Department of Veterinary Medicine
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Item Discovery and Biochemical Characterization of PlyP56, PlyN74, and PlyTB40—Bacillus Specific Endolysins(MDPI, 2018-05-21) Etobayeva, Irina; Linden, Sara B.; Alem, Farhang; Harb, Laith; Rizkalla, Lucas; Mosier, Philip D.; Johnson, Allison A.; Temple, Louise; Hakami, Ramin M.; Nelson, Daniel C.Three Bacillus bacteriophage-derived endolysins, designated PlyP56, PlyN74, and PlyTB40, were identified, cloned, purified, and characterized for their antimicrobial properties. Sequence alignment reveals these endolysins have an N-terminal enzymatically active domain (EAD) linked to a C-terminal cell wall binding domain (CBD). PlyP56 has a Peptidase_M15_4/VanY superfamily EAD with a conserved metal binding motif and displays biological dependence on divalent ions for activity. In contrast, PlyN74 and PlyTB40 have T7 lysozyme-type Amidase_2 and carboxypeptidase T-type Amidase_3 EADs, respectively, which are members of the MurNAc-LAA superfamily, but are not homologs and thus do not have a shared protein fold. All three endolysins contain similar SH3-family CBDs. Although minor host range differences were noted, all three endolysins show relatively broad antimicrobial activity against members of the Bacillus cereus sensu lato group with the highest lytic activity against B. cereus ATCC 4342. Characterization studies determined the optimal lytic activity for these enzymes was at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55 °C), and at low concentrations of NaCl (<50 mM). Direct comparison of lytic activity shows the PlyP56 enzyme to be twice as effective at lysing the cell wall peptidoglycan as PlyN74 or PlyTB40, suggesting PlyP56 is a good candidate for further antimicrobial development as well as bioengineering studies.Item Contributions of Net Charge on the PlyC Endolysin CHAP Domain(MDPI, 2019-05-28) Shang, Xiaoran; Nelson, Daniel C.Bacteriophage endolysins, enzymes that degrade the bacterial peptidoglycan (PG), have gained an increasing interest as alternative antimicrobial agents, due to their ability to kill antibiotic resistant pathogens efficiently when applied externally as purified proteins. Typical endolysins derived from bacteriophage that infect Gram-positive hosts consist of an N-terminal enzymatically-active domain (EAD) that cleaves covalent bonds in the PG, and a C-terminal cell-binding domain (CBD) that recognizes specific ligands on the surface of the PG. Although CBDs are usually essential for the EADs to access the PG substrate, some EADs possess activity in the absence of CBDs, and a few even display better activity profiles or an extended host spectrum than the full-length endolysin. A current hypothesis suggests a net positive charge on the EAD enables it to reach the negatively charged bacterial surface via ionic interactions in the absence of a CBD. Here, we used the PlyC CHAP domain as a model EAD to further test the hypothesis. We mutated negatively charged surface amino acids of the CHAP domain that are not involved in structured regions to neutral or positively charged amino acids in order to increase the net charge from -3 to a range from +1 to +7. The seven mutant candidates were successfully expressed and purified as soluble proteins. Contrary to the current hypothesis, none of the mutants were more active than wild-type CHAP. Analysis of electrostatic surface potential implies that the surface charge distribution may affect the activity of a positively charged EAD. Thus, we suggest that while charge should continue to be considered for future engineering efforts, it should not be the sole focus of such engineering efforts.Item Characterization of LysBC17, a Lytic Endopeptidase from Bacillus cereus(MDPI, 2019-09-19) Swift, Steven M.; Etobayeva, Irina V.; Reid, Kevin P.; Waters, Jerel J.; Oakley, Brian B.; Donovan, David M.; Nelson, Daniel C.Bacillus cereus, a Gram-positive bacterium, is an agent of food poisoning. B. cereus is closely related to Bacillus anthracis, a deadly pathogen for humans, and Bacillus thuringenesis, an insect pathogen. Due to the growing prevalence of antibiotic resistance in bacteria, alternative antimicrobials are needed. One such alternative is peptidoglycan hydrolase enzymes, which can lyse Gram-positive bacteria when exposed externally. A bioinformatic search for bacteriolytic enzymes led to the discovery of a gene encoding an endolysin-like endopeptidase, LysBC17, which was then cloned from the genome of B. cereus strain Bc17. This gene is also present in the B. cereus ATCC 14579 genome. The gene for LysBC17 encodes a protein of 281 amino acids. Recombinant LysBC17 was expressed and purified from E. coli. Optimal lytic activity against B. cereus occurred between pH 7.0 and 8.0, and in the absence of NaCl. The LysBC17 enzyme had lytic activity against strains of B. cereus, B. anthracis, and other Bacillus species.Item IDENTIFICATION AND ENGINEERING BACTERIOPHAGE ENDOLYSINS FOR INACTIVATION OF GRAM-POSITIVE SPORE-FORMING BACILLI(2018) Etobayeva, Irina V.; Nelson, Daniel C.; Veterinary Medical Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation concentrates on the study of the antibacterial potential of bacteriophage-encoded endolysins derived from phages that infect the Gram-positive Bacillus cereus sensu lato group. Bacteriophage-encoded endolysins are peptidoglycan hydrolases that have been identified as important factors in the phage life cycle. Endolysins are encoded by phage late genes during an intracellular infection cycle to lyse the bacterial cell wall from within and allow phage progeny release. Endolysins derived from phages of Gram-positive bacterial hosts are equipped with an enzymatic domain that hydrolyzes conserved covalent bonds in bacterial peptidoglycan, and a cell wall binding domain that ensures proper attachment of endolysins to bacilli. In this study three novel endolysins, PlyP56, PlyN74, and PlyTB40 have been discovered and identified. The biochemical analysis shows that all three endolysins have relatively broad antimicrobial activity against organisms of the B. cereus group with the optimal lytic activity at physiological pH (pH 7.0–8.0), over a broad temperature range (4–55°C), and at low concentrations of NaCl (<50 mM). The domain shuffling engineering studies were undertaken to observe enhancements of bacteriolytic properties of chimeric lysins that retained their specificity to B. cereus species. Finally, these studies have identified a new development in lysis of peptidoglycan of Gram-positive B. cereus group of organisms by phage-encoded endolysins. When grown to stationary phase, bacilli, especially, in overnight cultures become more resistant to lysis despite the evidence that the cell wall domains bind the bacterial surface. In light of these findings, I hypothesize that B. cereus group of species have evolved complex behaviors to interact with phage by modulating surface associated secondary polymers throughout the maturation of the bacilli in order to render them more resistant to the lytic action of phage encoded endolysins, which, contributes to bacterial survival from phage infection.