Cell Lytic Enzymes
Cell lytic enzymes are natural antimicrobials that depolymerize cell wall peptidoglycan of target bacterial cells and cause rapid cell lysis. 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 towards 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, being highly active against the targeted bacteria, are exquisitely selective with narrow killing spectra, and are environmental friendly. In addition, it is exceptionally difficult for targeted bacteria to gain resistance against lytic enzymes.
Figure CL-1. Different types of cell lytic enzymes shown in a single cell.
Bacillus anthracis is the causative agent of anthrax. In the B. anthracis strain Ames genome, we identified a cell lytic enzyme, termed AmiBA2446. Through biochemical analyses, we found that AmiBA2446 exists as a homodimer and is an N-acetylmuramoyl-L-alanine amidase. This enzyme shows significant activity against B. anthracis and B. cereus strains, and is also active against B. anthracis spores pretreated with germinants1. Interestingly, the activity of AmiBA2446 is dependent on cell age, which is also observed for another B. anthracis lytic enzyme, PlyPH. This phenomenon is hypothesized to be related to the distinct binding affinity of the enzyme binding domain to the cell surface. Indeed, we have also identified Plyβ, which has a homologous catalytic domain but a different binding domain compared with PlyPH, has strong binding to the B. anthracis cell surface at all the growth stages, and its activity is independent of cell growth (Figure CL-2)2.
Figure CL-2. Activity of PlyPH and Plyβ as a function of bacterial age. 1.5 μM PlyPH rapidly lost killing efficacy as the bacterial culture aged (a), whereas 1.5 μM Plyβ (b) was active against both stationary and rapidly proliferating cells. The results are representative of three independent experiments2.
Anti-Mycobacterium (Tuberculosis) Enzymes
Mycobacterium tuberculosis is the etiological agent of tuberculosis. The emergence of drug resistant mycobacteria and the lack of effective therapies against M. tuberculosis infections is a global health problem. We identified an enzyme, a LysB homolog from mycobacteriophage Bxz2, and compared its activity with a previously reported LysB from mycobacteriophage Ms6. While both enzymes are equally active in inhibiting the growth of Mycobacterium smegmatis, a common non-pathogenic substitute of M. tuberculosis, Bxz2 LysB has a 10-fold higher esterase activity but a significantly lower lipolytic activity compared to that of Ms6 LysB, and the enzyme activity is enhanced in the presence of surfactants such as Tween 80 and Triton X-1003.
Figure CL-3. (Left) LysB cleavage of ester bond between mycolic acids and arabinogalactan. (Right) Growth inhibition of M. smegmatis with LysB: In presence of 0.05% (v/v) Tween 80.
Using in silico analysis in the genome of C. difficile with PlyG serving as a probe, we identified CDG with a catalytic domain homologous to that of PlyG, and CD11 with a binding domain highly similar to that of CDG. The heterologously expressed CDG and CD11 are highly active against a wide range of clinical isolates of C. difficile, with > 3-log killing in 5 h treatment in buffer (Figure CL-4). LC-MS analysis indicates that both enzymes are N-acetylmuramoyl-L-alanine amidase4. 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. We took CD11 as an example, and found that the inability of this enzyme to function in rich media is caused by the low binding of the enzyme to the cell surface. By partially blocking the biosynthesis of wall teichoic acids (WTAs) in C. difficile using tunicamycin, the enzyme binging capability and cell killing activity were restored greatly5. This work provides an evidence of the role of WTAs in regulating cell responses and resistance to macromolecular antimicrobials (Figure CL-5). The lytic enzymes identified in this work could be encapsulated in enteric polymers, thereby protecting the lytic enzymes from the acidic environment in the stomach while delivering the enzymes to the colon to target C. difficile. Other approaches that could be explored in the future include the administration of probiotics engineered to express these lytic enzymes for the treatment of C. difficile associated diarrhea (CDAD). Such approaches may be very useful in combating the growing concern posed by bacterial pathogens such as C. difficile.
