Antonio Pellegrini

Isolation and identification of three bactericidal domains in the bovine α-lactalbumin molecule

Abstract Proteolytic digestion of α-lactalbumin by pepsin, trypsin and chymotrypsin yielded three polypeptide fragments with bactericidal properties. Two fragments were obtained from the tryptic digestion. One was a pentapeptide with the sequence EQLTK (residues 1–5) and the other, GYGGVSLPEWV CTTF ALC SEK (residues (17–31)S-S(109–114)), was composed of two polypeptide chains held together by a disulfide bridge. Fragmentation of α-lactalbumin by chymotrypsin yielded CKDDQNPH ISC DKF (residues (61–68)S-S(75–80)), also a polypeptide composed of two polypeptide chains held together by a disulfide bridge. The three polypeptides were synthesized and found to exert antimicrobial activities. The polypeptides were mostly active against Gram-positive bacteria. Gram-negative bacteria were only poorly susceptible to the bactericidal action of the polypeptides. GYGGVSLPEWV CTTF ALC SEK was most, EQLTK least bactericidal. Replacement of leucine (23) with isoleucine, having a similar chemical structure but higher hydrophobicity, in the sequence GYGGVSLPEWV CTTF ALC SEK significantly reduced the bactericidal capacity of the polypeptide. Digestion of α-lactalbumin by pepsin yielded several polypeptide fragments without antibacterial activity. α-Lactalbumin in contrast to its polypeptide fragments was not bactericidal against all the bacterial strains tested. Our results suggest a possible antimicrobial function of α-lactalbumin after its partial digestion by endopeptidases.

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin

Proteolytic digestion of bovine beta-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15-20), AASDISLLDAQSAPLR (residues 25-40), IPAVFK (residues 78-83) and VLVLDTDYK (residues 92-100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55-64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of beta-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of beta-lactoglobulin could be useful to increase its antimicrobial function.

Identification and isolation of a bactericidal domain in chicken egg white lysozyme

Chicken egg white lysozyme exhibits antimicrobial activity against both Gram‐positive and Gram‐negative bacteria. Fractionation of clostripain‐digested lysozyme yielded a pentadecapeptide with antimicrobial activity but without muramidase activity. The peptide was isolated and its sequence found to be I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R (amino acids 98–112 of chicken egg white lysozyme). A synthesized peptide of identical sequence had the same bactericidal activity as the natural peptide. Replacement of Trp 108 with tyrosine significantly reduced the antibacterial capacity of the peptide. By replacement of Trp 111 with tyrosine the antibacterial activity was lost. Replacement of Asn 106 with the positively charged arginine strongly increased the antibacterial capacity of I‐V‐S‐D‐G‐N‐G‐M‐N‐A‐W‐V‐A‐W‐R. The peptide I‐V‐S‐D‐G‐N‐G‐M consisting of the eight amino acids of the N‐terminal side had no bactericidal properties, whereas the peptide N‐A‐W‐V‐A‐W‐R of the C‐terminal side retained some bactericidal activity. Replacement of asparagine 106 by arginine (R‐A‐W‐V‐A‐W‐R) increased the bactericidal activity considerably. The D enantiomer of R‐A‐W‐V‐A‐W‐R was as active as the L form against five of the tested bacteria, but substantially less active against Serratia marcescens, Micrococcus luteus,Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus lentus. For these bacterial species some stereospecific complementarity between receptor structures of the bacteria and the peptide can be assumed.

Strategies for New Antimicrobial Proteins and Peptides: Lysozyme and Aprotinin as Model Molecules

The increasing development of bacterial resistance to traditional antibiotics has reached alarming levels, thus necessitating the strong need to develop new antimicrobial agents. These new antimicrobials should possess both novel modes of action as well as different cellular targets compared with the existing antibiotics. Lysozyme, muramidase, and aprotinin, a protease inhibitor, both exhibit antimicrobial activities against different microorganisms, were chosen as model proteins to develop more potent bactericidal agents with broader antimicrobial specificity. The antibacterial specificity of lysozyme is basically directed against certain Gram-positive bacteria and to a lesser extent against Gram-negative ones, thus its potential use as antimicrobial agent in food and drug systems is hampered. Several strategies were attempted to convert lysozyme to be active in killing Gram-negative bacteria which would be an important contribution for modern biotechnology and medicine. Three strategies were adopted in which membrane-binding hydrophobic domains were introduced to the catalytic function of lysozyme, to enable it to damage the bacterial membrane functions. These successful strategies were based on either equipping the enzyme with a hydrophobic carrier to enable it to penetrate and disrupt the bacterial membrane, or coupling lysozyme with a safe phenolic aldehyde having lethal activity toward bacterial membrane. In a different approach, proteolytically tailored lysozyme and aprotinin have been designed on the basis of modifying the derived peptides to confer the most favorable bactericidal potency and cellular specificity. The results obtained from these strategies show that proteins can be tailored and modelled to achieve particular functions. These approaches introduced, for the first time, a new conceptual utilization of lysozyme and aprotinin, and thus heralded a great opportunity for potential use in drug systems as new antimicrobial agent.

