA纳米珠表面。在纯培养中使用80 mg/mL的纳米珠时，如此定向展示在纳米珠上的溶菌酶可裂解高达79%的MC 155菌群体。为了确定纳米珠是否能作为个人防护装备（PPE）中的保护层有效，我们采用了一种基于织物的测试，并观察到在纺织品上暴露5小时后细胞群体最多减少1个对数（91%细胞裂解）。在纺织品表面试验中，溶菌酶B酶作为防护屏障的效果优于溶菌酶A酶。这些结果表明，细菌溶菌酶展示在PHA纳米珠上时作用高效，并且在短短45分钟内就能导致大量菌群体死亡。我们的结果提供了原理证明，即分枝杆菌噬菌体溶菌酶可用于功能化纳米珠上，在那里它们可以保护诸如经常接触雾化细菌的个人防护装备（PPE）等表面。
