PLAN-M; mycobacteriophage Endolysins Fused to Bioparticles Mitigate Mycobacterial Growth in Liquid and on Surfaces

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. M. tuberculosis and it is one of the most deadly pathogens on the planet; roughly 1.5 million people die every year of TB infections.

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The HHMI SEA-PHAGES programme has contributed to discovering bacteriophages that are able to infect Mycobacterium smegmatis MC2155, a close, non-pathogenic relative of M. tuberculosis. This collection of diverse Mycobacteriophages is an excellent resource for trialing bacteriophage-sourced enzymes in novel applications.

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Bacteriophages are the most abundant entity on the planet. There are an estimated 10 bacteriophages for every bacterial cell. Bacteriophages are a source of enzymes that have evolved for millions of years to destroy their bacterial hosts and there is no larger collection of host-specific bacteriophages than that for M. smegmatis MC2155.

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Development of Anti-mycobacterial Functionalised Bioparticles

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In a cooperation between Associate Professors Heather Hendrickson and Eric Altermann, biodegradable bioparticles displaying a set of eight mycobacteriophage endolysins were developed. These 'Plan-M' bioparticles were tested in liquid and on fabrics and observed to kill up to 80% of Mycobacteria . This is the sort of technology that can be applied further in the protection against Mycobacteria using Personal Protective Equipment (PPE).

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Synthesizing the PLAN-M Bioparticles

(A) Genetic maps of the empty PhaC pET-14b vector, pET-14b StarStuff-LysA PhaC, and pET-14b-D29-LysB-PhaC, respectively. (B) Representative PLAN-M bioparticles produced from the vectors shown in (A) under 20,000x transmission electron microscopy Scale bars are 3 nm.

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Textile test of PLAN-M Bioparticles against M. Smegmatis MC2155

Textile test of PLAN-M bioparticles against M. smegmatis MC2155. (A) The AATCC-100-2004 protocol involves using a treated textile, in this case, filter papers with 80 mg/mL of each of 8 PLAN-M nanobeads. A 1 mL aliquot of M. smegmatis MC2155 culture was applied for exposures of 45 min and 5 h. (B) Relative cell lysis of M. smegmatis MC2155 after treatment by lysin-free (PhaC) or PLAN-M bioparticles (n = 3). (C) Relative cell lysis of M. smegmatis MC2155 after treatment by two PLAN-M bioparticles; StarStuff-LysA and D29-LysB, in a 1:1 ratio and a final concentration of 80 mg/mL combined (40 mg/mL each) for either 45 min or 5 h. Surviving populations are reported as percent change compared to the no bioparticles control. Error bars represent the standard error based on three replicates of each experimental test. Significance test results reported above the bars as p-values from Student’s t-test as follows; ns > 0.05, * < 0.05, ** < 0.01, *** < 0.001.

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Observations on fold decrease in Mycobacterial populations

We have observed up to a 1 log fold decrease in Mycobacterial populations due to exposure to bioparticles displaying functional endolysins in our biocidal textile assay. This represents a significant reduction in potentially infectious particles in the PPE application that we are envisioning in which between 20,000 and 700 infectious particles can be transmitted in a droplet by a sneeze or a cough, respectively (Fernstrom and Goldblatt, 2013).

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[1]

Drug-resistant, Mycobacterium tuberculosis bacteria, the pathogen responsible for causing the disease tuberculosis (TB).

Protecting the health of community members

Droplet based pathogen spread is currently a significant issue in those settings where protecting the health of patients, community members, and medical staff are of paramount importance (Jones et al., 2020).

We are currently planning to investigate the efficacy of prototype PPE (i.e., Masks) with functionalized bioparticle enhancements in clinical model systems. A promising avenue for future work would be to consider the combined effects of the lysins described here. Lysin A kills the cells but may not be as efficient as Lysin B. Dual Fusion bioparticles that display both on the same PhaC enzyme will enable us to test the true synergy of these enzymes against M. smegmatis MC2155.

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