In vitro and in vivo antimicrobial activity of two α-helical cathelicidin peptides and of their synthetic analogs

Abstract Two α-helical antimicrobial peptides (BMAP-27 and -28) and four synthetic analogs were compared for in vitro and in vivo antimicrobial efficacy. All peptides proved active in vitro at micromolar concentrations against a range of clinical isolates, including antibiotic-resistant strains. BMAP-27 and two analogs were more effective towards Gram-negative, and BMAP-28 towards Gram-positive organisms. In addition, BMAP-28 provided some protection in vitro against human herpes simplex virus type 1 (HSV-1). The parent peptides and mBMAP-28 analog protected mice from lethal i.p. infections in an acute peritonitis model at peptide doses significantly lower than those toxic to the animals, suggesting a satisfactory therapeutic index.

Effect of lysozyme or modified lysozyme fragments on DNA and RNA synthesis and membrane permeability of Escherichia coli.

Previously we have shown that chicken egg white lysozyme, an efficient bactericidal agent, affects both gram-positive and gram-negative bacteria independently of its muramidase activity. More recently we reported that the digestion of lysozyme by clostripain yielded a pentadecapeptide, IVSDGNGMNAWVAWR (amino acid 98-112 of chicken egg white lysozyme), with moderate bactericidal activity but without muramidase activity. On the basis of this amino acid sequence three polypeptides, in which asparagine 106 was replaced by arginine (IVSDGNGMRAWVAWR, RAWVAWR, RWVAWR), were synthesized which showed to be strongly bactericidal. To elucidate the mechanisms of action of lysozyme and of the modified antimicrobial polypeptides Escherichia coli strain ML-35p was used. It is an ideal organism to study the outer and the inner membrane permeabilization since it is cryptic for periplasmic beta-lactamase and cytoplasmic beta-galactosidase unless the outer or inner membrane becomes damaged. For the first time we present evidence that lysozyme inhibits DNA and RNA synthesis and in contrast to the present view is able to damage the outer membrane of Escherichia coli. Blockage of macromolecular synthesis, outer membrane damage and inner membrane permeabilization bring about bacterial death. Ultrastructural studies indicate that lysozyme does not affect bacterial morphology but impairs stability of the organism. The bactericidal polypeptides derived from lysozyme block at first the synthesis of DNA and RNA which is followed by an increase of the outer membrane permeabilization causing the bacterial death. Inner membrane permeabilization, caused by RAWVAWR and RWVAWR, follows after the blockage of macromolecular synthesis and outer membrane damage, indicating that inner membrane permeabilization is not the deadly event. Escherichia coli bacteria killed by the substituted bactericidal polypeptides appeared, by electron microscopy, with a condensed cytoplasm and undulated bacterial membrane. So the action of lysozyme and its derived peptides is not identical.

The antiviral activity of naturally occurring proteins and their peptide fragments after chemical modification

Abstract Chemical modification of the proteins bovine serum albumin, α-lactalbumin, β-lactoglobulin and chicken lysozyme by 3-hydroxyphthalic anhydride (3-HP) yielded compounds which exerted antiviral activity in vitro as compared with the native unmodified proteins. Of the three enveloped viruses tested, human herpes simplex virus type 1 (HSV-1), bovine parainfluenza virus type 3 and porcine respiratory corona virus, only HSV-1 proved sensitive to the 3-HP-proteins. All of the chemically modified proteins presented antiviral activity against HSV-1 when assayed before, during or after infection. However, to achieve HSV-1 inhibition, significantly higher concentrations of the modified proteins were required if present before infection as compared to during or after infection. Our results suggest that multiple mechanisms are involved in the inhibition of HSV-1 infection. Proteolytical digestion of albumin, α-lactalbumin, β-lactoglobulin and lysozyme by trypsin, chymotrypsin and pepsin yielded several peptide fragments with antiherpetic activity. Chemical modification of these peptide fragments by 3-HP generated peptides with antiviral activity, however, this was almost always combined with a cytotoxic effect on the Vero cells. Overall, our results suggest that targeted chemical modification of some natural products might provide compounds effective against HSV-1 infection.

