Abstract
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.
Highlights
The genus Mycobacterium includes over 150 recognized species, many of which are human pathogens (King et al, 2017)
A set of seven novel bacteriophages, chosen from separate Mycobacteriophage clusters, as defined by average nucleotide dissimilarity were chosen for this study (Hatfull et al, 2010)
Endolysin Lysin B from phage D29 was included as a known positive control that had previously been demonstrated to have the ability to lyse M. smegmatis MC2 155 cells from without as a purified protein (Payne and Hatfull, 2012)
Summary
The genus Mycobacterium includes over 150 recognized species, many of which are human pathogens (King et al, 2017). M. tuberculosis (TB) is among the most serious; onefourth of the global population is estimated to carry latent tuberculosis infection (Houben and Dodd, 2016). In 2017, the WHO reported that active tuberculosis infections caused 1.3 million deaths, 558,000 people developed TB infections that were resistant to the frontline drug rifampicin, and 82% of reported cases were resistant to two of the most powerful anti-tuberculosis drugs (WHO, 2019). An individual can transmit tuberculosis to their community through aerosolised bacteria expelled in droplets from the lungs (Hannan et al, 2000). Airborne transmission is of special concern in clinical settings, where health care workers exposed to coughing patients are at high risk of infection (Escombe et al, 2007; Ruhl et al, 2020). Mitigating the risk of infection and safeguarding healthcare professionals and the community with bespoke Personal Protective Equipment (PPE) is a high priority
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