The lysozyme locus in Drosophila melanogaster: an expanded gene family adapted for expression in the digestive tract

Lysozyme has been studied in insects as part of the system of inducible antibacterial defence in the haemolymph. We recently found two Drosophila lysozyme genes that are constitutively expressed in the digestive tract, and are probably involved in the digestion of bacteria in the food. To obtain an overview of the lysozyme genes in this species and their possible roles in immunity and digestion, we have now characterized all six lysozyme genes in the cloned part of the lysozyme locus at 61F, and a seventh gene that maps to the same chromosomal location. The expression of the genes follows four different patterns: firstly, four closely related genes, LysB, C, D and E, are all strongly expressed in the midgut of larvae and adults; secondly, LysP is expressed in the adult salivary gland; thirdly, LysS is expressed mainly in the gastric caecae of larvae; and finally, LysX is primarily expressed in the metamorphosing midgut of late larvae and early pupae. The LysD-like genes and LysS are strongly repressed in artificially infected animals, possibly reflecting a malaise reaction in the digestive tract. None of the genes is expressed in the fat body or haemocytes. Thus rather than being a component of the haemolymph, the Drosophila lysozymes are found mainly in the digestive tract where they are expressed at a high level. Furthermore all genes, except LysP, encode acidic proteins, in contrast to the strongly basic typical lysozymes. This is highly reminiscent of the situation in ruminants, where the lysozymes have been recruited for the digestion of symbiotic bacteria in the stomach.     

Recombinant expression and characterization of a lysozyme from the midgut of Triatoma brasiliensis (Hemiptera,Reduviidae) in comparison with intestinal muramidase activity

Insect c‐type lysozymes are antibacterial proteins that are synthesized in different organs with high activity against Gram‐positive bacteria. Because lysozymes possess muramidase activity, they also play an important role in the digestion of bacteria in Diptera. Triatomines express lysozyme‐encoding genes constitutively in the anterior region (cardia and stomach) of the midgut and the fat body after injection of bacteria into the haemocoel. The present study describes the overexpression of the Triatoma brasiliensis lysozyme 1 (lys1) in Escherichia coli. Recombinant T. brasiliensis Lys1 (TbLys1) is purified after solubilization of the inclusion bodies. The protein refolds successfully, showing muramidase activity against Micrococcus lysodeikticus lyophilized cells, after enterokinase cleavage of its thioredoxin fusion protein. In in‐gel zymograms and turbidimetric liquid assays TbLys1 is broadly active under alkaline and acid conditions, indicating a possible digestive function in the two physiologically different midgut regions of the bug: the stomach and small intestine. Muramidase activity is shown in the stomach and small intestine content of unfed bugs and bugs at different days after feeding, respectively. Western blot analysis identifies TbLys1 as lysozyme.     

Cloning of cDNAs and hybridization analysis of lysozymes from two oyster species, Crassostrea gigas and Ostrea edulis

In bivalve molluscs including oysters, lysozymes play an important role in the host defense mechanisms against invading microbes. However, it remains unclear in which sites/cells the lysozyme genes are expressed and which subsequently produced the enzyme. This study cloned lysozyme cDNAs from the digestive organs of Pacific oyster Crassostrea gigas and European flat oyster Ostrea edulis. Both complete sequences of two oysters' lysozymes were composed of 137 amino acids. Two translated proteins present a high content in cysteine residues. Phylogenetic analyses showed that these oysters' lysozymes clustered with the invertebrate-type lysozymes of other bivalve species. In the Pacific oyster, lysozyme mRNA was expressed in all tissues except for those of the adductor muscle. In situ hybridization analyses revealed that lysozyme mRNA was expressed strongly in basophil cells in the digestive gland tubule of C. gigas, but not in digestive cells. Results indicated that the basophil cells of the oyster digestive gland are the sites of lysozyme synthesis.     

Characterization of six antibacterial response genes from the European corn borer (Ostrinia nubilalis) larval gut and their expression in response to bacterial challenge

Six cDNAs encoding putative antibacterial response proteins were identified and characterized from the larval gut of the European corn borer (Ostrinia nubilalis). These antibacterial response proteins include four peptidoglycan recognition proteins (PGRPs), one β-1,3-glucanase-1 (βglu-1), and one lysozyme. Tissue-specific expression analysis showed that these genes were highly expressed in the midgut, except for lysozyme. Analysis of expression of these genes in different developmental stage showed that they were expressed in larval stages, but little or no detectable expression was found in egg, pupa and adult. When larvae were challenged with Gram-negative bacteria (Enterobacter aerogenes), the expression of all six genes was up-regulated in the fatbodies. However, when larvae were challenged with Gram-positive bacteria (Micrococcus luteus), only PGRP-C and lysozyme genes were up-regulated. This study provides additional insights into the expression of antibacterial response genes in O. nubilalis larvae and helps us better understand the immune defense response in O. nubilalis.     