Natural protease inhibitors: qualitative and quantitative assay by fibrinogen-agarose electrophoresis.

An electrophoretic procedure for the qualitative and quantitative assay of protein protease inhibitors is reported. This assay is particularly suited for investigations of crude biological materials when specific antisera are not available. The supporting medium consists of agarose into which denatured fibrinogen is incorporated as the substrate for proteases. The processing then is divided into two steps: (1) electrophoretic resolution of the inhibitor containing material and (2) detection of the inhibitor bands through their protease inhibiting activity. The inhibitor position is thus made visible as a colored band of denatured fibrinogen which has escaped digestion by protease. By electrophoretic separation of multiple copies of a sample of biological fluid followed by soaking each of them in the solution of a distinct protease, the enzyme specificity of a particular inhibitor band can easily be established. The bands can in selected cases be quantitated accurately by densitometry and the inhibitor activity thus determined using a reference serum calibrated with Trasylol as a standard. The activity of alpha-1-protease inhibitor in healthy horses is reported.

Reevaluation of the effect of lysoyzme on Escherichia coli employing ultrarapid freezing followed by cryoelectronmicroscopy or freeze substitution

Lysozyme is able to lyse Gram‐positive bacteria acting as muramidase on the peptidoglycan polymer. Gram‐negative bacteria in vitro are not lysed by lysozyme. It was assumed that the peptido‐glycan is protected by the outer membrane and thus that Gram‐negative bacteria are not affected by lysozyme without the aid of other factors such as EDTA or complement which enable lysozyme to penetrate the outer membrane. Accidentally, Pellegrini et al. [(1992) J. Appl. Bacteriol., 72:180–187] found that lysozyme per se is able to kill some Gram‐negative bacteria. On the basis of morphological and immunocytochemical findings obtained from chemically fixed bacteria, it was concluded that lysozyme does not lyse Gram‐negative bacteria but affects the cytoplasm of for example, Escherichia coli, leading to its disintegration, whilst the membranes do not break down. In an attempt to clarify the action of lysozyme on E. coli, we employed cryotechniques including ultrarapid freezing, cryomicroscopy and freeze substitution, and immunolabeling. Bacteria that were immediately frozen after exposure to lysozyme remained morphologically intact. Individual bacteria plated on agar after exposure to lysozyme were mostly intact when frozen within a few seconds. However, inner and outer membranes of 80% of the bacteria were disrupted, whereas the cytoplasm of only a few bacteria showed signs of disintegration when bacteria were frozen with a delay of only 5 min of plating onto pure agar or agar containing growth medium. After a period of time of 15 min between plating onto agar and freezing, about 97% of the bacteria showed changes of disintegration of various extent. Immunolabeling showed that lysozyme binds to the outer cell membrane and may penetrate the membrane, reaching the periplasmic space and possibly the inner cell membrane. The ultrastructural findings and the results of antibacterial assays suggest that lysozyme is bactericidal for E. coli but is not able to induce disintegration. Disintegration is accomplished by changes of the environment starting at the cell membranes. The mechanism by which lysozyme penetrates the membrane, the way it acts to be bactericidal, and the way disintegration is initiated remain to be clarified. Microsc. Res. Tech. 39:297–304, 1997.

Fractionation and partial characterization of α-1-protease isoinhibitors of horse

The principal α-1-protease inhibitor of horse was fractionated by classical methods and analysed with a modified fibrinogen-agarose gel electrophoretic method of high sensitivity and resolving power. Starting with an electrophoretically homogeneous inhibitor in unfractionated serum, two isoinhibitor bands became apparent after fractionation with (NH4)2SO4 and DEAE-cellulose DE-52 ion-exchange chromatography. The isoinhibitors differed in electrophoretic migration and in the elution pattern from Sephadex G-100 gel filtration, but possessed identical antigenic determinants and enzyme specificity. The slower migrating isoinhibitor with an apparent molecular weight of 90 000 could be highly purified. In contrast the faster moving isoinhibitor (molecular weight 65 000) could not be completely freed from a contaminating α-2-protease inhibitor. The formation of the two isoinhibitors is discussed considering conformational changes analogous to phenomena observed with α-2-macroglobulin, or dimer formation in combination with altered conformations. The isoinhibitors described here are new additions to the different heterogeneities which exist in α-1-protease inhibitors in horse. They also supplement the different heterogeneities which exist among the α-1-protease inhibitors of mammals.