Bacteriolytic Enzymes Research Articles

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

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.

Sweetness characterization of recombinant human lysozyme.

Lysozyme, a bacteriolytic enzyme, is widely distributed in nature and is a component of the innate immune system. It is established that chicken egg lysozyme elicits sweetness. However, the sweetness of human milk lysozyme, which is vital for combating microbial infections of the gastrointestinal tract of breast-fed infants, has not been characterized. This study aimed to assess the elicitation of sweetness using recombinant mammalian lysozymes expressed in Pichia pastoris. Recombinant human lysozyme (h-LZ) and other mammalian lysozymes of mouse, dog, cat and bovine milk elicited similar sweetness as determined using a sensory test, whereas bovine stomach lysozyme (bs-LZ) did not. Assays of cell cultures showed that h-LZ activated the human sweet taste receptor hT1R2/hT1R3, whereas bs-LZ did not. Point mutations confirmed that the sweetness of h-LZ was independent of enzyme activity and substrate-binding sites, although acidic amino acid residues of bs-LZ played a significant role in diminishing sweetness. Therefore, we conclude that elicitation of sweetness is a ubiquitous function among all lysozymes including mammalian lysozymes. These findings may provide novel insights into the biological implications of T1R2/T1R3-activation by mammalian lysozyme in the oral cavity and gastrointestinal tract. However, the function of lysozyme within species lacking the functional sweet taste receptor gene, such as cat, is currently unknown.

Antibacterial metabolites and bacteriolytic enzymes produced by Bacillus pumilus during bacteriolysis of Arthrobacter citreus.

The marine isolate Bacillus pumilus SBUG 1800 is able to lyse living cells of Arthrobacter citreus on solid media as well as pasteurized A. citreus cells in liquid mineral salt medium. The cultivation of B. pumilus in the presence of pasteurized A. citreus is accompanied by an enhanced production of 2,5-diketopiperazines (DKPs). DKPs inhibit bacterial growth, but do not seem to cause bacteriolysis. This study shows that B. pumilus also lyses living cells of A. citreus in co-culture experiments as an intraguild predator, even if the inoculum of B. pumilus is low. In order to characterize the bacteriolytic process, more precisely changes in the extracellular metabolome and proteome have been analyzed under different culture conditions. Besides the known DKPs, a number of different pumilacidins and bacteriolytic enzymes are produced. Two lipopeptides with [M + H](+) = 1008 and [M + H](+) = 1022 were detected and are proposed to be pumilacidin H and I. While the lipopeptides lyse living bacterial cells in lysis test assays, a set of extracellular enzymes degrades the dead cell material. Two of the cell wall hydrolases involved have been identified as N-acetylmuramoyl-L-alanine amidase and beta-N-acetylglucosaminidase. These findings together with electron microscopic and cell growth monitoring during co-culture experiments give a detailed view on the bacteriolytic process.

Multistage selection of soil actinomycete Streptomyces albus as a producer of antimicrobial substances

Streptomycetes are known as the producers of various bioactive substances widely used in agriculture and many branches of industry. Different biotechnological methods and approaches are used to increased productivity of streptomycetes. In the present study, S. albus strains were subjected to mutagenesis associated with multistage selection and mutant strain with an increase of 3.5 times bacteriolytic enzyme complex production was obtained. Analysis of cell survival rate, antimicrobial and lytic activity as well as enzyme complex production in Streptomyces exposed to the various mutagen combinations (MNU, UV-irradiation, HNO2) were carried out. Mutant strains were characterized by high bacteriolytic enzyme activity and significant antibiotic activity against Kocuria (Micrococcus) variants and Candida albicans. To estimate genomic changes caused by different mutagens, PCR with primers to short nucleotide repeats was performed and amplicons distinguishing parental and mutant strains were defined. The efficacy of the sequential mutagenic treatment for the hyper-producer obtaining as well as the relationship between the mutagen nature and the genomic variability has been shown. The results obtained in our research can be used and applied to the others Streptomyces species.

Structure-based redesign of lysostaphin yields potent antistaphylococcal enzymes that evade immune cell surveillance

Staphylococcus aureus infections exert a tremendous burden on the health-care system, and the threat of drug-resistant strains continues to grow. The bacteriolytic enzyme lysostaphin is a potent antistaphylococcal agent with proven efficacy against both drug-sensitive and drug-resistant strains; however, the enzyme’s own bacterial origins cause undesirable immunogenicity and pose a barrier to clinical translation. Here, we deimmunized lysostaphin using a computationally guided process that optimizes sets of mutations to delete immunogenic T cell epitopes without disrupting protein function. In vitro analyses showed the methods to be both efficient and effective, producing seven different deimmunized designs exhibiting high function and reduced immunogenic potential. Two deimmunized candidates elicited greatly suppressed proliferative responses in splenocytes from humanized mice, while at the same time the variants maintained wild-type efficacy in a staphylococcal pneumonia model. Overall, the deimmunized enzymes represent promising leads in the battle against S. aureus.