Figure CL-4. 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-logs4.
Figure CL-5. Schematic illustration of the role of wall teichoic acids in regulating cellular responses to CD11 under different nutrient conditions5.
Exploiting Cell Lytic Specificity
Targeting infectious bacterial pathogens is important for reducing the evolution of antibiotic-resistant bacteria and preserving the endogenous human microbiome. Cell lytic enzymes including bacteriophage endolysins, autolysins, and peptidoglycan targeting enzymes are potentially useful alternatives to antibiotics, and genetic information of numerous cell lytic enzymes is currently available. Yet, the identification of their antimicrobial function and specificity has been limited due to time-intensive techniques to identify their specific targets.
We have used our cell chip platform to assess rapidly the function of diverse genes that are suggestive of encoding cell lytic enzymes (Figure CL-6). Such a platform can then be used to quantify antimicrobial activity in high-throughput by detecting bacterial cell growth in volumes as low as 40 nL in alginate gel spots on micropillar supports. To expand the capacity of this platform, we demonstrated that cell-free protein synthesis could be achieved in a complementary microwell chip. The combination of the chip-based antimicrobial assay with parallel cell-free protein synthesis enabled the rapid identification and evaluation of species-specific antimicrobial function of 11 genes encoding known and putative cell lytic enzymes6 (Figure CL-7).
Figure CL-6. Schematic of the bacterial cell chip for high-throughput functional analysis of genes encoding cell lytic enzymes.
Figure CL-7. Scanned image of the cell viability of four different pathogens treated with 11 cell lytic enzymes and a negative control (GFP) synthesized by in vitro TNT in the chip. The region of significant growth inhibition in the scanned image is highlighted in yellow boxes.
Emerging antibiotic resistance in pathogenic bacteria has been linked to generalized antimicrobial treatment strategies, primarily overuse of broad spectrum antibiotics. This has resulted in an emergent need for pathogen specific antimicrobial treatment. Species specific cell lytic enzymes have been shown to have specific lytic activity against specific bacteria, including in some cases, their individual species, thereby making them potential antimicrobial agents for therapeutic applications. Moreover, through careful choice of cell lytic enzymes, it is possible to control the composition of microbiomes in various environments, e.g., human gut and skin, environmental infrastructures, etc. Our current work is a preliminary step towards understanding the remodeling of the human skin microbiome in vitro caused by antimicrobial treatment with specific lytic enzymes (Figure CL-8). A typical skin bacterial community consists of bacteria belonging to Staphylococcus, Micrococcus, Propinonibacteria, and Bacillus species. The health of the microbiome depends on the complex interaction of commensal community members. We are using specific cell lytic enzymes to remove S. aureus (including MRSA strains) and B. cereus in an in vitro model microbiome. We are also examining the effect of selective pathogen removal on the dynamic responses of the microbiome with a goal of understanding microbiome resilience and its impact on human health.
Figure CL-8. Selective remodeling of skin microbiome using cell lytic enzymes.
Certain Gram-positive bacteria, such as Bacillus and Clostridia, form spores (also termed endospores) under nutrient starvation through a process called sporulation. Spores contain multiple layers – (from the outside-in) exosporium, coat, cortex, and core. As a result, spores are highly resistant to chemical, biological and mechanical stresses. When environmental conditions become favorable for cell growth, spores rapidly undergo germination, and within minutes can grow into vegetative, metabolically active cells.
