Antimicrobial activity of Mycobacteriophage D29 Lysin B during Mycobacterium ulcerans infection

Abstract

Buruli Ulcer (BU) is a cutaneous disease caused by Mycobacterium ulcerans . The pathogenesis of this disease is closely related to the secretion of the toxin mycolactone that induces extensive destruction of the skin and soft tissues. Currently, there are no effective measures to prevent the disease and, despite availability of antibiotherapy and surgical treatments, these therapeutic options are often associated with severe side effects. Therefore, it is important to develop alternative strategies for the treatment of BU. Endolysins (lysins) are phage encoded enzymes that degrade peptidoglycan of bacterial cell walls. Over the past years, lysins have been emerging as alternative antimicrobial agents against bacterial infections. However, mycobacteria have an unusual outer membrane composed of mycolylarabinogalactan-peptidoglycan. To overcome this complex barrier, some mycobacteriophages encode a lipolytic enzyme, Lysin B (LysB). In this study, we demonstrate for the first time that recombinant LysB displays lytic activity against M . ulcerans isolates. Moreover, using a mouse model of M . ulcerans footpad infection, we show that subcutaneous treatment with LysB prevented further bacterial proliferation, associated with IFN-γ and TNF production in the draining lymph node. These findings highlight the potential use of lysins as a novel therapeutic approach against this neglected tropical disease.

Deciphering the Role of Holin in Mycobacteriophage D29 Physiology

“…Indeed, mycobacteriophages have not only shown their potential to treat Mycobacterium abscessus infection (Dedrick et al, 2019), but they have also been used for the development of phage-based diagnostics (Jacobs et al, 1993;Pearson et al, 1996;Park et al, 2003). Among the many mycobacteriophages, D29, which is a virulent phage and is capable of infecting and killing both slow and fast growing mycobacterial species (Ford et al, 1998;Rybniker et al, 2006), and its lysis enzymes have been used to kill M. tuberculosis and other mycobacteria in various animal-based experiments (Carrigy et al, 2019;Fraga et al, 2019). The host cell lysis is governed by the protein products of the "Lytic cassette" genes.…”

“…The host cell lysis is governed by the protein products of the "Lytic cassette" genes. In D29, the lytic cassette comprises gp10 coding for an endolysin LysA that targets the peptidoglycan cell wall and gp12 producing LysB that targets the outer mycolic acid layer of mycobacteria (Payne et al, 2009;Fraga et al, 2019). Additionally, gp11, which is sandwiched between gp10 and gp12, produces holin that perforates the cell membrane of mycobacteria thus allowing for the diffusion of the hydrolytic enzymes to the periplasm and ensuing cell lysis (Catalao et al, 2013;Kamilla and Jain, 2016).…”

Mycobacteriophages as Potential Therapeutic Agents against Drug-Resistant Tuberculosis

“…LysB also degrades trehalose dimycolate (TDM), a glycolipid that plays an important role in mycobacterial pathogenesis [ 104 , 105 ] ( Figure 1 B). Recently, the antimicrobial activity of phage D29 LysB against M. ulcerans has been demonstrated in a mouse model, showing synergistic inhibitory effects with other antimycobacterial drugs in vivo and inducing an immune response by increasing the levels of pro-inflammatory cytokines IFN-γ and TNF in the DLN (draining lymph node) [ 106 ]. Besides the lysis as a primary mechanism of mycobacterial death, other authors have suggested the existence of secondary mechanisms, such as the production of superoxide radicals by lysed cells, or the induction of programmed cell death by mycobacteriophages [ 107 ] ( Figure 1 B).…”

“…Phage-derived lytic enzymes such as endolysins are being studied as potential antimicrobial agents to combat infections caused by different gram-negative and -positive pathogens [ 34 , 109 ], with progresses made optimizing lysins through bioengineering [ 106 , 110 , 111 ]. Indeed, a recent study describes the novel concept of “innolysins”, an engineering approach that allows customization of endolysins by combining them with phage Receptor Binding Proteins (RBPs) to target specific bacteria [ 112 ].…”

Bacteriophage Therapy of Bacterial Infections: The Rediscovered Frontier

“…Endolysins are bacteriophage-encoded peptidoglycan-disrupting enzymes synthesized at the last stage of the phage life cycle in the infected bacteria [83]. One endolysin, lysine B, was found to lyse M. ulcerans infecting the footpad of experimental mice [84].…”