Fusion with a cell wall binding domain renders autolysin LytM a potent anti-Staphylococcus aureus agent.

Despite intense efforts by the medical and pharmaceutical communities, Staphylococcus aureus continues to be a pervasive pathogen that causes a myriad of diseases and a high level of morbidity and mortality among infected patients. Thus, discovering or designing novel therapeutics able to kill both drug-resistant and drug-sensitive S. aureus remains a top priority. Bacteriolytic enzymes, mostly from phage, have shown great promise in preclinical studies, but little consideration has been given to cis-acting autolytic enzymes derived from the pathogen itself. Here, we use the S. aureus autolysin LytM as a proof of principal to demonstrate the antibacterial potential of endogenous peptidoglycan-degrading enzymes. While native LytM is only marginally bactericidal, fusion of LytM to the lysostaphin cell wall binding domain enhances its anti-staphylococcal activity approximately 540-fold, placing it on par with many phage lysins currently in preclinical development. The potential to therapeutically co-opt a pathogen's endogenous peptidoglycan recycling machinery opens the door to a previously untapped reservoir of antibacterial drug candidates.

The draft genome, transcriptome, and microbiome of Dermatophagoides farinae reveal a broad spectrum of dust mite allergens

A sequenced house dust mite (HDM) genome would advance our understanding of HDM allergens, a common cause of human allergies. We sought to produce an annotated Dermatophagoides farinae draft genome and develop a combined genomic-transcriptomic-proteomic approach for elucidation of HDM allergens. A D farinae draft genome and transcriptome were assembled with high-throughput sequencing, accommodating microbiome sequences. The allergen gene structures were validated by means of Sanger sequencing. The mite's microbiome composition was determined, and the predominant genus was validated immunohistochemically. The allergenicity of a ubiquinol-cytochrome c reductase binding protein homologue was evaluated with immunoblotting, immunosorbent assays, and skin prick tests. The full gene structures of 20 canonical allergens and 7 noncanonical allergen homologues were produced. A novel major allergen, ubiquinol-cytochrome c reductase binding protein-like protein, was found and designated Der f 24. All 40 sera samples from patients with mite allergy had IgE antibodies against rDer f 24. Of 10 patients tested, 5 had positive skin reactions. The predominant bacterial genus among 100 identified species was Enterobacter (63.4%). An intron was found in the 13.8-kDa D farinae bacteriolytic enzyme gene, indicating that it is of HDM origin. The Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed a phototransduction pathway in D farinae, as well as thiamine and amino acid synthesis pathways, which is suggestive of an endosymbiotic relationship between D farinae and its microbiome. An HDM genome draft produced from genomic, transcriptomic, and proteomic experiments revealed allergen genes and a diverse endosymbiotic microbiome, providing a tool for further identification and characterization of HDM allergens and development of diagnostics and immunotherapeutic vaccines.

The transcriptional signatures of Sodalis glossinidius in the Glossina palpalis gambiensis flies negative for Trypanosoma brucei gambiense contrast with those of this symbiont in tsetse flies positive for the parasite: Possible involvement of a Sodalis-hosted prophage in fly Trypanosoma refractoriness?

Tsetse flies, such as Glossina palpalis gambiensis, are blood-feeding insects that could be subverted as hosts of the parasite Trypanosoma brucei gambiense: initiated in the tsetse fly mid gut, the developmental program of this parasite further proceeds in the salivary glands. The flies act as vectors of this human-invasive parasite when their salivary glands sustain the generation of metacyclic trypomastigotes, the exclusive morphotypes pre-programmed to further develop in the human individuals. Briefly, once the metacyclic trypomastigotes have been deposited in the skin of humans from whom the parasite-hosting tsetse flies are taking their blood meals, the complex developmental program of this Trypanosoma brucei subspecies can result in a severe disease named sleeping sickness. Unveiling the processes that could prevent, in tsetse flies, the developmental program of T. b. gambiense could contribute reducing the prevalence of the disease.Using a global approach, we were curious to extract transcriptional signatures of Sodalis glossinidius, a symbiont hosted by three distinct groups of tsetse flies. To meet this objective, the transcriptome of S. glossinidius from susceptible and refractory tsetse flies was analyzed at 3, 10 and 20days after flies blood feed on T. b. gambiense-hosting mice. Within this temporal window, 176 trypanosome responsive genes were shown to interact in well-defined patterns making it possible to distinguish flies susceptible to the parasite infection from refractory flies. Among the modulated transcripts in the symbiont population of flies refractory to trypanosome infection many were shown to cluster within the following networks: “lysozyme activity, bacteriolytic enzyme, bacterial cytolysis, cell wall macromolecule catabolic process”. These novel data are further delineated in the following questions: could the activation of prophage hosted by S. glossinidius lead to the release of bacterial agonists that trigger the tsetse fly immune system along a profile that no more allows the parasite developmental program?

