Involvement of apolipophorin III in antibacterial defense of Galleria mellonella larvae

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. We investigated involvement of apoLp-III in the antibacterial response in Galleria mellonella larvae. Immune challenge with Gram-negative (Escherichia coli, Klebsiella pneumoniae) and Gram-positive (Micrococcus luteus) bacteria led to an increase in the level of apoLp-III in G. mellonella hemolymph, 0.5-2h and 8h after treatment, respectively. ApoLp-III purified from larval hemolymph as well as that present in hemolymph extracts adsorbed on the surface of different bacteria. The adsorption capacity of apoLp-III on bacterial cells prompted us to investigate the effect of this phenomenon on bacterial growth. Our results demonstrate antibacterial activity of apoLp-III against selected Gram-positive and Gram-negative bacteria in vitro. Among bacteria tested, Salmonella typhimurium and K. pneumoniae were the most sensitive to apoLp-III. LIVE/DEAD staining of bacteria incubated with purified apoLp-III revealed their growth inhibition; however, neither morphological changes in the cell shape nor formation of cell aggregates was noticed. The results suggest that apoLp-III is a multifunctional protein in G. mellonella hemolymph.     

Metabolism of Stored Reserves in Insect Fat Body: Hormonal Signal Transduction Implicated in Glycogen Mobilization and Biosynthesis of the Lipophorin System*

The mobilization of carbohydrate and lipid reserves from the insect fat body as fuels for migratory flight activity is controlled by adipokinetic hormone (AKH), of which in Locusta migratoria three different forms occur: AKH-I, -II and -III. In fat body in vitro, each AKH is capable of activating glycogen phosphorylase and of stimulating cAMP production, but only in the presence of extracellular Ca²⁺. The hormones stimulate both the influx and the efflux of Ca²⁺, the higher influx probably causing an increase in intracellular [Ca²⁺]. AKH enhances the production of inositol phosphates among which inositol 1,4,5-triphosphate may mediate the mobilization of Ca²⁺ from intracellular stores. Evidence is presented in favor of the occurrence of a capacitative calcium entry mechanism. Results suggest that transduction of the AKH signal occurs through stimulatory G protein-coupled receptor(s). A tentative model is presented for the interactions between the AKH signaling pathways in the locust fat body cell. AKH-induced lipid mobilization during flight requires the presence in the insect blood of high-density lipophorin (HDLp) particles and apolipophorin III (apoLp-III). Both protein components are synthesized in the fat body. In the locust, the two integral, nonexchangeable HDLp apolipophorins (apoLp-I and -II) were shown to originate from a common precursor; an mRNA of 10.3 kb seems to code for this precursor protein. The models proposed for lipophorin assembly and secretion in a number of insects are not in agreement. The exchangeable apoLp-III may occur in two or more isoforms; locust apoLp-III is secreted from the fat body as one of the two isoforms and in the hemolymph converted into the truncated second one. The rationale for this process is as yet unknown.     

Purification and Characterization of Apolipohorin-III in Cabbage Butterfly, Artogeia rapae

ABSTRACT The apoLp-III in the adult hemolymph of Artogeia rapae can associate reversibly with lipophorin. The apoLp-III was purified from the adult and larval hemolymph by KBr density gradient ultracentrifugation, gel permeation chromatography anion exchange chromatography and preparative electrophoresis (Prep Cell). ApoLp-I, ApoLp-II and apoLp-III have the molecular weights of 212 kDa, 80 kDa respectively. N-terminal sequence of apoLp-III were determined. The N-terminal amino acid sequence of apoLp-III shows 50-57% identity with those of other lepidopteran insects. apoLp-III has the antibacterial activity. Injection of bacteria increase the concentration of apoLp-III in the hemolymph, indicating that apoLp-III plays a role in insect immunity. Immunological analysis was also investigated with the anti-apoLp-III.     

An atomic force microscopy study of Galleria mellonella apolipophorin III effect on bacteria

Apolipophorin III (apoLp-III) is an abundant hemolymph protein involved in lipid transport and immune response in insects. As revealed by LIVE/DEAD staining, incubation of Gram-negative and Gram-positive bacteria in the presence of Galleria mellonella apoLp-III led to growth inhibition of selected bacteria. An atomic force microscopy (AFM) study of bacterial cells after apoLp-III treatment showed considerable alterations in the cell surface of Bacillus circulans, Klebsiella pneumoniae and Salmonella typhimurium. Our results clearly demonstrate that apoLp-III disturbed the proper structure of the bacterial cell surface. The alterations were dissimilar to those caused by cationic antimicrobial peptide, cecropin B, suggesting a different mode of action against bacteria. The present results indicate that AFM provides a powerful tool for studying the interactions of apoLp-III with microbial cells.     

