Newly made enzymes determine ongoing cell wall synthesis and the antibacterial effects of cell wall synthesis inhibitors.

Cell wall synthesis can continue with less than the total complement of cell wall synthetic enzymes present in normal growing cells. A method was developed to investigate whether there exists an excess of cell wall-synthesizing enzymes (penicillin-binding proteins [PBPs]) which all remain functional or whether a mixed population of functional and nonfunctional enzymes characterize normal cells. Surprisingly, cells in which less than 10% of the PBPs were functional could grow at a normal rate, as evidenced by increases in viable counts, culture turbidity, and rates of peptidoglycan, protein, and RNA synthesis. This subset of functional enzymes was biosynthetically new. Penicillin-induced lysis occurred contingent on the acylation of this same small fraction of PBPs, the copy number and affinities of which were below the level of detection by current fluorographic assay techniques. We propose that PBPs have a short functional half-life and that cell wall synthesis and bacterial lysis reflect the activity of newly synthesized PBPs.     

LambdaSa2 prophage endolysin requires Cpl-7-binding domains and amidase-5 domain for antimicrobial lysis of streptococci

Streptococcal pathogens contribute to a wide variety of human and livestock diseases. The routine use of antibiotics to battle these pathogens has produced a new class of multidrug-resistant streptococci. Thus, there is a need for new antimicrobials. Bacteriophage endolysins (peptidoglycan hydrolases) comprise one group of new antimicrobials that are reportedly refractory to resistance development. The LambdaSa2 prophage endolysin gene was recently isolated from a Group B streptococcal genome, expressed on an Escherichia coli plasmid, and shown by homology screening and biochemical analysis to harbor an amidase-5 (endopeptidase) domain, an amidase-4 (glycosidase) domain, and two Cpl-7 cell wall-binding domains. In this study, turbidity reduction and plate lysis assays indicate that this hydrolase shows strong lytic activity toward Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus uberis, Streptococcus equi, GES, and GGS. Deletion analysis indicates that the N-terminal endopeptidase domain with both Cpl-7 domains can lyse with a higher specific activity than the full-length protein (against some strains). This dual Cpl-7 domain truncated version also shows weak lytic activity against methicillin-resistant Staphylococcus aureus (MRSA) and the coagulase negative staphylococci, Staphylococcus xylosus. The truncated constructs harboring the glycosidase domain are virtually inactive, showing only minimal activity on plate lysis assays.     

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion-associated peptidoglycan hydrolase: fusions, deletions, and synergy with LysH5

Virion-associated peptidoglycan hydrolases have potential as antimicrobial agents due to their ability to lyse Gram-positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion-associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88. Full-length HydH5 and two truncated derivatives containing only the CHAP (cysteine, histidine-dependent amidohydrolase/peptidase) domain exhibited high lytic activity against live S. aureus cells. In addition, three different fusion proteins were created between lysostaphin and HydH5, each of which showed higher staphylolytic activity than the parental enzyme or its deletion construct. Both parental and fusion proteins lysed S. aureus cells in zymograms and plate lysis and turbidity reduction assays. In plate lysis assays, HydH5 and its derivative fusions lysed bovine and human S. aureus strains, the methicillin-resistant S. aureus (MRSA) strain N315, and human Staphylococcus epidermidis strains. Several nonstaphylococcal bacteria were not affected. HydH5 and its derivative fusion proteins displayed antimicrobial synergy with the endolysin LysH5 in vitro, suggesting that the two enzymes have distinct cut sites and, thus, may be more efficient in combination for the elimination of staphylococcal infections.     

Effects of Detergents on Lysis of Micrococcus lysodeikticus by Lysozyme

The effects of detergents on the lysozyme-catalyzed hydrolysis of Micrococcus lysodeikticus cells were investigated by changing the concentration of Na-phosphate buffer and pH in the presence or absence of sucrose. Also, a parallel study of the hydrolysis of glycolchitin by lysozyme was conducted and compared to the lytic reaction. Electron microscopy was utilized to follow the changes in cell morphology during the various treatments.

None of the detergents changed turbidity of the cell suspension. However, they did affect the change in turbidity during lysis in unique ways. SDS, which is an anionic detergent, inhibited lysozyme activity and its addition to the reaction mixture caused a rapid and large decrease in the turbidity. Brij 35 and Triton X-100, which are non-ionic detergents, did not inhibit lysozyme activity, but their presence in the reaction mixture changed the rate of turbidity change. Apparently non-ionic detergents disrupt only the protoplast, while anionic detergents disrupt both the protoplast and the damaged cell. The lytic mechanism of M. lysodeikticus by lysozyme was discussed in detail.     