Mitrecin A , an endolysin‐like bacteriolytic enzyme from a newly isolated soil streptomycete

An open reading frame with homology to known endolysin genes was identified in the genome of Streptomyces sp. strain 212, which is a newly isolated soil bacterium. The heterologously expressed gene product of this endolysin-like gene, called Mitrecin A, demonstrated bacteriolytic activity against several Gram-negative bacteria. The genome of the bacterial strain was sequenced to draft quality using pyrosequencing followed by genome assembly and gene annotation. Within the sequence, a chromosomally located endolysin-like open reading frame was predicted. The gene product, designated Mitrecin A, was heterologously expressed and isolated from contaminating proteins as a fusion protein to a 6-histidine tag. Mitrecin A consists of 127 amino acids arranged in modular domains of activity. It has an estimated molecular weight of 14·3 kDa and retains sequence homology to the M15C peptidase subfamily of zinc metallocarboxypeptidases. The heat-labile purified recombinant protein has an overall positive charge, has optimal catalytic activities at 26°C in solution of pH 9 with 1% saline and has bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia.Significance and Impact of the StudyThe gene of a new protein antimicrobial, Mitrecin A, was discovered in the genome of a soil bacterium. The purified recombinant enzyme, resulting from heterologous over expression of the gene, was found to be tolerant of increased pH conditions and to have bacteriolytic activity against Gram-negative bacteria of the medically important genera Aeromonas, Escherichia, Salmonella, Shigella, Vibrio and Yersinia. Characterization of enzymes such as Mitrecin A from previously uncharacterized bacteria provides potential options for new biocontrol agents in medically and economically important applications like therapeutics, disinfectants, food preservatives, agricultural livestock antimicrobials, and inhibitors of biofilm production.

Biofilm-degrading enzymes from Lysobacter gummosus

Biofilm-degrading enzymes could be used for the gentle cleaning of industrial and medical devices and the manufacture of biofilm-resistant materials. We therefore investigated 20 species and strains of the bacterial genus Lysobacter for their ability to degrade experimental biofilms formed by Staphylococcus epidermidis, a common nosocomial pathogen typically associated with device-related infections. The highest biofilm-degradation activity was achieved by L. gummosus. The corresponding enzymes were identified by sequencing the L. gummosus genome. Partial purification of the biofilm-degrading activity from an extract of extracellular material followed by peptide mass fingerprinting resulted in the identification of two peptidases (α-lytic protease and β-lytic metalloendopeptidase) that were predicted to degrade bacterial cell walls. In addition, we identified two isoforms of a lysyl endopeptidase and an enzyme similar to metalloproteases from Vibrio spp. Potential peptidoglycan-binding C-terminal fragments of two OmpA-like proteins also co-purified with the biofilm-degrading activity. The L. gummosus genome was found to encode five isoenzymes of α-lytic protease and three isoenzymes of lysyl endopeptidase. These results indicated that the extracellular digestion of biofilms by L. gummosus depends on multiple bacteriolytic and proteolytic enzymes, which could now be exploited for biofilm control.

