Our long-term goal is to develop potent agents for prevention and treatment of anthrax and methicillinresistant S. aureus (MRSA) by engineering phage endolysins. PlyG, a lysin of 25 kDa molecular weight encoded by gamma phage. PlyG contains a T7 lysozyme-like catalytic domain capable of hydrolyzing B. anthracis cell wall peptidoglycan, resulting in bacterial cell lysis, attached to a -75 amino acid C- terminal domain. The C-terminal domain is a dimeric carbohydrate recognition module that targets the enzyme specifically to vegetative B. anthracis cells and germinating spores. We hypothesize that full- length PlyG exists in a monomeric inactive state stabilized by specific contacts between the N- and C-terminal domains, and that binding of the C-terminal (regulatory) domain to carbohydrates unique to the B. anthracis cell wall releases the autoinhibitory interaction and promotes formation of the fully active, dimeric PlyG enzyme. Phage endolytic enzymes like PlyG have the potential to serve as novel and powerful antibiotic agents, termed 'enzybiotics'. We broadened this approach to include MRSA by assembling an enzybiotic from other phage lysins with specific S. aureus activity. ClyS is a chimeric protein containing an N-terminal catalytic domain and a C-terminal cell wall targeting domain. The N-terminal catalytic domain is an endopeptidase of 184 amino acids and the C-terminal cell wall targeting domain is 94 residues.
In aim 1, we will determine the NMR structure of full-length (inactive) PlyG to reveal the molecular basis of lysin autoinhibition for this class of antibacterial enzymes, and solve structures of the ClyS lysin and its component domains.
In Aim 2, the basis for specific anthrax and MRSA targeting will be elucidated using NMR to monitor interactions between cell wall components and the PlyG and ClyS binding domains.
Aim 3 will exploit this structural knowledge to engineer isoforms of PlyG and ClyS with enhanced stability in vivo. Because the bacterium must alter the basic construction of the cell wall to evade an enzybiotic, the probability that PlyG- and ClyS- resistant strains will emerge is low. These studies will provide important mechanistic insights into a novel class of antibacterial compounds moving toward clinical application.

Public Health Relevance

Methicillin-resistant S. aureus (MRSA) and anthrax pose serious threats to public health. The enzyme PlyG possesses unique anti- anthrax activity that can be exploited for treating infection and detecting germinating spores. A similar enzyme, ClyS, was engineered to kill MRSA strains. The proposed studies will reveal at a molecular level the manner in which each protein recognizes a specific type of bacteria and kills it by dismantling the cell wall, and enable the development of improved PlyG and ClyS formulations for treatment of bacterial infections in human patients.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Specialized Center--Cooperative Agreements (U54)
Project #
5U54AI057153-08
Application #
8233343
Study Section
Special Emphasis Panel (ZAI1)
Project Start
2011-03-01
Project End
2014-02-28
Budget Start
2011-03-01
Budget End
2012-02-29
Support Year
8
Fiscal Year
2011
Total Cost
$372,362
Indirect Cost
Name
University of Chicago
Department
Type
DUNS #
005421136
City
Chicago
State
IL
Country
United States
Zip Code
60637
Sloup, Rudolph E; Konal, Ashley E; Severin, Geoffrey B et al. (2017) Cyclic Di-GMP and VpsR Induce the Expression of Type II Secretion in Vibrio cholerae. J Bacteriol 199:
Chen, Grischa Y; McDougal, Courtney E; D'Antonio, Marc A et al. (2017) A Genetic Screen Reveals that Synthesis of 1,4-Dihydroxy-2-Naphthoate (DHNA), but Not Full-Length Menaquinone, Is Required for Listeria monocytogenes Cytosolic Survival. MBio 8:
Coulson, Garry B; Johnson, Benjamin K; Zheng, Huiqing et al. (2017) Targeting Mycobacterium tuberculosis Sensitivity to Thiol Stress at Acidic pH Kills the Bacterium and Potentiates Antibiotics. Cell Chem Biol 24:993-1004.e4
Hollands, Andrew; Corriden, Ross; Gysler, Gabriela et al. (2016) Natural Product Anacardic Acid from Cashew Nut Shells Stimulates Neutrophil Extracellular Trap Production and Bactericidal Activity. J Biol Chem 291:13964-73
Kuhn, Misty L; Alexander, Evan; Minasov, George et al. (2016) Structure of the Essential Mtb FadD32 Enzyme: A Promising Drug Target for Treating Tuberculosis. ACS Infect Dis 2:579-591
Duckworth, Benjamin P; Wilson, Daniel J; Aldrich, Courtney C (2016) Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay. Methods Mol Biol 1401:53-61
Park, Sung Ryeol; Tripathi, Ashootosh; Wu, Jianfeng et al. (2016) Discovery of cahuitamycins as biofilm inhibitors derived from a convergent biosynthetic pathway. Nat Commun 7:10710
Agostoni, Marco; Waters, Christopher M; Montgomery, Beronda L (2016) Regulation of biofilm formation and cellular buoyancy through modulating intracellular cyclic di-GMP levels in engineered cyanobacteria. Biotechnol Bioeng 113:311-9
Völlger, Lena; Akong-Moore, Kathryn; Cox, Linda et al. (2016) Iron-chelating agent desferrioxamine stimulates formation of neutrophil extracellular traps (NETs) in human blood-derived neutrophils. Biosci Rep 36:
Le, J; Dam, Q; Schweizer, M et al. (2016) Effects of vancomycin versus nafcillin in enhancing killing of methicillin-susceptible Staphylococcus aureus causing bacteremia by human cathelicidin LL-37. Eur J Clin Microbiol Infect Dis 35:1441-7

Showing the most recent 10 out of 513 publications