Structure of the induced antibacterial protein from tasar silkworm, Antheraea mylitta. Implications to molecular evolution

The crystal structure of an antibacterial protein of immune origin (TSWAB), purified from tasar silkworm (Antheraea mylitta) larvae after induction by Escherichia coli infection, has been determined. This is the first insect lysozyme structure and represents induced lysozymes of innate immunity. The core structure of TSWAB is similar to c-type lysozymes and alpha-lactalbumins. However, TSWAB shows significant differences with respect to the other two proteins in the exposed loop regions. The catalytic residues in TSWAB are conserved with respect to the chicken lysozyme, indicating a common mechanism of action. However, differences in the noncatalytic residues in the substrate binding groove imply subtle differences in the specificity and the level of activity. Thus, conformational differences between TSWAB and chicken lysozyme exist, whereas functional mechanisms appear to be similar. On the other hand, alpha-lactalbumins and c-type lysozymes exhibit drastically different functions with conserved molecular conformation. It is evident that a common molecular scaffold is exploited in the three enzymes for apparently different physiological roles. It can be inferred on the basis of the structure-function comparison of these three proteins having common phylogenetic origin that the conformational changes in a protein are minimal during rapid evolution as compared with those in the normal course of evolution.     

The influence of silkworm species on cellular interactions with novel PVA/silk sericin hydrogels

Sericin peptides and PVA are chemically modified with methacrylate groups to produce a covalent PVA/sericin hydrogel. Preservation of the sericin bioactivity following methacrylation is confirmed, and PVA/sericin hydrogels are fabricated for both B. mori and A. mylitta sericin. Cell adhesion studies confirm the preservation of sericin bioactivity post incorporation in PVA gels. PVA/A. mylitta gels are observed to facilitate cell adhesion to a significantly greater degree than PVA/B. mori gels. Overall, the incorporation of sericin does not alter the physical properties of the PVA hydrogels but does result in significantly improved cellular interaction, particularly from A. mylitta gels.     

Antibacterial proteins in rainbow trout, Oncorhynchus mykiss

Antibacterial proteins are an important part of the innate immune system for all animals. They have been extensively studied in mammals, amphibians and invertebrates, but have received only scant attention in fish. Their expression and processing, however, provide a way of monitoring defence vigour during development or with seasonal changes in physiology. The aim of the present work was to identify and characterise antibacterial proteins in rainbow trout. In vitro analyses of extracts of the peripheral blood leucocytes, head kidney leucocytes and mucus from adult unstimulated (non-immune) fish showed marked antibacterial activity against Gram positive bacteria. Fractionation by ion exchange chromatography and RP-HPLC of head kidney extracts showed the presence of two forms of lysozyme but no constitutively expressed antimicrobial proteins of < 10 kDa. By contrast, chromatographic analyses of mucus revealed at least four antibacterial proteins. Two are conventional lysozymes, a third is an unusual lysozyme-like protein with a low isoelectric point, and the fourth is a highly hydrophobic, cationic peptide of c. 3 kDa.     

The P5 protein from bacteriophage phi-6 is a distant homolog of lytic transglycosylases

Peptidases are classical objects of enzymology and structural studies. However, a few protein families with experimentally characterized proteolytic activity, but unknown catalytic mechanism and three-dimensional structures, still exist. Using comparative sequence analysis, we deduce spatial structure for one of such families, namely, U40, which contains just one P5 protein from bacteriophage phi-6. We show that this singleton sequence possesses conserved sequence motifs characteristic of lysozymes and is a distant homolog of lytic transglycosylases that cleave bacterial peptidoglycan. The structure of the P5 protein is therefore predicted to adopt the lysozyme-like fold shared by T4, lambda, C-type, G-type lysozymes, and lytic transglycosylases. Since previous biochemical experiments with P5 of phi-6 have indicated that the purified enzyme possesses endopeptidase activity and not glycosidase activity, our results point to the possibility of a newly evolved molecular function and call for further experimental characterization of this unusual P5 protein.     

Minimum structure of peptidoglycan required for induction of antibacterial protein synthesis in the silkworm, Bombyx mori

Various peptidoglycan fragments, different in mode of cross-linking and molecular size, were isolated, and the elicitor activity was tested for induction of antibacterial protein synthesis in larvae of Bombyx mori. Linear uncross-linked peptidoglycans from Bacillus licheniformis and Micrococcus luteus were effective elicitors, similar to the directly cross-linked peptidoglycan from B. licheniformis cell wall. The fragments of uncross-linked peptidoglycan with a sugar chain length of four or more were active elicitors, but the disaccharide unit had no elicitor activity. The minimum structure of peptidoglycan required for induction of antibacterial protein synthesis was determined to be two repeating N-acetylglucosamine-N-acetylmuramic acid units with peptide side chains.     