Ecdysone-binding proteins in haemolymph and tissues of Drosophila hydei larvae

An α-ecdysone-binding protein fraction, approx. mol. wt. 120,000, has been demonstrated in haemolymph of Drosophila hydei late third instar larvae. The protein has been partly characterized by Sephadex G-25 filtration, hydroxylapatite chromatography, density gradient centrifugation, and ezyme digestion experiments. The protein-steroid complex appears to be heat stable. Binding of labelled ecdysone to the protein fraction is significantly reduced in competition experiments using unlabelled ecdysones.An ecdysone-binding protein fraction has been detected in hand-isolated total alimentary tract tissues (predominantly midgut, Malpighian tubules, and salivary glands) and in mass-isolated midgut and Malpighian tubules. The sedimentation properties of this protein-hormone complex are similar to those of the complex found in haemolymph.     

黑水虻消化道形态及组织结构的观察

本文比较了不同发育阶段黑水虻Hermetia illucens消化道的形态学差异,掌握了幼虫消化系统的组织学特征。利用体视镜观察黑水虻5龄幼虫、预蛹及成虫的消化道形态,利用光学显微镜和扫描电镜观察幼虫消化道各段(前肠、中肠、后肠)的显微及超微结构。结果表明：黑水虻幼虫及预蛹的消化道均由前肠(食道和前胃)、中肠及后肠组成,从幼虫到成虫,消化道的长度不断缩短。与幼虫和预蛹相比,成虫消化道形态变化明显,前胃消失,出现了嗉囊及胃盲囊,中肠进一步缩短,后肠分化为回肠、结肠和直肠。组织学观察结果显示,幼虫的唾液腺开口于口腔,由膨大的管状腺体和腺管组成。食道由特化为角质刺突的内膜层及发达的肌层组成,其末端延伸至前胃。前胃膨大为球状,包括三层组织结构。根据上皮细胞形态的差异,中肠可分为四个区段。后肠薄,肠腔内褶丰富,肠壁可见数量较多的杆状细菌。马氏管开口于中、后肠交界处,包括4支盲管,管内壁密布微绒毛。黑水虻消化道形态随发育阶段的变化,反映了各阶段摄食及消化生理的差异。幼虫消化道各段具有各自典型的组织学特征,其前、中、后肠可能分别承担了食物接纳与初步消化、消化与吸收以及重吸收功能。本研究结果为进一步了...     

Lysozymes in the animal kingdom

Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme types have been identified — the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes.     

Bacteria Associated with the Ectoperitrophic Space in the Midgut of the Larva of the Midge Xylotopus par (Diptera: Chironomidae)

An ectoperitrophic association of bacteria with the midgut of Xylotopus par larvae was investigated by scanning electron microscopy and transmission electron microscopy. The bacteria attached to the epithelium as a well-defined band in the posterior midgut. They were morphotypically uniform and formed short filaments with endosporelike structures. The consistent presence and well-defined location of the bacteria in a region of the insect digestive tract usually void of microbes indicates a highly evolved symbiotic association, the nature of which is unknown at present.     

Isolation and characterization of a cDNA encoding for a lysozyme from the gut of the reduviid bug Triatoma infestans

We have isolated and characterised a Triatoma infestans cDNA encoding a lysozyme. A 174-bp fragment was amplified by PCR using degenerate oligodeoxyribonucleotide primers derived from the known amino acid sequences of lysozyme from other insects. This PCR fragment was used to screen a cDNA gut library of T. infestans. A clone containing the 3'-end of the lysozyme cDNA (219 bp) was isolated and sequenced. RACE was used to amplify the 5'-end of the lysozyme cDNA. After sequencing the complete lysozyme cDNA, the deduced 417 amino acid sequence showed high identity (40-50%) with other chicken-type lysozymes. The amino acid residues responsible for the catalytic activity and the binding of the substrate were essentially conserved. The expression pattern of the lysozyme gene in bugs at different molting and feeding states showed that this gene was upregulated in the digestive tract directly after the molt and after feeding. Additionally, this lysozyme gene was expressed differently in the different regions of the digestive tract, strongly in the cardia and stomach, the anterior regions of the midgut, and only traces of lysozyme mRNA could be detected in the small intestine, the posterior region of the midgut.     

