Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

Abstract

To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Agrobacterium tumefaciens. Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens. The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ϳ42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative phage peptidoglycan hydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N-acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. IMPORTANCEThe characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium tumefaciens, may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The specificity of bacteriophage treatment for the host is an asset in complex communities, such as in orchards where it would be detrimental to harm the symbiotic bacteria in the environment. Second, bacteriophages are potential sources of enzymes that efficiently lyse bacterial cells. These phage proteins may have a broad specificity, but since proteins do not replicate as phages do, their effect is highly localized, providing an alternative to traditional antibiotic treatments. Thus, studies of lytic bacteriophages that infect A. tumefaciens may provide insights for designing preventative strategies against bacterial pathogens.

A Phage Lysin Fused to a Cell-Penetrating Peptide Kills Intracellular Methicillin-Resistant Staphylococcus aureus in Keratinocytes and Has Potential as a Treatment for Skin Infections in Mice

“…The potent bactericidal effect of phages is mainly attributed to their genome-encoded cell wall hydrolase, also called lysin, which exhibits a lethal effect by forming holes in the cell wall through peptidoglycan digestion (14). Evidence has shown that lysins exhibit a high level of antibacterial activity, and they have proven their effectiveness for the treatment of infections caused by bacteria resistant to conventional chemical antibiotics in diverse animal models (15)(16)(17). A number of studies have revealed the enormous potential of the use of phage lysins, which even surpasses that of intact phage, as therapeutics for MRSA infections (16,18).…”

Bacteriophages: Protagonists of a Post-Antibiotic Era

“…In addition, it has been shown that they can be useful against multiresistant bacterial strains [ 71 ]. Recent research proposed VAPGHs activity to treat plant diseases, such as those provoked by Agrobacterium tumefaciens [ 72 ]. Finally, to improve their bactericidal capacities, chimeric proteins can be created by combining several enzymes or by exchanging their functional domains.…”

Potential for Bacteriophage Endolysins to Supplement or Replace Antibiotics in Food Production and Clinical Care

“…Attai et al [ 161 ] recently characterized an endolysin from bacteriophages Atu_ph02 and Atu_ph03 for the biocontrol of Agrobacterium tumefaciens. A. tumefaciens is a Gram-negative soil-borne bacterium that is the etiologic agent of crown gall disease in a variety of orchard and vineyard crops [ 162 ].…”

“…Its severity and widespread impact has contributed to it to becoming the subject of many recent studies [ 163 ]. The lytic protein displayed interesting properties, with the ability to not only rapidly lyse the cell, but to also block cell division, ensuring potent antimicrobial activity [ 161 ]. Therefore, the enzyme is a candidate for biocontrol of A. tumefaciens ; however, the method of implementation needs to be researched before a viable strategy for crop protection can be developed.…”