Data_Sheet_1_PLAN-M; Mycobacteriophage Endolysins Fused to Biodegradable Nanobeads Mitigate Mycobacterial Growth in Liquid and on Surfaces.docx

The Mycobacteria are a genus of Actinobacteria that include human pathogens such as Mycobacterium tuberculosis (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 M. smegmatis MC2 155, a close relative of M. tuberculosis. 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 M. smegmatis MC2 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.

分枝杆菌属（Mycobacteria）是放线菌门（Actinobacteria）下的一个菌属，包含多种人类致病菌，例如结核分枝杆菌（Mycobacterium tuberculosis，TB）。活动性结核病可通过空气传播，感染医护人员及其所在社区人群。霍华德·休斯医学研究所（HHMI）的SEA-PHAGES项目已成功发掘出能够感染耻垢分枝杆菌MC2 155（Mycobacterium smegmatis MC2 155，结核分枝杆菌的近缘菌株）的噬菌体。这一多样化的分枝杆菌噬菌体（Mycobacteriophages）集合，为在新型应用中测试噬菌体来源酶类提供了优质资源。
本研究中，我们测试了分枝杆菌噬菌体溶素（Mycobacteriophage endolysins）在与可生物降解聚羟基脂肪酸酯（polyhydroxyalkanoate，PHA）纳米珠功能融合后裂解宿主菌株的能力。PHA纳米珠既可促进酶类在表面的表达与应用，又已被证实可在实际应用中稳定多种蛋白质，同时规避了传统蛋白质纯化的诸多难题。
我们选取了2个溶素A（Lysin A）同源蛋白与6个溶素B（Lysin B）同源蛋白，使其与聚羟基脂肪酸酯合酶C（polyhydroxyalkanoate synthase C，PhaC）实现功能融合。上述重组载体的表达，成功使功能性溶素展示于PHA纳米珠表面。在纯培养体系中，以80 mg/mL的纳米珠用量，定向展示于纳米珠表面的溶素可裂解高达79%的耻垢分枝杆菌MC2 155菌群。
为验证纳米珠能否作为个人防护装备（personal protective equipment，PPE）中的有效防护层，我们改良了织物基检测方法，结果显示：在纺织品表面暴露5小时后，菌群可出现最大1个对数级的数量下降（细胞裂解率达91%）。在纺织品表面防护性能检测中，溶素B类酶的表现优于溶素A类酶。
上述结果表明，细菌溶素在展示于PHA纳米珠后仍可保持高效裂解活性，最短仅需45分钟即可造成菌群的显著死亡。本研究结果证实了分枝杆菌噬菌体溶素可应用于功能化纳米珠，进而可用于保护诸如个人防护装备（PPE）这类经常接触气溶胶化细菌的表面，为相关应用提供了原理性验证。