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

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.

Endolysin B as a new archetype inM. tuberculosistreatment

“…This would promote cell wall hydrolysis and the inhibition of M. tuberculosis growth, thus further supporting LysBFL antimycobacterial effect. In this regard, because LysBFL acts on non-genomic encoded targets (i.e., mycolic acid), diversely from the clinically used antimicrobials, the rise of resistance to their lytic activity is rare and could be exploited to generate innovative and specific antimicrobial therapies 18,[38][39][40] . On the other hand, one of the main limitations to the use of phage and/or phage-derived proteins for therapy stands in the possible immune response that the host could develop in response to exogenous proteins 41,42 .…”

“…The concerted degrading action of both LysA and LysB finally leads to mycobacterial lysis, which occurs following the loss of communication between the bacillus cell wall the outer membrane 32 . Therefore, the endolysin-mediated lytic activity towards host bacteria, combined with the pressing need to counterbalance the exponential rise of antibiotic-resistant pathogens, are posing these lytic-derived enzymes as new potential antibacterial agents 18,23,32 .…”

Inhibition of Listeria monocytogenes by Phage Lytic Enzymes Displayed on Tailored Bionanoparticles

“…The results show an initial proof-of-concept for the application of these tailored PHA_BNPs in the inhibition of L. monocytogenes . Future experiments will determine if the tailored PHA_BNPs can be applied to inhibit L. monocytogenes present on surfaces in food-processing plants in an approach similar to that used by Davies et al 2021 [ 17 ], where Mycobacteriophage endolysins fused to biodegradable nanobeads were applied to solid surfaces (filter paper). As the PHA_BNPs are active in liquid suspensions, as indicated in this study, a potential option for their application includes spraying onto food-contact surfaces, as with traditional sanitizers.…”

“…Effective uses of these BNPs have previously been demonstrated by Altermann et al [ 14 ] wherein tailored BNPs lysed a range of rumen methanogen strains and reduced methane production by 97%. Similarly, Davies et al [ 17 ] reported that tailored BNPs could act as a successful protective layer in PPE against Mycobacteria after a one log (91%) reduction was reported.…”

“…These include the covalent binding and stabilization of the protein in a uniform direction to the surface of the nanobead. The stabilizing matrix on the nanobeads enables ready deployment of proteins and enzymes in liquids or on surfaces, the expression of proteins in a one-step process, and the resulting high yield of product [ 17 ]. Effective uses of these BNPs have previously been demonstrated by Altermann et al [ 14 ] wherein tailored BNPs lysed a range of rumen methanogen strains and reduced methane production by 97%.…”