Elena E. Paskaleva

Enzyme-Based Listericidal Nanocomposites

Cell lytic enzymes represent an alternative to chemical decontamination or use of antibiotics to kill pathogenic bacteria, such as listeria. A number of phage cell lytic enzymes against listeria have been isolated and possess listericidal activity; however, there has been no attempt to incorporate these enzymes onto surfaces. We report three facile routes for the surface incorporation of the listeria bacteriophage endolysin Ply500: covalent attachment onto FDA approved silica nanoparticles (SNPs), incorporation of SNP-Ply500 conjugates into a thin poly(hydroxyethyl methacrylate) film; and affinity binding to edible crosslinked starch nanoparticles via construction of a maltose binding protein fusion. These Ply500 formulations were effective in killing L. innocua (a reduced pathogenic surrogate) at challenges up to 105 CFU/ml both in non-growth sustaining PBS as well as under growth conditions on lettuce. This strategy represents a new route toward achieving highly selective and efficient pathogen decontamination and prevention in public infrastructure.

Enzyme‐driven bacillus spore coat degradation leading to spore killing

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, increasing coat permeability towards cortex lytic enzymes such as lysozyme and SleB, thereby initiating germination. Specifically in the presence of lysozyme, proteinase K resulted in 14‐fold faster enzyme induced germination and exhibited significantly shorter lag times, than spores without protease pretreatment. 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. Biotechnol. Bioeng. 2014;111: 654–663.

Growth inhibition of Mycobacterium smegmatis by mycobacteriophage-derived enzymes.

We report the ability of mycobacteriophage-derived endolysins to inhibit the growth of Mycobacterium smegmatis. We expressed and purified LysB from mycobacteriophage Bxz2 and compared its activity with that of a previously reported LysB from mycobacteriophage Ms6. The esterase activity of Bxz2 LysB with pNP esters was 10-fold higher than that of the previously reported LysB but its lipolytic activity was significantly lower. The presence of surfactant - Tween 80 or Triton X-100 - significantly increased the activity of LysB. Characterization of LysB-treated M. smegmatis cells and LysB-treated purified cell wall by mass spectroscopy confirmed the hydrolytic activity of the enzyme. Both enzymes were equally effective in inhibiting the growth of M. smegmatis, demonstrating their potential as bacteriostatic agents.

Characterization of AmiBA2446, a Novel Bacteriolytic Enzyme Active against Bacillus Species

ABSTRACT 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. In this work, we performed in silico analysis of the genome of Bacillus anthracis strain Ames, using a consensus binding domain amino acid sequence as a probe, and identified a novel lytic enzyme that we termed AmiBA2446. This enzyme exists as a homodimer, as determined by size exclusion studies. It possesses N-acetylmuramoyl-l-alanine amidase activity, as determined from liquid chromatography-mass spectrometry (LC-MS) analysis of muropeptides released due to the enzymatic digestion of peptidoglycan. Phylogenetic analysis suggested that AmiBA2446 was 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 Bacillus cereus 4342, a surrogate for B. anthracis. 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 also stable in solution, retaining its activity after 4 months of storage at room temperature.

Characterization of the activity of the spore cortex lytic enzyme CwlJ1

The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of Bacillus anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report 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. CwlJ1 was active on both decoated spores and spore cortical fragments. 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. Unlike most amidases, CwlJ1 did not appear to contain a divalent cation, as it retained its activity in the presence of EDTA. This study shines some light on the mechanism of spore germination, and provides increased insight into the development of sporicidal enzyme systems for decontamination of B. anthracis and other related bacteria. Biotechnol. Bioeng. 2015;112: 1365–1375.

Newly identified bacteriolytic enzymes that target a wide range of clinical isolates of Clostridium difficile.

