Date of Award
phage lytic enzymes, drug-resistant bacteria, viral metagenomics, bacteriophages
Recently, phage lytic enzymes (also known as endolysins or, simply, lysins) have received considerable attention as potential antibacterial agents. During the infective cycle of double-stranded DNA phage, these peptidoglycan hydrolases are responsible for digesting the cell wall of the host bacterium and freeing newly-assembled viral particles. At the same time, an increasing body of evidence has demonstrated that recombinantly-purified phage lysinsâ€”when added exogenouslyâ€”can potently kill Gram-positive bacteria, whose peptidoglycan is accessible from the extracellular space. Consequently, lysins have been proposed as novel enzybiotic (i.e. enzyme-antibiotic) molecules that could serve as novel weapons in the fight against drug-resistant bacteria. Most lysins characterized to date were initially identified through either recombinant screening or DNA-sequencing of phage genomes. Recent technological and methodological advances, however, have drastically increased the potential avenues for lysin identification. The goal of the work presented here to exploit and expand upon these advances so that the identification of new lysins is increasingly rapid and straightforward. This thesis is subdivided into four interrelated sections, each of which represents a distinct study into a novel approach/method for cloning phage lysins. The first study (Chapter 2) addresses the issue of bacterial genomic sequencing and how the rapidly expanding database of bacterial genomes represents a vast source of proviral lysins. Focusing on the anaerobic pathogen Clostridium perfringens, the genomes of 9 recently-sequenced strains were computationally mined for prophage lysins and lysin-like ORFs (open reading frames), revealing several dozen proteins of various enzymatic classes. Of these lysins, a muramidase (termed PlyCM) from strain ATCC 13124 was chosen for recombinant analysis based on its dissimilarity to the only other previouslycharacterized C. perfringens lysin. Following expression and purification, various biochemical properties of PlyCM were determined in vitro, including pH/saltdependence and temperature stability. The enzyme exhibited activity at low ïg/ml concentrations, and it was active against 23/24 strains of C. perfringens tested. Chapters 3 and 4 focus on the emerging field of viral metegenomics, a term which refers to the bulk extraction and analysis of DNA from environmental phage without prior laboratory culture of any particular virus. Phage metagenomes have been shown to be incredibly complex and diverse, and the goal of these chapters was to tap into this diversity through functional metagenomic screens for lytic enzymes. Chapter 3 first addresses a preliminary methodological issue, namely the fact that uncultured phage samples generally do not provide sufficient quantities of DNA for ready screening. A novel ELASL protocol (for expressed linker amplified shotgun library) was developed that combines linker amplification of enzyme-digested DNA with subsequent topoisomerase cloning into linearized expression plasmids. As proof-ofprinciple, genomic and metagenomic E-LASLs were constructed and screened for antibacterial and hemolytic activity in an Escherichia coli host. Six Bacillus anthracis phage lysins were cloned in the process, along with a virulence factor of the aerolysin gene family. Chapter 4 proceeds to address an additional methodological issue surrounding metagenomic lysin identification: the question of how to identify lysin-encoding clones in a functional screen when the targeted bacteria are not pre-defined. A novel two-step screening technique was devised for this purpose. It involves a primary screen in which transformed E. coli clones were identified that demonstrated colony lysis following exposure to nebulized inducing agent. This effect, which can be due to the expression of membrane-permeabilizing phage holins, was discerned by the development a hemolytic-effect in surrounding blood agar. The selected clones were then overlaid with autoclaved Gram-negative bacteria (specifically Pseudomonas aeruginosa) to assay directly for recombinant expression of lytic enzymes, which are often encoded proximally to holins in phage genomes. This method was combined with the aforementioned E-LASL technique and applied to a viral metagenomic library constructed from mixed animal feces. Twenty-six lytic enzymes were cloned in this screen, including both Gram-positive-like and Gram-negative-like enzymes, as well as several atypical lysins whose predicted structures are less common among known phage. Finally, Chapter 5 takes the above techniques and reapplies them outside the context of metegenomics, returning to individual genomes as sources of lytic enzymes. Specifically, 2 lysins were cloned from prophage of Streptococcus suis, an important veterinary and emerging zoonotic pathogen. One of these S. suis enzymes (PlySs1) was identified by applying the two-step screen to the genome of an unsequenced clinical strain. The other (PlySs2) was identified in a manner similar to the clostridial lysin PlyCM, by analyzing the published genomes of various sequenced strains. Finally, PlySs1 was subject to chromatographic purification and in vitro analysis against numerous suis and non-suis strains of streptococci. Currently, both PlySs1 and PlySs2 are involved in a collaboratorâ€™s ongoing in vivo trial employing experimentally-infected pigs.
Schmitz, Jonathan Edward, "Expanding the Horizons of Enzybiotic Identification" (2011). Student Theses and Dissertations. 138.