蛇毒纤维蛋白（原）溶解酶的研究进展

蛇毒纤溶酶能直接溶解纤维蛋白（原）,具有作为强力溶栓剂的潜在价值。对蛇毒纤溶酶的深入研究,不仅有助于阐明蛇伤中毒患者的凝血病理机制,而且为其开发应用提供了理论基础。文章综述蛇毒纤溶酶的研究进展及应用前景,重点阐述其分子结构、酶学特性及其与出血活性的关系     

LysK CHAP endopeptidase domain is required for lysis of live staphylococcal cells

LysK is a staphylococcal bacteriophage endolysin composed of three domains: an N-terminal cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) endopeptidase domain, a midprotein amidase 2 domain, and a C-terminal SH3b_5 (SH3b) cell wall-binding domain. Both catalytic domains are active on purified peptidoglycan by positive-ion electrospray ionization MS. The cut sites are identical to LytA (phi11 endolysin), with cleavage between d -alanine of the stem peptide and glycine of the cross-bridge peptide, and N -acetylmuramoyl- l -alanine amidase activity. Truncations of the LysK containing just the CHAP domain lyse Staphylococcus aureus cells in zymogram analysis, plate lysis, and turbidity reduction assays but have no detectable activity in a minimal inhibitory concentration (MIC) assay. In contrast, truncations harboring just the amidase lytic domain show faint activity in both the zymogram and turbidity reduction assays, but no detectable activity in either plate lysis or MIC assays. A fusion of the CHAP domain to the SH3b domain has near full-length LysK lytic activity, suggesting the need for a C-terminal binding domain. Both LysK and the CHAP-SH3b fusion were shown to lyse untreated S. aureus and the coagulase-negative strains. In the checkerboard assay, the CHAP-SH3b fusion achieves the same level of antimicrobial synergy with lysostaphin as the full-length LysK.     

Complete lysis of glutamic acid-producing bacteria by the use of antibiotics which inhibit the biosynthesis of cell walls.

A novel method for obtaining complete lysis of coryneform bacteria has been devised. Bacteria in the late-exponential phase of growth are treated with antibiotics which inhibit biosynthesis of the cell wall. Organisms treated in this way show increased sensitivity to lysis by added lytic enzymes. The preparation of DNA from this group of organisms is thus greatly facilitated.     

Cell wall degradation in the autolysis of filamentous fungi

A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes.Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present.In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.     

A structured mechanistic model of the kinetics of enzymatic lysis and disruption of yeast cells

A structured, mechanistic model has been built for the kinetics of yeast cell lysis by microbial cell lytic enzymes, based on an understanding of the two-layer yeast cell wall structure and the properties of yeast-lytic enzyme systems. The model predicts the release of protein, peptides and carbohydrates from four cell structures: the outer and inner wall layers, the cytosol and organelles or proteins present in particles; it also predicts organelle or particle lysis or solubilization and the breakdown of released proteins to peptides. Applications of the model to design and optimization of selective product release are discussed.     

Kinetics of enzymatic lysis and disruption of yeast cells: I. Evaluation of two lytic systems with different properties

Many microorganisms produce enzymes which lyse the walls of yeasts, fungi, and bacteria. The proportions of different enzyme activities present in the lytic system, their action patterns, synergism, and dependence on inhibitors, constitute the activity profile of the lytic system. Taken together, the activity profile and process conditions for lysis determine the reaction rate and the distribution of products from lysis of any given type of cells. Kinetics of glucan hydrolysis, proteolysis, and lysis of brewer's yeast were compared for two extracellular yeast-lytic enzyme systems with different properties. The enzyme sources used were filtered culture broths from Cytophaga sp. NCIB 9497 grown in batch culture and from Oerskovia xanthineolytica LL-G109, grown under carbon limitation in continuous culture. Rate and extent of cell hydrolysis, and the accumulation of soluble proteins, peptides, and carbohydrates from the lysed yeast cells, are discussed in terms of the activity profiles and potential applications of the two enzyme systems.     

Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin

Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.     

The function of lymphocyte proteases. Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors.