Clostridium difficile has emerged as a major cause of infectious diarrhea in hospitalized patients, with increasing mortality rate and annual healthcare costs exceeding

Nanotechnologies for biosensor and biochip

3 billion. Since C. difficile infections are associated with the use of antibiotics, there is an urgent need to develop treatments that can inactivate the bacterium selectively without affecting commensal microflora. Lytic enzymes from bacteria and bacteriophages show promise as highly selective and effective antimicrobial agents. These enzymes often have a modular structure, consisting of a catalytic domain and a binding domain. In the current work, using consensus catalytic domain and cell‐wall binding domain sequences as probes, we analyzed in silico the genome of C. difficile, as well as phages infecting C. difficile. We identified two genes encoding cell lytic enzymes with possible activity against C. difficile. We cloned the genes in a suitable expression vector, expressed and purified the protein products, and tested enzyme activity in vitro. These newly identified enzymes were found to be active against C. difficile cells in a dose‐dependent manner. We achieved a more than 4‐log reduction in the number of viable bacteria within 5 h of application. Moreover, we found that the enzymes were active against a wide range of C. difficile clinical isolates. We also characterized the biocatalytic mechanism by identifying the specific bonds cleaved by these enzymes within the cell wall peptidoglycan. These results suggest a new approach to combating the growing healthcare problem associated with C. difficile infections. Biotechnol. Bioeng. 2016;113: 2568–2576.

Binding domains of Bacillus anthracis phage endolysins recognize cell culture age-related features on the bacterial surface

1Department of BioNano Technology, Gachon University, 1342 Seongnamdae-ro, Sujeong-gu, Seongnam, Gyeonggi 461-701, Republic of Korea 2Department of Chemistry, Chung-Ang University, 84 Heukseok-ro, Dongjak-gu, Seoul 06974, Republic of Korea 3Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180, USA 4College of Life Information Science and Instrument Engineering, Hangzhou Dianzi University, Xiasha, Hangzhou, Zhejiang 310018, China 5Departement Materiaalkunde (MTM), KU Leuven, Kasteelpark Arenberg 44, Bus 2450, 3001 Leuven, Belgium 6Pivotal Purification Process Development, Amgen, Inc., Cambridge, MA 02142, USA

Wall Teichoic Acids Are Involved in the Medium-Induced Loss of Function of the Autolysin CD11 against Clostridium difficile.

Bacteriolytic enzymes often possess a C‐terminal binding domain that recognizes specific motifs on the bacterial surface and a catalytic domain that cleaves covalent linkages within the cell wall peptidoglycan. PlyPH, one such lytic enzyme of bacteriophage origin, has been reported to be highly effective against Bacillus anthracis, and can kill up to 99.99% of the viable bacteria. The bactericidal activity of this enzyme, however, appears to be strongly dependent on the age of the bacterial culture. Although highly bactericidal against cells in the early exponential phase, the enzyme is substantially less effective against stationary phase cells, thus limiting its application in real‐world settings. We hypothesized that the binding domain of PlyPH may differ in affinity to cells in different Bacillus growth stages and may be primarily responsible for the age‐restricted activity. We therefore employed an in silico approach to identify phage lysins differing in their specificity for the bacterial cell wall. Specifically we focused our attention on Plyβ, an enzyme with improved cell wall‐binding ability and age‐independent bactericidal activity. Although PlyPH and Plyβ have dissimilar binding domains, their catalytic domains are highly homologous. We characterized the biocatalytic mechanism of Plyβ by identifying the specific bonds cleaved within the cell wall peptidoglycan. Our results provide an example of the diversity of phage endolysins and the opportunity for these biocatalysts to be used for broad‐based protection from bacterial pathogens.

Evaluation of Potential Genotoxicity of HIV Entry Inhibitors Derived from Natural Sources

Bacterial lysins are potent antibacterial enzymes with potential applications in the treatment of bacterial infections. Some lysins lose activity in the growth media of target bacteria, and the underlying mechanism remains unclear. Here we use CD11, an autolysin of Clostridium difficile, as a model lysin to demonstrate that the inability of this enzyme to kill C. difficile in growth medium is not associated with inhibition of the enzyme activity by medium, or the modification of the cell wall peptidoglycan. Rather, wall teichoic acids (WTAs) appear to prevent the enzyme from binding to the cells and cleaving the cell wall peptidoglycan. By partially blocking the biosynthetic pathway of WTAs with tunicamycin, cell binding improved and the lytic efficacy of CD11 was significantly enhanced. This is the first report of the mechanism of lysin inactivation in growth medium, and provides insights into understanding the behavior of lysins in complex environments, including the gastrointestinal tract.