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Endolysin enzymes from bacteriophage cause bacterial lysis by degrading the peptidoglycan cell wall. The streptococcal C1 phage endolysin PlyC, is the most potent endolysin described to date and can rapidly lyse group A, C, and E streptococci. PlyC is known to bind the Group A streptococcal cell wall, but the specific molecular target or the binding site within PlyC remain uncharacterized. Here we report for the first time, that the polyrhamnose backbone of the Group A streptococcal cell wall is the binding target of PlyC. We have also characterized the putative rhamnose binding groove of PlyC and found four key residues that were critical to either the folding or the cell wall binding action of PlyC. Based on our results, we suggest that the interaction between PlyC and the cell wall may not be a high-affinity interaction as previously proposed, but rather a high avidity one, allowing for PlyC's remarkable lytic activity. Resistance to our current antibiotics is reaching crisis levels and there is an urgent need to develop the antibacterial agents with new modes of action. A detailed understanding of this potent endolysin may facilitate future developments of PlyC as a tool against the rise of antibiotic resistance.

The genome of Escherichia coli O157:H7 bacteriophage vB_EcoM_CBA120 encodes four distinct tailspike proteins (TSPs). The four TSPs, TSP1-4, attach to the phage baseplate forming a branched structure. We report the 1.9 Å resolution crystal structure of TSP2 (ORF211), the TSP that confers phage specificity towards E. coli O157:H7. The structure shows that the N-terminal 168 residues involved in TSPs complex assembly are disordered in the absence of partner proteins. The ensuing head domain contains only the first of two fold modules seen in other phage vB_EcoM_CBA120 TSPs. The catalytic site resides in a cleft at the interface between adjacent trimer subunits, where Asp506, Glu568, and Asp571 are located in close proximity. Replacement of Asp506 and Asp571 for alanine residues abolishes enzyme activity, thus identifying the acid/base catalytic machinery. However, activity remains intact when Asp506 and Asp571 are mutated into asparagine residues. Analysis of additional site-directed mutants in the background of the D506N:D571N mutant suggests engagement of an alternative catalytic apparatus comprising Glu568 and Tyr623. Finally, we demonstrate the catalytic role of two interacting glutamate residues of TSP1, located in a cleft between two trimer subunits, Glu456 and Glu483, underscoring the diversity of the catalytic apparatus employed by phage vB_EcoM_CBA120 TSPs.

The prevalence of multidrug-resistant Pseudomonas aeruginosa has stimulated development of alternative therapeutics. Bacteriophage peptidoglycan hydrolases, termed lysins, represent an emerging antimicrobial option for targeting Gram-positive bacteria. However, lysins against Gram-negatives are generally deterred by the outer membrane and their inability to work in serum. One solution involves exploiting evolved delivery systems used by colicin-like bacteriocins (e.g., S-type pyocins of P. aeruginosa) to translocate through the outer membrane. Following surface receptor binding, colicin-like bacteriocins form Tol- or TonB-dependent translocons to actively import bactericidal domains through outer membrane protein channels. With this understanding, we developed lysocins, which are bioengineered lysin-bacteriocin fusion molecules capable of periplasmic import. In our proof-of-concept studies, components from the P. aeruginosa bacteriocin pyocin S2 (PyS2) responsible for surface receptor binding and outer membrane translocation were fused to the GN4 lysin to generate the PyS2-GN4 lysocin. PyS2-GN4 delivered the GN4 lysin to the periplasm to induce peptidoglycan cleavage and log-fold killing of P. aeruginosa with minimal endotoxin release. While displaying narrow-spectrum antipseudomonal activity in human serum, PyS2-GN4 also efficiently disrupted biofilms, outperformed standard-of-care antibiotics, exhibited no cytotoxicity toward eukaryotic cells, and protected mice from P. aeruginosa challenge in a bacteremia model. In addition to targeting P. aeruginosa, lysocins can be constructed to target other prominent Gram-negative bacterial pathogens.

Bacteriophage tailspike proteins mediate virion absorption through reversible primary receptor binding, followed by lipopolysaccharide or exopolysaccharide degradation. The Escherichia coli O157:H7 bacteriophage CBA120 genome encodes four distinct tailspike proteins, annotated as ORFs 210 through 213. Previously, we reported the crystal structure of ORF210 (TSP1). Here we describe the crystal structure of ORF212 (TSP3) determined at 1.85 Å resolution. As observed with other tailspike proteins, TSP3 assembles into a trimer. Each subunit of TSP3 has an N-terminal head domain that is structurally similar to that of TSP1, consistent with their high amino acid sequence identity. In contrast, despite sharing a β-helix fold, the overall structure of the C-terminal catalytic domain of TSP3 is quite different when compared to TSP1. The TSP3 structure suggests that the glycosidase active site resides in a cleft at the interface between two adjacent subunits where three acidic residues, Glu362 and Asp383 on one subunit, and Asp426 on a second subunit, are located in close proximity. Comparing the glycosidase activity of wild-type TSP3 to various point mutants revealed that catalysis requires the carboxyl groups of Glu362 and Asp426, and not of Asp383, confirming the enzyme employs two carboxyl groups to degrade lippopolysaccharide using an acid/base mechanism.