The Tape Measure Protein of the Staphylococcus aureus Bacteriophage vB_SauS-phiIPLA35 Has an Active Muramidase Domain

Tailed double-stranded DNA (dsDNA) bacteriophages frequently harbor structural proteins displaying peptidoglycan hydrolytic activities. The tape measure protein from Staphylococcus aureus bacteriophage vB_SauS-phiIPLA35 has a lysozyme-like and a peptidase_M23 domain. This report shows that the lysozyme-like domain (TG1) has muramidase activity and exhibits in vitro lytic activity against live S. aureus cells, an activity that could eventually find use in the treatment of infections.     

Purification and biochemical characterization of a 70 kDa sericin from tropical tasar silkworm, Antheraea mylitta

Sericin isolated from the cocoon of the tropical tasar silkmoth Antheraea mylitta showed three major bands, with the lowest 70 kDa. This band was purified by anion exchange chromatography. Immunoblotting with concanavalin-A suggests a glycoprotein and CD analysis of secondary structure includes beta-sheet. Amino acid analysis shows that the protein is enriched in glycine and serine while the mole percentages of these two amino acids are different from sericin of mulberry silkworm. An anti A. mylitta sericin antibody was able to cross-react with sericin from A. assamensis but not the sericin of Bombyx mori and Philosamia ricini. Immunoblot analysis with proteins isolated from middle silk gland of A. mylitta at different developmental stages of larva showed that the 70 kDa sericin is developmentally regulated. These data extend the range of biochemical features found in this unusual family of proteins and may help in developing an improved understanding of their role in forming environmentally stable fibroin fiber-sericin composite structures (cocoons).     

Peptidoglycan recognition proteins kill bacteria by activating protein-sensing two-component systems

Mammalian peptidoglycan recognition proteins (PGRPs), similar to antimicrobial lectins, bind the bacterial cell wall and kill bacteria through an unknown mechanism. We show that PGRPs enter the Gram-positive cell wall at the site of daughter cell separation during cell division. In Bacillus subtilis, PGRPs activate the CssR-CssS two-component system that detects and disposes of misfolded proteins that are usually exported out of bacterial cells. This activation results in membrane depolarization, cessation of intracellular peptidoglycan, protein, RNA and DNA synthesis, and production of hydroxyl radicals, which are responsible for bacterial death. PGRPs also bind the outer membrane of Escherichia coli and activate the functionally homologous CpxA-CpxR two-component system, which kills the bacteria. We exclude other potential bactericidal mechanisms, including inhibition of extracellular peptidoglycan synthesis, hydrolysis of peptidoglycan and membrane permeabilization. Thus, we reveal a previously unknown mechanism by which innate immunity proteins that bind the cell wall or outer membrane exploit the bacterial stress defense response to kill bacteria.     

COG3926 and COG5526: a tale of two new lysozyme-like protein families

We have identified two new lysozyme-like protein families by using a combination of sequence similarity searches, domain architecture analysis, and structural predictions. First, the P5 protein from bacteriophage phi8, which belongs to COG3926 and Pfam family DUF847, is predicted to have a new lysozyme-like domain. This assignment is consistent with the lytic function of P5 proteins observed in several related double-stranded RNA bacteriophages. Domain architecture analysis reveals two lysozyme-associated transmembrane modules (LATM1 and LATM2) in a few COG3926/DUF847 members. LATM2 is also present in two proteins containing a peptidoglycan binding domain (PGB) and an N-terminal region that corresponds to COG5526 with uncharacterized function. Second, structure prediction and sequence analysis suggest that COG5526 represents another new lysozyme-like family. Our analysis offers fold and active-site assignments for COG3926/DUF847 and COG5526. The predicted enzymatic activity is consistent with an experimental study on the zliS gene product from Zymomonas mobilis, suggesting that bacterial COG3926/DUF847 members might be activators of macromolecular secretion.     

Protist-type lysozymes of the nematode Caenorhabditis elegans contribute to resistance against pathogenic Bacillus thuringiensis

Pathogens represent a universal threat to other living organisms. Most organisms express antimicrobial proteins and peptides, such as lysozymes, as a protection against these challenges. The nematode Caenorhabditis elegans harbours 15 phylogenetically diverse lysozyme genes, belonging to two distinct types, the protist- or Entamoeba-type (lys genes) and the invertebrate-type (ilys genes) lysozymes. In the present study we characterized the role of several protist-type lysozyme genes in defence against a nematocidal strain of the Gram-positive bacterium Bacillus thuringiensis. Based on microarray and subsequent qRT-PCR gene expression analysis, we identified protist-type lysozyme genes as one of the differentially transcribed gene classes after infection. A functional genetic analysis was performed for three of these genes, each belonging to a distinct evolutionary lineage within the protist-type lysozymes (lys-2, lys-5, and lys-7). Their knock-out led to decreased pathogen resistance in all three cases, while an increase in resistance was observed when two out of three tested genes were overexpressed in transgenic lines (lys-5, lys-7, but not lys-2). We conclude that the lysozyme genes lys-5, lys-7, and possibly lys-2 contribute to resistance against B. thuringiensis, thus highlighting the particular role of lysozymes in the nematode's defence against pathogens.     