To kill other cells, lymphocytes can exocytose granules that contain serine proteases and pore-forming proteins (perforins). We report that mechanism-based isocoumarin inhibitors inhibited the proteases and inactivated lysis. When inhibited proteases were restored, lysis was also restored, indicating that the proteases were essential for lysis. We found three new lymphocyte protease activities, "Asp-ase,"Met-ase," and "Ser-ase," which in addition to ly-tryptase and ly-chymase, comprise five different protease activities in rat RNK-16 granules. The general serine protease inhibitor 3,4-dichloroisocoumarin (DCI) inhibited all five protease activities. Essentially all protease molecules were inactivated by DCI before lysis was reduced, as determined from DCI's second order inhibition rate constants for the proteases, the DCI concentrations, and the times of pretreatment needed to block lysis. The pH favoring DCI inhibition of lysis was the pH optimum for protease activity. Isocoumarin reagents acylate, and may sometimes secondarily alkylate, serine protease active sites. Granule proteases, inhibited by DCI acylation, were deacylated with hydroxylamine, restoring both the protease and lytic activities. Hydroxylamine does not restore alkylated proteases and did not restore the lytic activities after inhibition with 4-chloro-7-guanidino-3-(2-phenylethoxy)-isocoumarin, a more alkylating mechanism-based inhibitor designed to react with tryptases. It is improbable that isocoumarin reagents directly inactivated pore-forming proteins because 1) these reagents require protease activation, 2) their nonspecific effects are alkylating, and 3) alkylated proteins are not restored by hydroxylamine. We conclude that serine proteases participate in lysis when lysis is mediated by the complete assembly of granule proteins.     

Cell-free transmission of human adenovirus by passive mass transfer in cell culture simulated in a computer model

Viruses spread between cells, tissues, and organisms by cell-free and cell-cell transmissions. Both mechanisms enhance disease development, but it is difficult to distinguish between them. Here, we analyzed the transmission mode of human adenovirus (HAdV) in monolayers of epithelial cells by wet laboratory experimentation and a computer simulation. Using live-cell fluorescence microscopy and replication-competent HAdV2 expressing green fluorescent protein, we found that the spread of infection invariably occurred after cell lysis. It was affected by convection and blocked by neutralizing antibodies but was independent of second-round infections. If cells were overlaid with agarose, convection was blocked and round plaques developed around lytic infected cells. Infected cells that did not lyse did not give rise to plaques, highlighting the importance of cell-free transmission. Key parameters for cell-free virus transmission were the time from infection to lysis, the dose of free viruses determining infection probability, and the diffusion of single HAdV particles in aqueous medium. With these parameters, we developed an in silico model using multiscale hybrid dynamics, cellular automata, and particle strength exchange. This so-called white box model is based on experimentally determined parameters and reproduces viral infection spreading as a function of the local concentration of free viruses. These analyses imply that the extent of lytic infections can be determined by either direct plaque assays or can be predicted by calculations of virus diffusion constants and modeling.     

Calibration of bioaffinity assays using kinetic data

Bioaffinity assays are usually calibrated by using a set of standard measurements fitted to a simple empirical model. In this paper, a new calibration approach based on mechanistic model of reaction kinetics is presented. When the calibration assay is known in terms of reaction mechanism, incubation time, initial concentration, and rate constants, one can back-calculate concentrations of unknown samples measured in a nonequilibrium time point. This paper describes a calculation method of unknown sample concentrations based on kinetically measured single calibration assay point. The theoretical results are verified by two common in-vitro diagnostic assays.     

Optimization of enzymatic lysis of yeast

In enzymatic lysis of yeast for the recovery of intracellular proteins, the rupture of whole cells is caused by the action of a lytic system consisting primarily of protease and glucanase. A first-principles mechanism for the lytic reaction based on a two-layer model of the wall structure and a burst model for the disruption of cells is pre sented. The fed-batch model results in a dynamic optimization problem, with the enzymes, activities being the control variables. Orthogonal collocation is applied to discretize the state equations, and the resulting non linear program is solved using successive quadratic pro gramming to determine the enzyme and protease inhibitor add-in rates and pH control profiles that maximize the recovery of intracellular protein. Applying the proposed approach, optimal profiles were determined such that a significant increase of the production of proteins in a fed-batch reactor is realized. Also, the optimal control policies in a series of continuous-flow stirred tank reactors (CFSTRs) are determined.     

Enzymes of the yeast lytic system produced by Arthrobacter GJM-1 bacterium and their role in the lysis of yeast cell walls.

Yeast lytic system produced by Arthrobacter GJM-1 bacterium during growth on baker's yeast cell walls contains a complete set of enzymes which can hydrolyze all structural components of cell walls of Saccharomyces cerevisiae. Chromatographic fractionation of the lytic system showed the presence of two types of endo-beta-1,3-glucanase. Rapid lysis of isolated cell walls of yeast was induced only by endo-beta-1,3-glucanase exhibiting high affinity to insoluble beta-1,3-glucans and releasing laminaripentaose as the main product of hydrolysis of beta-1,3-glucans. This enzyme was able to lyse intact cells of S. cerevisiae only in the presence of an additional factor present in the Arthrobacter GJM-1 lytic system, which was identified as an alkaline protease. This enzyme possesses the lowest molecular weight among other identified enzyme components present in the lytic system. Its role in the solubilization of yeast cell walls from the outer surface by endo-beta-1,3-glucanase could be substituted by preincubation of cells with Pronase or by allowing the glucanase to act on cells in the presence of thiol reagents. The mechanism of lysis of intact cells and isolated cell walls by the enzymes of Arthrobacter GJM-1 is discussed in the light of the present conception of yeast cell wall structure.     