Digestive function of lysozyme in synanthropic acaridid mites enables utilization of bacteria as a food source

The activity of lysozyme, the enzyme that hydrolyzes peptidoglycan in G⁺ bacterial cell walls, was detected in whole mite extracts (WME) and in spent growth medium extracts (SGME) of 14 species of synanthropic mites (Acari: Acaridida). The adaptation of lysozyme for digestive activity and bacteriophagy was based on: (i) high lysozyme activity in SGME, and (ii) the correlation of maximum lysozyme activity at acidic pH values, corresponding to pH in the ventriculus and caeca. We show that the digestion of fluorescein-labeled Micrococcus lysodeikticus cells began in ventriculus and continued during the passage of a food bolus through the gut. The fluorescein was absorbed by midgut cells and penetrated to parenchymal tissues. Eight species showed a higher rate of population growth on a M. lysodeikticus diet than on a control diet. The lysozyme activity in SGME was positively correlated to the standardized rate (r _s) of population growth, although no correlation was found between r _s and lysozyme activity in WME. The lysozyme activity in WME was negatively correlated to that in SGME. The highest activity of digestive lysozyme was found in Lepidoglyphus destructor, Chortoglyphus arcuatus and Dermatophagoides farinae. All of these findings indicate that lysozyme in acaridid mites possesses both defensive and digestive functions. The enzymatic properties of mite lysozyme are similar to those of the lysozymes present in the ruminant stomach and in the insect midgut.     

Two Goose-Type Lysozymes in Mytilus galloprovincialis: Possible Function Diversification and Adaptive Evolution

Two goose-type lysozymes (designated as MGgLYZ1 and MGgLYZ2) were identified from the mussel Mytilus galloprovincialis. MGgLYZ1 mRNA was widely expressed in the examined tissues and responded sensitively to bacterial challenge in hemocytes, while MGgLYZ2 mRNA was predominately expressed and performed its functions in hepatopancreas. However, immunolocalization analysis showed that both these lysozymes were expressed in all examined tissues with the exception of adductor muscle. Recombinant MGgLYZ1 and MGgLYZ2 could inhibit the growth of several Gram-positive and Gram-negative bacteria, and they both showed the highest activity against Pseudomonas putida with the minimum inhibitory concentration (MIC) of 0.95–1.91 µM and 1.20–2.40 µM, respectively. Protein sequences analysis revealed that MGgLYZ2 had lower isoelectric point and less protease cutting sites than MGgLYZ1. Recombinant MGgLYZ2 exhibited relative high activity at acidic pH of 4–5, while MGgLYZ1 have an optimum pH of 6. These results indicated MGgLYZ2 adapted to acidic environment and perhaps play an important role in digestion. Genomic structure analysis suggested that both MGgLYZ1 and MGgLYZ2 genes are composed of six exons with same length and five introns, indicating these genes were conserved and might originate from gene duplication during the evolution. Selection pressure analysis showed that MGgLYZ1 was under nearly neutral selection while MGgLYZ2 evolved under positive selection pressure with three positively selected amino acid residues (Y¹⁰², L²⁰⁰ and S²⁰²) detected in the mature peptide. All these findings suggested MGgLYZ2 perhaps served as a digestive lysozyme under positive selection pressure during the evolution while MGgLYZ1 was mainly involved in innate immune responses.     

Potential role for gut microbiota in cell wall digestion and glucoside detoxification in Tenebrio molitor larvae

Tenebrio molitor larvae were successfully reared free of cultivatable gut lumen bacteria, yeasts and fungi using two approaches; aseptic rearing from surface sterilized eggs and by feeding larvae with antibiotic-containing food. Insects were reared on a rich-nutrient complete diet or a nutrient-poor refractory diet. A comparison of digestive enzyme activities in germ free and conventional insects containing a gut microbiota did not reveal gross differences in enzymes that degrade cell walls from bacteria (lysozyme), fungi (chitinase and laminarinase) and plants (cellulase and licheninase). This suggested that microbial-derived enzymes are not an essential component of the digestive process in this insect. However, more detailed analysis of T. molitor midgut proteins using an electrophoretic separation approach showed that some digestive enzymes were absent and others were newly expressed in microbiota-free larvae. Larvae reared in antibiotic-containing refractory wheat bran diet performed poorly in comparison with controls. The addition of saligenin, the aglycone of the plant glucoside salicin, has more deleterious effects on microbiota-free larvae than on the conventionally reared larvae, suggesting a detoxifying role of midgut microbiota. Analysis of the volatile organic compounds released from the faecal pellets of the larvae shows key differences in the profiles from conventionally reared and aseptically reared larvae. Pentadecene is a semiochemical commonly found in other beetle species. Here we demonstrate the absence of pentadecene from aseptically reared larvae in contrast to its presence in conventionally reared larvae. The results are discussed in the light of the hypothesis that microbial products play subtle roles in the life of the insect, they are involved in the digestion of refractory food, detoxification of secondary plant compounds and modify the volatile profiles of the insect host.     