2′-N-Acetylation of Butirosin A by Mutants of Streptomyces fradiae

To reveal the mechanism of reducing sugar-induced polymerization of proteins, monomeric lysozymes were isolated at various stages of storage from whole lysozyme (WL) being kept with glucose at 75% relative humidity and 50°C for up to 30 days, and their chemical properties were investigated and compared with the corresponding WL. The impairment of Lys, Arg, and Trp residues was observed in all the isolated monomeric lysozymes (IM) as well as in the WL.

When the IM were stored for another 10 days without glucose, they polymerized and had an additional impairment of Arg and Lys but not Trp residues. All IM exhibited almost the same polymerization rate, but the sum of additional losses of Lys and Arg residues varied. The IM was also found to cross-link untreated lysozymes even in the absence of glucose.

On basis of the results obtained hitherto, it is suggested that the glucose-induced polymerization of lysozymes proceeds through the following paths. At the first step, some bifunctional agents (BF), probably α-dicarbonyl compounds, generated from the reaction between ?-amino groups of lysine residues and glucose, attach to Arg, Lys, and Trp residues through one of their two functional sites. At the second step, some of those proteins with BF attached polymerize by binding of the other unoccupied functional site with the remaining Lys and Arg (not Trp) residues of the other protein molecules. The other of the proteins with BF attached polymerize through the combination between the other unoccupied functional sites themselves with no loss of amino acid residues.     

PGRP-SB1: an N-acetylmuramoyl L-alanine amidase with antibacterial activity

The peptidoglycan recognition protein (PGRP) family is conserved from insects to mammals and is involved in immune regulation and bacterial clearance. They form at least three functional classes; receptors required for immune gene expression; amidases that degrade peptidoglycan and scavenge the tissues from immune-stimulating peptidoglycan; and as proteins with antibacterial activity. We here report that PGRP-SB1 is an N-acetylmuramoyl l-alanine amidase, which (in contrast to the previously described PGRP-amidases) shows antibacterial activity. PGRP-SB1 is highly active against peptidoglycans that have a diaminopimelic acid (DAP) residue in the cross-linking peptide, but lack activity to most lysine-containing peptidoglycans. The antibacterial activity is pronounced against Bacillus megaterium with an LD(50) of 1.5microg ml(-1). The bactericidal effect of PGRP-SB1 is dependent on its enzymatic activity, as the zinc co-factor is essential. The bactericidal mode of action is thus different from non-enzymatic vertebrate PGRPs that have been reported to be antibacterial.     

Expression analysis of proteins encoded by genes of the tag7/tagL (PGRP-S,L) family in human peripheral blood cells

Various types of human blood cells were tested for expression of the Tag7/PGRP-SA and TagL/PGRP-L proteins, which belong to the family of proteins possessing the lysozyme-like peptidoglycan recognition protein (PGRP) domain. Expression regulation by several factors was demonstrated.     

O-Acetylated peptidoglycan: controlling the activity of bacterial autolysins and lytic enzymes of innate immune systems

The O-acetylation of peptidoglycan is now known to occur in 50 different bacterial species, both Gram positive and Gram negative, including a number of important human pathogens. This modification to the essential cell wall component of bacteria provides both a level of control over endogenous autolysins and protection from the lysozymes of innate immune systems. In this review, we describe the details of the pathways for peptidoglycan O-acetylation that are now beginning to emerge and we explore the possibility that the associated enzymes may present new candidates for antibacterial targets.     

Expression Analysis of Proteins Encoded by Genes of the tag7/tagL (PGRP-S, L) Family in Human Peripheral Blood Cells

Various types of human blood cells were tested for expression of the Tag7/PGRP-SA and TagL/PGRP-L proteins, which belong to the family of proteins possessing the lysozyme-like peptidoglycan recognition protein (PGRP) domain. Expression regulation by several factors was demonstrated.     

Thermal stability and enzymatic activity of a smaller lysozyme from silk moth (Bombyx mori)

Bombyx mori lysozyme is 10 amino acids shorter than hen egg-white lysozyme, which is a typical c-type lysozyme. It was expressed by using the methylotrophic yeast Pichia pastoris. The thermal stability and the enzymatic activity of the Bombyx mori lysozyme were estimated and compared with those of human and hen egg-white lysozymes. The denaturation temperature was 17-26°C lower than those of human and hen egg-white lysozymes. Further, the enthalpy change and the heat capacity change for unfolding were smaller than those of human lysozyme. It was also confirmed that the stability against guanidine hydrochloride was lower than those of the other two lysozymes. The enzymatic activity toward a simple synthetic substrate was measured and compared with those of human and hen egg-white lysozymes. The B-F binding mode was obviously dominant, although the A-E binding mode was preferred in human and hen egg-white lysozymes.