Lytic transglycosylases: bacterial space-making autolysins

Lytic transglycosylases are an important class of bacterial enzymes that act on peptidoglycan with the same substrate specificity as lysozyme. Unlike the latter enzymes, however, the lytic transglycosylases are not hydrolases but instead cleave the glycosidic linkage between N-actetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anydromuramoyl product. They are ubiquitous in bacteria which produce a compliment of different forms that are responsible for creating space within the peptidoglycan sacculus for its biosynthesis and recycling, cell division, and the insertion of cell-envelope spanning structures, such as flagella and secretion systems. As well, the lytic transglyosylases may have a role in pathogenesis of some bacterial species. Given their important role in bacterial cell wall metabolism, the lytic transglycosylases may present an attractive new target for the development of broad-spectrum antibiotics.     

Enzymology of elongation and constriction of the murein sacculus of Escherichia coli

Multiple deletions in murein hydrolases revealed that predominantly amidases are responsible for cleavage of the septum during cell division. Endopeptidases and lytic transglycosylases seem also be involved. In the absence of these enzymes E. coli grows normally but forms chains of adhering cells. Surprisingly, mutants lacking up to eight different murein hydrolases still grow with almost unaffected growth rate. Therefore it is speculated that general enlargement of the murein sacculus may differ from cell division by using transferases rather than the two sets of hydrolytic and synthetic enzymes as seems to be the case for the constriction process. A model is presented that describes growth of the murein of both Gram-positive and -negative bacteria by the activity of murein transferases. It is speculated that enzymes exist that catalyze a transpeptidation of the pre-existing murein onto murein precursors or nascent murein by using the chemical energy present in peptide cross-bridges. Such enzymes would at the same time cleave bonds in the murein net and insert new material into the growing sacculus.     

Bacteriophage-based biocontrol of biological sludge bulking in wastewater

In a previous paper, the first ever application of lytic bacteriophage (virus)-mediated biocontrol of biomass bulking in the activated sludge process using Haliscomenobacter hydrossis as a model filamentous bacterium was demonstrated. In this work we extended the biocontrol application to another predominant filamentous bacterium, Sphaerotilus natans, notoriously known to cause filamentous bulking in wastewater treatment systems. Very similar to previous study, one lytic bacteriophage was isolated from wastewater that could infect S. natans and cause lysis. Significant reduction in sludge volume index and turbidity of the supernatant was observed in batches containing S. natans biomass following addition of lytic phages. Microscopic examination confirmed that the isolated lytic phage can trigger the bacteriolysis of S. natans. This extended finding further strengthens our hypothesis of bacteriophage-based biocontrol of overgrowth of filamentous bacteria and the possibility of phage application in activated sludge processes, the world's widely used wastewater treatment processes.     

Positively selected Leu-11a (CD16+) cells require the presence of accessory cells or factors for the lysis of herpes simplex virus-infected fibroblasts but not herpes simplex virus-infected Raji

Previous studies from our laboratory indicated that human NK activity against HSV-infected fibroblasts (HSV-Fs) but not K562 targets was sensitive to treatment with anti-HLA-DR plus C. In the current study, we have selected Leu-11a+ (CD-16) cells by fluorescence activated cell sorting and found that although Leu-11a enriched populations lysed K562 targets in 14-h 51Cr-release assays, they were unable to kill HSV-Fs targets unless a Leu-11a-depleted population was added back to the effectors or unless known activators of NK cells (IFN-alpha or IL-2) were added to the assays. In contrast, Leu-11a-enriched populations were able to mediate ADCC against HSV-Fs in the presence of sera from HSV-seropositive individuals without the requirement for accessory cells. We have begun preliminary characterization of the accessory cells which allow lysis of HSV-Fs by NK cells: they are HLA-DR+ cells which enrich in the light density fractions of Metrizamide density gradients. They need be present in very small numbers for lysis to take place and are not MHC restricted in that heterologous add-backs between anti-HLA-DR plus C and anti-Leu-11b plus C-treated populations are capable of target cell lysis at levels similar to those achieved with the autologous add-backs. Further, the levels of lysis in heterologous add-back experiments reflected the lytic potential of the effector rather than the accessory cell donor. Finally, although the requirement for accessory cells for NK lysis has been demonstrated for fibroblasts infected with HSV-1, CMV, and VZV, lysis of HSV-infected Raji lymphoblastoid cells is relatively accessory-cell independent, indicating that the requirement for accessory cells for lysis by NK cells is not a property of all herpesvirus-infected targets.