Structural engineering of a phage lysin that targets Gram-negative pathogens

Abstract

Bacterial pathogens are becoming increasingly resistant to antibiotics. As an alternative therapeutic strategy, phage therapy reagents containing purified viral lysins have been developed against Grampositive organisms but not against Gram-negative organisms due to the inability of these types of drugs to cross the bacterial outer membrane. We solved the crystal structures of a Yersinia pestis outer membrane transporter called FyuA and a bacterial toxin called pesticin that targets this transporter. FyuA is a β-barrel membrane protein belonging to the family of TonB dependent transporters, whereas pesticin is a soluble protein with two domains, one that binds to FyuA and another that is structurally similar to phage T4 lysozyme. The structure of pesticin allowed us to design a phage therapy reagent comprised of the FyuA binding domain of pesticin fused to the N-terminus of T4 lysozyme. This hybrid toxin kills specific Yersinia and pathogenic E. coli strains and, importantly, can evade the pesticin immunity protein (Pim) giving it a distinct advantage over pesticin. Furthermore, because FyuA is required for virulence and is more common in pathogenic bacteria, the hybrid toxin also has the advantage of targeting primarily disease-causing bacteria rather than indiscriminately eliminating natural gut flora.colicin | muramidase | TonB-dependent transport | plague

Blocking Yersiniabactin Import Attenuates Extraintestinal Pathogenic Escherichia coli in Cystitis and Pyelonephritis and Represents a Novel Target To Prevent Urinary Tract Infection

“…Furthermore, we demonstrate that genes encoding bacterial systems for iron acquisition, including the yersiniabactin system, are highly expressed during natural, uncomplicated cystitis in women. Overall, our data support the yersiniabactin system as a therapeutic target to prevent or treat UPEC-mediated UTI and reinforce the potential of the yersiniabactin system as a common target for several, increasingly multidrug-resistant, Gram-negative enteric pathogens (38,39).…”

Uropathogenic enterobacteria use the yersiniabactin metallophore system to acquire nickel

“…The two other HPI operons encode Ybt biosynthesis-associated proteins and the transcription factor, YbtA, which is necessary for expression of all four HPI operons (19). FyuA is a β-barrel outer membrane protein which imports Ybt complexes into the periplasm using energy from the proton gradient across the inner membrane by coupling to the inner membrane TonB/ExbB/ExbD complex (20). YbtP and YbtQ are putative ABC transporters that are thought to form a heterodimer (termed YbtPQ) in the inner membrane and are necessary for cellular utilization of Ybt-bound iron (8,18,21).…”

Physiology and Pathology of Multidrug-Resistant Bacteria: Phage-Related Therapy

“…This hybrid toxin killed Yersinia and Escherichia coli strains and also bypassed the pesticin immunity (PIM) [111]. Furthermore, Ply187-derived CD, when fused to non-SH3b CBD from phage phi13 endolysin NM3, protects mice against MRSA [112].…”

“…In 2012, Lukacik et al showed that the fusion of FyuA-binding domain of pesticin and T4 lysozyme utilizes FyuA for transport across the OM of Gram-negative Yersinia pestis [111]. This hybrid toxin killed Yersinia and Escherichia coli strains and also bypassed the pesticin immunity (PIM) [111].…”