Lysostaphin-mediated fragmentation of microbial peptidoglycans for label-free electrochemical impedance immunoanalysis of Staphylococcus aureus

Lysostaphin is a Staphylococcus aureusspecific bacteriolytic enzyme that recognizes and cleaves the pentaglycine linkage of peptidoglycan layers in the cell wall of S. aureus. Its target-specific lytic property to the staphylococcal cell wall has attracted interest for the application of lysostaphin as natural antibiotics against S. aureus. However, studies that consider its applicability in the detection of S. aureus have not been reported yet. Based on this, lysostaphin-mediated peptidoglycan fragmentation principles were implemented in the label-free immunoassay of S. aureus. In this study, peptidoglycan layers of S. aureus were fragmented by lysostaphin and the obtained fragments were quantitatively analyzed by electrochemical impedance spectroscopy (EIS). A linear relationship between the logarithmic value of the S. aureus concentration and the increase in the EIS signal was acquired in a range between 102 and 107 colony-forming units (CFU)/mL. Compared to the whole cell-based analysis, the developed lysostaphin-mediated assay for S. aureus showed a 90% increase in sensitivity because the fragments can interact with the immunoanalytical surface more efficiently than the whole cell. In contrast to previous EIS-based bacterial immunoassays that utilize complex electrodes and/or additional signaling probes such as labeled antibodies for signal amplification, we could detect signals with high sensitivity on conventional electrodes without any signaling probes. In addition, by using the developed method, S. aureus was selectively determined in bacterial samples that also contained 106 CFU/mL of Escherichia coli and Corynebacterium glutamicum.

Outer Membrane Permeabilization Is an Essential Step in the Killing of Gram-Negative Bacteria by the Lectin RegIIIβ

The C-type lectin RegIIIβ can kill certain Gram-positive and Gram-negative bacteria. The susceptibility of S. Typhimurium depends on the bacterial growth phase, i.e., bacteria from the logarithmic growth phase do bind RegIIIβ and are subsequently killed. Lipid A is one of the bacterial targets for RegIIIβ. However, at the molecular level, it is not understood how RegIIIβ interacts with and kills Gram-negative bacteria. Here, we show that RegIIIβ interacts with Gram-negative bacteria in two distinct steps. Initially, it binds to surface-exposed lipid A. The lipid A can be shielded by the O-antigen of lipopolysaccharide (LPS), as indicated by the exquisite susceptibility of wbaP mutants to RegIIIβ-mediated killing. Increased cell viability after incubation with an anti-lipid A antibody also supports this conclusion. This RegIIIβ-binding permeabilizes the outer membrane to hydrophobic dyes like Ethidium bromide or to bulky bacteriolytic enzymes like lysozyme. Conversely, compromising the outer membrane integrity by the mild detergent Triton X-100 enhances the antibacterial effect of RegIIIβ. Based on our observations, we conclude that RegIIIβ interacts with Gram-negative bacteria in two subsequent steps. Initially, it binds to the outer membrane thus leading to outer membrane permeabilization. This initial step is necessary for RegIIIβ to reach a second, still not well understood target site (presumably localized in the periplasm or the cytoplasmic membrane), thereby triggering bacterial death. This provides novel insights into the outer membrane-step of the bactericidal mechanism of RegIIIβ.

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

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.

Skin-associated Bacillus, staphylococcal and micrococcal species from the house dust mite, Dermatophagoides pteronyssinus and bacteriolytic enzymes

Dust mites produce bacteriolytic enzymes, one of which belongs to the NlpC/P60 superfamily comprising bacterial and fungal proteins. Whether this enzyme is derived from the mite or from mite-associated microbes is unclear. To this end, the bacteriology of mites per se, and carpet and mattress dust from a group of asthmatic children and their parents was investigated. Dust from parents' and children's mattresses yielded significantly more colony forming units compared with dust from their corresponding carpets. Zymography demonstrated some dusts contained bacteriolytic enzymes, and in nine of the twelve dust samples from three of five houses examined, a prominent bacteriolytic band was obtained that corresponded to the mite band, although in one home, other lytic bands were detected. Fifty bacterial isolates were obtained from surface-sterilised, commercially obtained Dermatophagoides pteronyssinus. 16S rRNA, tuf and rpoB gene sequencing of nine Gram-positive isolates identified them as Bacillus cereus, B. licheniformis, Staphylococcus aureus, S. epidermidis, S. capitis and Micrococcus luteus, known human skin commensals. 16S rRNA sequence homologies of four of the nine isolates identified as B. licheniformis formed a distinct phylogenetic cluster. All species secreted lytic enzymes during culture although the lytic profiles obtained differed between the rods and the cocci, and none of the bands detected corresponded to those observed in dust or mites. In conclusion, mites harbour a variety of bacterial species often associated with human skin and house dusts contain bacteriolytic enzymes that may be mite-derived. The identification of a novel cluster of B. licheniformis isolates suggests an ecological adaptation to laboratory-reared D. pteronyssinus. It remains to be determined whether the previously described mite-associated 14K lytic enzyme is derived from a microbial source.

