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ANTIMICROBIAL SYSTEMS

We are exploiting nature’s biocatalytic defense mechanisms to combat microbial and viral infections, overcome bacterial resistance mechanisms, and applying our approach to surfaces within common infrastructures, including hospitals, schools, food processing facilities, etc. Our approach involves two classes of enzymes; peptidoglycan hydrolases and oxidative biocatalysts. The former are lytic enzymes, which are extremely selective and do not require reagents apart from water to act. The oxidative enzymes, including oxidases, peroxidases and perhydrolases, are generally nonselective and require addition of reagents to catalyze their microbicidal activity. In both classes, these enzymes can be used in their soluble form or embedded into materials that can be used to coat surfaces and kill bacteria on contact.

Cell Lytic Enzymes – Fundamentals and Applications

Broad-spectrum antibiotics indiscriminately kill bacteria, removing non-pathogenic microorganisms and leading to evolution of antibiotic resistant strains. Specific antimicrobials that could selectively kill pathogenic bacteria without targeting other bacteria in the natural microbial community or microbiome may be able to address this concern. Cell lytic enzymes are natural antimicrobial agents that depolymerize cell wall peptidoglycans of target bacteria and cause rapid cell lysis [Wu et al. Annu. Rev. Chem. Biomol. Eng. 8, 87-113 (2017)]. There are four types of cell lytic enzymes – endolysins, autolysins, virion-associated lysins (VALs), and bacteriolysins (Figure CL-1). Endolysins and VALs are bacteriophage-associated lytic enzymes. VALs act externally at the beginning of phage infection for the local decomposition of target cell wall and injection of phage genome into the host cell, whereas endolysins degrade cell wall from inside the host cell at the end of the infection cycle for release of phage progeny. Autolysins and bacteriolysins are generated by bacterial cells. While autolysins function on the producing cells and take part in cell wall remodeling and cell division, bacteriolysins lyse the species that compete for nutrients and growth space, and hence protect the producing cells. These enzymes are exquisitely selective with narrow killing spectra and are environmentally friendly. In addition, it is exceptionally difficult for targeted bacteria to gain resistance against lytic enzymes.

 

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Figure CL-1.  Different types of cell lytic enzymes with actions against a microbial target [Wu et al. Annu. Rev. Chem. Biomol. Eng. 8, 87-113 (2017)].

We are discovering and mechanistically characterizing a wide range of lytic enzymes that target key human pathogens. In addition to identifying new enzymes and uncovering enzyme mechanisms, we are exploiting these enzymes for applications in therapeutics, infrastructure decontamination and pathogen detection.

Threrapeutics – Anti-Clostridium Enzymes 

Clostridium difficile (Cdiff) has emerged as a major hospital-acquired infection, which results in severe morbidity and mortality. Using in silico analysis in the genome of C. difficile with PlyG serving as a probe, we identified the enzyme CDG with a catalytic domain homologous to that of PlyG, and CD11 with a binding domain highly similar to that of CDG [Mehta et al. Biotechnol. Bioeng. 113, 2568-2576 (2016)]. The heterologously expressed CDG and CD11 are active against a wide range of clinical isolates of C. difficile, with > 3-6 log kill in 5 h treatment in buffer (Figure CL-2). LC-MS analysis indicates that both enzymes are N-acetylmuramoyl-L-alanine amidases. An interesting observation is that although these enzymes are highly active in buffer, they are almost inactive in rich media that support cell growth and metabolism. In this light, we have elucidated the role of cell wall teichoic acids (WTAs) in regulating cell responses and resistance to macromolecular antimicrobials (Figure CL-3). Specifically, by partially blocking the biosynthesis of WTAs in C. difficile using tunicamycin, enzyme binding to the cell and cell killing activity were largely restored [Wu et al. Sci. Rep. 6, 35616 (2016)]. Lytic enzymes could be encapsulated in enteric polymers, thereby protecting them from the acidic environment of the stomach while delivering the enzymes to the intestine and colon to target C. difficile.

 

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Figure CL-2. Enzyme activity against clinical isolates of C. difficile for CD11 (light grey), CDG (dark grey), and PBS (black). Absence of gray and/or white bars for some clinical isolates indicates no surviving bacterial cells and corresponds to a reduction in cell viability of more than 5-logs [Mehta et al. Biotechnol. Bioeng. 113, 2568-2576 (2016)].

 

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Figure CL-3. Schematic illustration of the role of wall teichoic acids in regulating cellular responses to CD11 under different nutrient conditions [Wu et al. Sci. Rep. 6, 35616 (2016)].