Through the life cycles of sporulation and germination, the bacterial species can tolerate harsh treatments, such as the normal disinfection procedures, and regain virulence at a later time. To overcome the challenge of spore decontamination, we developed a three-step strategy that potentially can be applied to eradicate spores and spore-forming bacteria (Figure CL-9)7. In the first step, we treat spores with proteases for the partial degradation and permeabilization of the spore coat, which serves as a barrier to large molecules. The resulting partially decoated spores are then exposed to germination enzymes, or cortex lytic enzymes (CLEs), for the depolymerization of cortex peptidoglycan and transition into vegetative cells. In the last step, the germinating or germinated spores can be efficiently killed by cell lytic enzymes. Using B. cereus and B. anthracis spores as model systems, we have shown that spores decoated with proteinase K and subtilisin Calsberg could be germinated by lysozyme and SleB, and were then susceptible to PlyPH7.
Figure CL-9. 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.
A key step in spore killing is the degradation of spore cortex by germination enzymes, and there are two major germination enzymes in B. anthracis and B. cereus; SleB and CwlJ1. We heterologously expressed both enzymes in E. coli. Through liquid chromatography-mass spectrometry analysis of the digested cortical fragments, we determined that CwlJ1 was a thermostable N-acetylmuramoyl-L-alanine amidase. 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 (Figure CL-10). Also, CwlJ1 maintains cortex-binding capability despite the absence of a peptidoglycan binding domain8. The ability of SleB and CwlJ1 to degrade spore cortex is depicted in Figure CL-11. Release of calcium dipicolinic acid (Ca2+-DPA) from the spore occurs upon cortex degradation and can be detected using a fluorescent terbium chloride assay.
Figure CL-10. 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. The supernatant of the final digestion products was analyzed by reversed-phase HPLC-mass spectrometry.
Figure CL-11. Cortex-lytic activity of purified SUMO-CwlJ1, as measured using the terbium fluorescence kinetic assay to monitor Ca2+-DPA release associated with cortex lysis of decoated B. anthracis Sterne spores incubated at 23°C.
- K.K. Mehta, E.E. Paskaleva, S. Azizi-Ghannad, D.J. Ley, M.A. Page, J.S. Dordick, and R.S. Kane (2013), "Characterization of AmiBA2446, a novel bacteriolytic enzyme active against Bacillus species," Appl. Environ. Microbiol. 79, 5899-5906.
- E.E. Paskaleva, R.V. Mundra, K.K. Mehta, R.C. Pangule, X. Wu, W.S. Glatfelter, Z. Chen, J.S. Dordick, and R.S. Kane (2015), "Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface," Biotechnol. Prog. 31, 1487-1493.
- N. Grover, E.E. Paskaleva, K.K. Mehta, J.S. Dordick, and R.S. Kane (2014), "Growth inhibition of Mycobacterium smegmatis by mycobacteriophage-derived enzymes," Enzyme Microb. Technol. 63, 1-6.
- K.K. Mehta, E.E. Paskaleva, X. Wu, N. Grover, R.V. Mundra, K. Chen, Y. Zhang, H. Feng, J.S. Dordick, and R.S. Kane (2016), "Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile," Biotechnol. Bioeng. 113, 2568-2576.
- X. Wu, E.E. Paskaleva, K.K. Mehta, J.S. Dordick, and R.S. Kane (2016), "Wall teichoic acids are involved in the medium-induced loss of function of the autolysin CD11 against Clostridium difficile," Sci. Rep. 6, 35616.
- S.J. Kwon, D. Kim, I. Lee, J. Kim, and J.S. Dordick (2017), "In vitro gene expression-coupled bacterial cell chip for screening species-specific antimicrobial enzymes," Biotechnol. Bioeng. (in press).
- R.V. Mundra, K.K. Mehta, X. Wu, E.E. Paskaleva, R.S. Kane, and J.S. Dordick (2013), “Enzyme-driven bacillus spore coat degradation leading to spore killing”, Biotechnol. Bioeng. 111, 654-663.
- X. Wu, N. Grover, E.E. Paskaleva, R.V. Mundra, M.A. Page, R.S. Kane, and J.S. Dordick (2015), “Characterization of the activity of the spore cortex lytic enzyme CwlJ1,” Biotechnol. Bioeng. 112, 1365-1375.