The increasing rate of resistance of pathogenic bacteria, such as Staphylococcus aureus, to classical antibiotics has driven research toward identification of other means to fight infectious disease. One particularly viable option is the use of bacteriophage-encoded peptidoglycan hydrolases, called endolysins or enzybiotics. These enzymes lyse the bacterial cell wall upon direct contact, are not inhibited by traditional antibiotic resistance mechanisms, and have already shown great promise in the areas of food safety, human health, and veterinary science. We have identified and characterized an endolysin, PlyGRCS, which displays dose-dependent antimicrobial activity against both planktonic and biofilm S. aureus, including methicillin-resistant S. aureus (MRSA). The spectrum of lytic activity for this enzyme includes all S. aureus and Staphylococcus epidermidis strains tested, but not other Gram-positive pathogens. The contributions of the PlyGRCS putative catalytic and cell wall binding domains were investigated through deletion analysis. The cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) catalytic domain displayed activity by itself, though reduced, indicating the necessity of the binding domain for full activity. In contrast, the SH3_5 binding domain lacked activity but was shown to interact directly with the staphylococcal cell wall via fluorescent microscopy. Site-directed mutagenesis studies determined that the active site residues in the CHAP catalytic domain were C29 and H92, and its catalytic functionality required calcium as a co-factor. Finally, biochemical assays coupled with mass spectrometry analysis determined that PlyGRCS displays both N-acetylmuramoyl-l-alanine amidase and d-alanyl-glycyl endopeptidase hydrolytic activities despite possessing only a single catalytic domain. These results indicate that PlyGRCS has the potential to become a revolutionary therapeutic option to combat bacterial infections.

Alginate is a polysaccharide produced by certain seaweeds and bacteria that consists of mannuronic acid and guluronic acid residues. Seaweed alginate is used in food and industrial chemical processes, while the biosynthesis of bacterial alginate is associated with pathogenic Pseudomonas aeruginosa. Alginate lyases cleave this polysaccharide into short oligo-uronates and thus have the potential to be utilized for both industrial and medicinal applications. An alginate lyase gene, algMsp, from Microbulbifer sp. 6532A, was synthesized as an E.coli codon-optimized clone. The resulting 37 kDa recombinant protein, AlgMsp, was expressed, purified and characterized. The alginate lyase displayed highest activity at pH 8 and 0.2 M NaCl. Activity of the alginate lyase was greatest at 50°C; however the enzyme was not stable over time when incubated at 50°C. The alginate lyase was still highly active at 25°C and displayed little or no loss of activity after 24 hours at 25°C. The activity of AlgMsp was not dependent on the presence of divalent cations. Comparing activity of the lyase against polymannuronic acid and polyguluronic acid substrates showed a higher turnover rate for polymannuronic acid. However, AlgMSP exhibited greater catalytic efficiency with the polyguluronic acid substrate. Prolonged AlgMsp-mediated degradation of alginate produced dimer, trimer, tetramer, and pentamer oligo-uronates.

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis(1). The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened(2). One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential(3-5). The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits(6). When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS(7). Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 °C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 °C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 °C, 43.5 °C and 50.2 °C, respectively(8-10). According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy(11). Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

Bacteriophages deploy lysins that degrade the bacterial cell wall and facilitate virus egress from the host. When applied exogenously, these enzymes destroy susceptible microbes and, accordingly, have potential as therapeutic agents. The most potent lysin identified to date is PlyC, an enzyme assembled from two components (PlyCA and PlyCB) that is specific for streptococcal species. Here the structure of the PlyC holoenzyme reveals that a single PlyCA moiety is tethered to a ring-shaped assembly of eight PlyCB molecules. Structure-guided mutagenesis reveals that the bacterial cell wall binding is achieved through a cleft on PlyCB. Unexpectedly, our structural data reveal that PlyCA contains a glycoside hydrolase domain in addition to the previously recognized cysteine, histidine-dependent amidohydrolases/peptidases catalytic domain. The presence of eight cell wall-binding domains together with two catalytic domains may explain the extraordinary potency of the PlyC holoenyzme toward target bacteria.

Endolysins are lytic enzymes encoded by bacteriophage that represent an emerging class of protein therapeutics. Considering macromolecular thermoresistance correlates with shelf life, PlyG, a Bacillus anthracis endolysin, was thermally characterized to further evaluate its therapeutic potential. Results from a biophysical thermal analysis revealed full-length PlyG and its isolated domains comprised thermal denaturation temperatures exceeding 63°C. In the absence of reducing agent, PlyG was determined to be kinetically unstable, a finding hypothesized to be attributable to the chemical oxidation of cysteine and/or methionine residues. The presence of reducing agent kinetically stabilized the endolysin, with PlyG retaining at least ~50% residual lytic activity after being heated at temperatures up to 80°C and remaining enzymatically functional after being boiled. Furthermore, the endolysin had a kinetic half-life at 50°C and 55°C of 35 and 5.5h, respectively. PlyG represents a thermostable proteinaceous antibacterial with subsequent prolonged therapeutic shelf life expectancy.