The crystal structure of a lysozyme c from housefly Musca domestica, the first structure of a digestive lysozyme

Lysozymes from family 22 of glycoside hydrolases are usually part of the defense system against bacteria. However in ruminant artiodactyls and saprophagous insects, lysozymes are involved in the digestion of bacteria. Here, we report the first crystallographic structure of a digestive lysozyme in its native and complexed forms, the structure of lysozyme 1 from Musca domestica larvae midgut (MdL1). Structural and biochemical data presented for MdL1 are analyzed in light of digestive lysozymes' traits. The structural core is similar, but a careful analysis of a structural alignment generated with other lysozymes c reveals that significant differences occur in coil regions. The loop from MdL1 defined by residues 98-100 has one deletion previous to residue Gln100, which leads to a less exposed conformation and might justify the resistance to proteolysis observed for MdL1. In addition, Gln100 is directly involved in a few hydrogen bonds to the ligand in a yet unobserved substrate binding mode. The pK(a)s of the MdL1 catalytic residues (Glu32 and Asp50) are lower (6.40 and 3.09, respectively) than those from Gallus gallus egg lysozyme (GgL, hen egg white lysozyme-HEWL) (6.61 and 3.85, respectively). A unique feature of MdL1 is a hydrogen bond between Thr107 Ogamma and Glu32 carboxylate group, which combined with the presence of Ser106 contributes to decrease the pK(a) of Glu32. Furthermore, in MdL1 the presence of Asn46 preventing the occurrence of an electrostatic repulsion with Asp50 and the increment in the solvent exposition of Asp50 due to Pro42 insertion contribute to reduce the pK(a) of Asp50. These structural elements affecting the pK(a)s of the catalytic residues should contribute to the acidic pH optimum presented by MdL1.     

Evolution of the bovine lysozyme gene family: Changes in gene expression and reversion of function

Recruitment of lysozyme to a digestive function in ruminant artiodactyls is associated with amplification of the gene. At least four of the approximately ten genes are expressed in the stomach, and several are expressed in nonstomach tissues. Characterization of additional lysozymelike sequences in the bovine genome has identified most, if not all, of the members of this gene family. There are at least six stomachlike lysozyme genes, two of which are pseudogenes. The stomach lysozyme pseudogenes show a pattern of concerted evolution similar to that of the functional stomach genes. At least four nonstomach lysozyme genes exist. The nonstomach lysozyme genes are not monophyletic. A gene encoding a tracheal lysozyme was isolated, and the stomach lysozyme of advanced ruminants was found to be more closely related to the tracheal lysozyme than to the stomach lysozyme of the camel or other nonstomach lysozyme genes of ruminants. The tracheal lysozyme shares with stomach lysozymes of advanced ruminants the deletion of amino acid 103, and several other adaptive sequence characteristics of stomach lysozymes. I suggest here that tracheal lysozyme has reverted from a functional stomach lysozyme. Tracheal lysozyme then represents a second instance of a change in lysozyme gene expression and function within ruminants. Correspondence to: D.M. Irwin     

The physiological role of the peritrophic membrane and trehalase: Digestive enzymes in the midgut and excreta of starved larvae of Rhynchosciara

Amylase, cellulase, trehalase, aminopeptidase and trypsin were determined using the midgut and trehalose using the haemolymph of starved and of subsequently fed larvae of Rhynchosciara americana. Midgut trehalase activity decreases steadily during starvation and increases again on feeding, whereas haemolymph trehalose titres remain constant, suggesting that trehalase is a true digestive enzyme. The decrease in amylase, cellulase and trypsin activity in the midgut during starvation is of the same order as that recovered from the excreta. Since this finding is exactly what one would expect if enzyme production stops in response to starvation, this supports the hypothesis that synthesis that synthesis of these enzymes is controlled. The excretion rate of amylase, cellulase and trypsin is very low in comparison to their activity inside the peritrophic membrane and the travel time of the food bolus through the gut. It is proposed that the peritrophic membrane separates two extracellular sites for digestion as an adaptation to conserve secreted enzymes. This could be accomplished by the existence of an endo-ectoperitrophic circulation of the enzymes involved in the initial attack on the food and by restricting to the ectoperitrophic fluid the enzymes which participate only in intermediary digestion of food.