Antimicrobial activity of Mycobacteriophage D29 Lysin B during Mycobacterium ulcerans infection

Abstract

16Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans. The 17 pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that 18 induces extensive destruction of the skin and soft tissues. Although the World Health Organization 19 recommends a combination of rifampicin and streptomycin for the treatment of BU, clinical 20 management of advanced stages often requires extensive surgical resection of the infected 21 tissue. Therefore, it is important to develop alternative strategies for the treatment of BU. 22 Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial 23 cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents 24 against Gram-positive bacteria. Amongst Gram-positive bacteria, mycobacteria have an unusual 25 outer membrane that is covalently attached to the mycolylarabinogalactan-peptidoglycan 26 complex. To overcome this additional barrier to phage-mediated lysis, some mycobacteriophages 27 encode a lipolytic enzyme, Lysin B (Lys B). 28In this study, we demonstrated for the first time that recombinant Lys B displays lytic 29 activity against M. ulcerans isolates. Moreover, using a mouse model of M. ulcerans footpad 30 infection, we show that subcutaneous treatment with Lys B leads to a reduction in bacterial 31 burdens, associated with IFN-γ and TNF production in the draining lymph node. These findings 32 highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical 33 disease. AUTHOR SUMMARY 35Buruli Ulcer (BU) is a necrotizing skin disease caused by Mycobacterium ulcerans. Standard 36 treatment for BU lesions consists of a combination of rifampicin and streptomycin for 8 weeks. 37However, clinical management of advanced stages of the disease often requires extensive 38 surgical resection of the infected tissue. Therefore, it is important to develop alternative strategies 39 for the treatment of BU. In that sense, we tested the efficacy of Lysin B (Lys B), a phage encoded 40 lipolytic enzyme that degrades the mycolylarabinogalactan-peptidoglycan complex present in the 41 mycobacterial cell wall. In this study, we show that Lys B not only displays lytic activity against M. 42 ulcerans isolates in vitro, but also leads to a reduction in bacterial burdens in M. ulcerans-infected 43 mouse footpads. These findings highlight the potential use of lysins as a novel therapeutic 44 approach against this neglected tropical disease. mycolactone that presents cytotoxic and immunosuppressive properties [4][5][6][7][8]. Early presentations 51 of active BU include a painless pre-ulcerative nodule, papule, plaque or edematous lesion, which 52 can evolve into typical ulcers or, in the most extreme cases, may result in extensive skin 53 destruction, multifocal lesions or bone involvement [1,3,7,9,10]. 54The antibiotic regimen recommended by the World Health Organization (WHO) (daily 55 administration of rifampicin (RIF) and streptomycin (STR) for 8 weeks) was p...