 

Infrastructure Decontamination-Anti-Bacillus Enzymes

There continues to be a need for developing efficient and environmentally friendly treatments for Bacillus anthracis, the causative agent of anthrax. One emerging approach for inactivation of vegetative B. anthracis is the use of bacteriophage endolysins or lytic enzymes encoded by bacterial genomes (autolysins) with highly evolved specificity toward bacterium-specific peptidoglycan cell walls. We performed in silico analysis of the genome of B. anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446 (Figure CL-4) [Mehta et al. Appl. Environ. Microbiol. 79, 5899-5906 (2013)]. This enzyme exists as a homodimer, as determined by size exclusion studies, and possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from LC-MS analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 is an autolysin of bacterial origin. We characterized the effects of enzyme concentration and phase of bacterial growth on bactericidal activity and observed close to a 5-log reduction in the viability of cells of B. cereus 4342, a surrogate for B. anthracis (Figure CL-5). We further tested the bactericidal activity of AmiBA2446 against various Bacillus species and demonstrated significant activity against B. anthracis and B. cereus strains. We also demonstrated activity against B. anthracis spores after pretreatment with germinants. AmiBA2446 enzyme was highly stable in solution, retaining its activity after 4 months of storage at room temperature.

 

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 Figure CL-4. Bioinformatics-based discovery of new bacterial autolysin, AmiBa2446. The modularity of Gram-positive cell lytic enzymes was exploited by searching the bacillus genome for protein sequences that have similar binding domains to that of PlyPH (an anti-bacillus phage endolysin). This resulted in identification of several candidate putative anti-bacillus enzymes, including AmiBa2446 [Mehta et al. Appl. Environ. Microbiol. 79, 5899-5906 (2013)].

 

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Figure CL-5. Biocatalytic properties of AmiBA2446. Upper left, purification from E. coli cell lysate following cloning the gene and expressing the enzyme. Upper right, activity of AmiBa2446 against B. cereus in PBS after 3 h. Lower left; stability of AmiBa2446 during storage in PBS and room temperature. Lower right, characterization of AmiBa2446 as an amidase [Mehta et al. Appl. Environ. Microbiol. 79, 5899-5906 (2013)].

   

The Bacillus spore coat confers chemical and biological resistance, thereby protecting the core from harsh environments. The primarily protein-based coat consists of recalcitrant protein crosslinks that endow the coat with such functional protection. Proteases are present in the spore coat, which play a putative role in coat degradation in the environment. However, these enzymes are poorly characterized. Nonetheless given the potential for proteases to catalyze coat degradation, we screened 10 commercially available proteases for their ability to degrade the spore coats of B. cereus and B. anthracis. Proteinase K and subtilisin Carlsberg, for B. cereus and B. anthracis spore coats, respectively, led to a morphological change in the otherwise impregnable coat structure [Mundra et al. Biotechnol. Bioeng. 111, 654-663 (2013)], increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination (Figure CL-6). Furthermore, the germinated spores were shown to be vulnerable to a lytic enzyme (PlyPH) resulting in effective spore killing. The spore surface in response to proteolytic degradation was probed using scanning electron microscopy (SEM), which provided key insights regarding coat degradation. The extent of coat degradation and spore killing using this enzyme-based pretreatment approach is similar to traditional, yet far harsher, chemical decoating methods that employ detergents and strong denaturants. Thus, the enzymatic route reduces the environmental burden of chemically mediated spore killing and demonstrates that a mild and environmentally benign biocatalytic spore killing is achievable. 

   

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Figure CL-6. Pathway to kill bacterial spores using lytic enzymes. Enzymatically decoated spores germinated by germination enzymes through degradation of the spore cortex grow into vegetative cells, which are susceptible to cell lytic enzymes for cell wall degradation and cell lysis [Mundra et al. Biotechnol. Bioeng. 111, 654-663 (2013)]. The SEM images show before (rough surface) and after (smooth surface) proteolytic permeabilization of the spore coat.

 

The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of B. anthracis spores. However, the specific function and catalytic activity of CwlJ1 has remained elusive. We have reported for the first time a detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B. anthracis when added exogenously (Figure CL-7) [Wu et al. Biotechnol. Bioeng. 112, 1365-1375 (2015)]. CwlJ1 was active on both decoated spores and spore cortical fragments. Through LC-MS analysis of the digested cortical fragments, we determined that CwlJ1 was a thermostable N-acetylmuramoyl-L-alanine amidase. As a result of this study, insight was gleaned into the mechanism of spore germination and may enable development of sporicidal enzyme systems for decontamination of B. anthracis and other spore-forming bacteria.