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 °C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 °C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 °C, 43.5 °C and 50.2 °C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 °C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 °C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 °C, 43.5 °C and 50.2 °C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

Bacteriolytic activity of human interleukin-2

In this paper we report the discovery of bacteriolytic activity of an immune system cytokine mediator, interleukin-2. Bacteriolytic activity of interleukin-2 was compared with a well-known bacteriolytic enzyme - chicken egg white lysozyme - by monitoring the lysis of the Gram-negative bacterium Escherichia coli, the Gram-positive coccus Micrococcus luteus, and the Gram-positive spore-forming bacillus Bacillus subtilis. It was found that interleukin-2 has greater specificity to the Gram-negative bacterium E. coli than does lysozyme. In contrast to chicken egg white lysozyme, interleukin-2 does not lyse the Gram-positive coccus M. luteus and the Gram-positive spore-forming bacillus B. subtilis. These results give a new understanding of the biological functions of interleukin-2, a regulatory protein that plays a role in oncological and infectious diseases.

Serum Lysozyme (Muramidase) Levels in Intra-Abdominal Abscesses: An Experimental Study.

The role of serum lysozyme in the presence of intra-abdominal sepsis was studied to act as an adjunct to various other modalities used for diagnosing intra-abdominal abscesses. Lysozyme (muramidase) is a bacteriolytic enzyme located within phagocytic cells including leukocytes. We measured serum concentrations of lysozyme by a standard turbidimetric method on three groups of murine models. One group underwent caecal ligation and puncture (CLP), the second group underwent a sham operation, and the third group was used as controls. In mice with intra-abdominal abscesses secondary to caecal ligation and perforation (n = 30), the serum lysozyme levels were increased compared to the sham-operated mice (n = 30) and controls (n = 30) (p = <0.001). In this study, serum lysozyme levels have a high sensitivity and specificity related to the presence of an intra-abdominal abscess in mice. Clinical studies are required to demonstrate its role in humans.

Analysis of bacterial and fungal community structure in replant strawberry rhizosphere soil with denaturing gradient gel electrophoresis

High quality DNA is the basis of analyzing bacterial and fungal community structure in replant strawberry rhizosphere soil with the method of denaturing gradient gel electrophoresis (DGGE). DNA of soil microorganisms was extracted from the rhizosphere soil of strawberries planted in different replanted years (0, two, six and seven), respectively, and crude DNA was purified after extraction. Three methods were established to evaluate the effects of cetyl trimethylammonium bromide (CTAB), polyvinylpolypyrolidone (PVPP), proteinase K and bacteriolytic enzymes on DNA extraction. DNA fragments above 23 kb in size were isolated well by method 1 (1% CTAB, proteinase K, no PVPP, no bacteriolytic enzyme) and method 3 (no CTAB, no proteinase K, 3% PVPP, bacteriolytic enzyme). Method 3 got the best yields 43.06 μg/g, and A260/A280 and A260/A230 were 1.1623 and 0.8135, respectively, which could ensure the veracity of subsequent DGGE analysis. Method 2 (3% CTAB, no proteinase K, no PVPP, no bacteriolytic enzyme) could not extract enough DNA to do the next PCRDGGE analysis. F341/R534 and FR1/FF390 primers were used to amplify the 16S rDNA V3 region of bacteria and 18S rDNA of fungi, and the expected fragments of 230 bp 16S rDNA V3 region and 390 bp 18S rDNA were amplified. The results of DGGE analysis showed that there were common and specific bacterial and fungal communities in different replant soils of strawberry. There were 84 and 54% similarity of bacterial and fungal communities between different replant soils. The numbers of both bacterial and fungal communities increased in the replant strawberry soil, they were positively correlated with the replant years. As the number of replant years increased from two to seven years, while the ratio of bacteria/fungi was decreased from 2.29 to 1.46 in the rhizosphere soils planted with strawberries.

Bacteriolytic enzymes from the blood plasma of the sheep

In the present work the studies ofbacteriolytic factors from sheep blood plasma have been performed. Three novel enzymes have been identified and characterized. Two of them have a molecular weight 15 +/- 2 kDa and able to lyse the gram-negative Escherichia coli bacteria. The third enzyme has a molecular weight 34 +/- 4 kDa and is able to lyse both gram-negative Escherichia coli and gram-positive Micrococcus luteus bacteria. The bacteriolytic reactions have been studied for all three enzymes; particularly, pH-optima have been identified with respect to the substrate. To identify the enzymes trypsinolysis and consequent MALDI-TOF mass spectrometry studies were performed. The results were compared to data from publicly available databases, such as Swiss-Prot, NCBI, MSDB.