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Figure CL-7. Simultaneous and sequential digestion of B. anthracis spore cortical fragments with CwlJ1 and lysozyme. Schematic of double digestion (crosslinking was omitted for clarity). Before the addition of the second enzyme, the digestion products of the first round of reaction were heated at 75°C for 30 min. The supernatant of the final digestion products was analyzed by reversed-phase LC-MS. CwlJ1 mainly recognized large segments of glycan chains in the cortex instead of the minimal structural unit tetrasaccharide, with specificity for muramic acid-δ-lactam-containing glycan chains and preference for the tetrapeptide side chain [Wu et al. Biotechnol. Bioeng. 112, 1365-1375 (2015)].

 

Exploiting Cell Wall Binding Domains – Expanding the Breadth of Lytic Enzymes

The lytic enzymes that target Gram-positive bacteria nearly universally have a two-domain structure, with the N-terminal catalytic domain bound through a short linker to a cell wall binding domain. We have exploited this modularity in designing new enzyme systems. We have exploited the extraordinary specificity of lytic enzyme cell wall binding domains (CBD) when coupled with silver nanoparticles to kill target bacteria (Figure CL-8) [Kim et al. ACS Appl. Mater. Interfaces 10, 13,317-13,324 (2018)]. As a relevant example, CBDBA (binding domain from Bacillus anthracis) selectively bound to B. anthracis in a mixture with B. subtilis, as well as in a mixture with Staphylococcus aureus. As a result, the nonselective antimicrobial silver was converted into a highly selective antimicrobial targeting organisms that are bound selectively by a specific CBD. This new biologically-assisted hybrid strategy, therefore, has the potential to provide selectivedecontamination of pathogenic bacteria with minimal impact on normal microflora.

 

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Figure CL-8. Schematic representation of cell wall binding domain-fluorescent protein-silver binding peptide complexation with AgNPs for selective recognition and specific killing of target pathogenic bacteria; cell wall binding domain CBDSA against S. aureus and CBDBA against B. anthracis. Antimicrobial Activity of various CBD-AgNP hybrids. (a) Inhibitory activity of EGFP-BP-AgNPs and CBDBA-mRUBY-BP-AgNPs on B. anthracis cell growth on BHI agar plate; (b) CFU assay showing dose-dependent growth inhibition of B. anthracis; (c) Effects of EGFP-BP-AgNPs and CBDBA-mRUBY-BP-AgNPs inhibition of S. aureus cell growth on BHI agar plate; (d) CFU assay showing dose-dependent growth inhibition of S. aureus [Kim et al. ACS Appl. Mater. Interfaces 10, 13,317-13,324 (2018)].

 

The modular nature of cell lytic enzymes can be exploited through unique assemblies. Cell lytic enzymes consist of catalytic and cell wall binding domains, which can be swapped among those of other lytic enzymes to produce unnatural chimeric enzymes. We have shown that microbially-generated biotinylated catalytic domains (CD) and cell wall binding domains (CBD) from the bacteriocin lysostaphin (Lst) and a putative autolysin from Staphylococcus aureus (SA1) can be assembled with streptavidin to form chimeric lytic enzymes

 

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 Figure CL-9. (A) Vector construction for enhanced green fluorescent protein (EGFP)-cell wall binding domain (CBDS) from SA1 and cell wall binding domain (CBDL) from lysostaphin fusions with Avi Tag at the N-terminus. (B) Vector construction for the CDS from SA1 and CDL from lysostaphin with Avi Tag at the Cā€terminus. (C) Schematic representation of the modular assembly of recombinant catalytic domains (CDs) and cell wall binding domains (CBDs) with a biotinylated on streptavidin. Blue and red colors indicate catalytic and cell wall binding domains of lysostaphin, respectively, and magenta and brown colors indicate catalytic and cell wall binding domains of SA1, respectively. Gray color indicates linker regions. Cyan color indicates streptavidin.

 

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Figure CL-10. Bactericidal effects of CDL and CBDS on streptavidin versus Lst-SA and free Lst:  0.5 μM CDL-CBDS (3:1) on streptavidin, 0.5 μM C-terminal biotinylated Lst on streptavidin, and 0.5 μM free Lst were incubated together with Staphylococcus aureus (106 CFU/mL), respectively. Control indicates incubation without enzyme construct in PBS. Bacterial killing was determined at 3 h based on a CFU count assay. ** indicates no colonies were detected. The values were plotted with error bars representing standard deviation from triplicate assays.

 

Collaborators:

Ravi Kane - Georgia Tech

Jungbae Kim - Korea University

Cynthis Collins - Rensselaer Polytechnic Institute