Apolipophorin III is dramatically up-regulated during the programmed death of insect skeletal muscle and neurons

The intersegmental muscles (ISMs) of the tobacco hawkmoth Manduca sexta, participate in the emergence behavior of the adult moth and then die during the subsequent 30 hours. In addition, several populations of interneurons and uniquely identified motor neurons also die after adult emergence. The trigger for all of these deaths is a decline in the circulating titer of the insect molting hormone 20-hydroxyecdysone. The ability of the muscles and neurons to die requires de novo gene expression. A differential hybridization screen of a “condemned” ISM cDNA library permitted the isolation of clones encoding four new up-regulated mRNAs. On sequencing, one of these recombinants was found to encode apolipophorin III (apoLp-III), a component of lipophorin, the major hemolymph lipoprotein of insects, previously shown to be synthesized in fat body. Although apoLp-III mRNA and protein were expressed at all stages of ISM development, levels of both molecules were dramatically elevated with the commitment of the cells to die. When ISM cell death was delayed by injection of 20-hydroxyecdysone, expression of apoLp-III at both the RNA and protein levels was markedly reduced at the normal time of cell death. Immunocytochemistry demonstrated that apoLp-III protein was abundantly expressed in the cytoplasm of dying muscles, interneurons, and identified motor neurons at the time of cell death. Apolipoproteins I and II, required components of lipophorin, were not expressed at detectable levels in the muscles or neurons. Furthermore, Western blots of native gels suggest that apoLp-III was not associated with any other proteins. These data suggest that apoLp-III has activities independent of lipid transport that may play a role in programmed cell death. ApoLp-III joins apolipoproteins E and J (clusterin, sulfated glycoprotein-2) as a group of proteins that function in both lipid transfer and cell death. © 1995 John Wiley & Sons, Inc.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Cloning and expression of apolipophorin-III from the common cutworm,Spodoptera litura

We have cloned apolipophorin-III (apoLp-III) cDNA from adult fat body of Spodoptera litura. The sequence encodes a 188 amino acid polypeptide including a 22 amino acid leader peptide. The circular dichroism spectrum from the purified apoLp-III indicated a considerable content of α-helix. Sequence alignment showed that S. Litura apoLp-III has a relatively high degree of sequence identity with the apoLps-III of lepidopteran, Manduca sexta (72%), Galleria mellonella (67%), Bombyx mori (60%). These alignments with four lepidopteran apoLps-III showed highly identical residues and conservative replacements at a degree of 86%. Levels of mRNA from last instar larval fat body and adult fat body were compared through Northern blot analysis using ³²P-labeled 704 bp apoLp-III cDNA probe. A 850 bp mRNA was detected in both stages and mRNA level of day 1 adult fat body was much higher than that of last instar larval fat body. The tissue-distribution of apoLp-III mRNA in adult ovary and testis was also examined and we confirmed the presence of apoLp-III mRNA in ovary and testis although apoLp-III was expressed in these tissues at very low levels compared with the adult fat body. Arch. Insect Biochem. Physiol. 39:166–173, 1998. © 1998 Wiley-Liss, Inc.     

RNA interference mediated knockdown of apolipophorin-III leads to knockdown of manganese superoxide dismutase in Hyphantria cunea

Apolipophorin-III (apoLp-III), a hemolymph protein that facilitates lipid transport in aqueous media in insects was recently shown to play a role in insect immune activation. Here, we report another novel possible function of apoLp-III in insects. To identify genes affected by apoLp-III expression in larvae, we decreased endogenous apoLp-III mRNA in Hyphantria cunea (Hc) through RNA interference; subsequently, we observed lower levels of antioxidant enzymes, including manganese superoxide dismutase (MnSOD), glutathione S-transferase, and immune proteins. Knockdown of Hc apoLp-III led to decreased MnSOD expression in fat body tissues and elevated superoxide anion levels in Hc fat body cells, suggesting that Hc apoLp-III is involved in the action and/or expression of antioxidant enzymes, especially MnSOD.     

Localization of injected apolipophorin III in vivo - new insights into the immune activation process directed by this protein

A few years ago, it was shown that intrahemocoelic injection of the insect apolipoprotein apolipophorin III (apoLp-III) stimulates an immune response in larvae of the greater wax moth, Galleria mellonella. Since the mode of action of this activation process is unknown, we followed apoLp-III's pathway in the early phase of the immune-stimulating process, using biotin as a probe. Biotinylated apoLp-III was injected and localized using avidin-coupled horseradish peroxidase. The labeled protein was fully functional; the added amount of biotin per apoLp-III molecule used in this study only slightly decreased its ability to associate with phospholipase C-treated human low-density lipoprotein, as well as the immune-stimulating capability of apoLp-III.Gel electrophoresis with subsequent staining of biotin moieties and lipids revealed that apoLp-III undergoes lipid association in vivo within the first few minutes after injection. After two hours, no biotinylated apoLp-III was detectable in cell-free hemolymph. At this time, a subpopulation of hemocytes showed a distinct peroxidase staining. Control injections of biotinylated bovine serum albumin did not lead to similar results, giving evidence for the specificity of the phenomena observed. The results indicate that lipid association of apoLp-III occurs prior to endocytosis by immune-competent hemocytes, which is followed by the induction of a humoral immune response.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Apolipophorin III: role model apolipoprotein

It has been one-quarter century since the identification of apolipophorin III (apoLp-III) as an important component of insect hemolymph lipid transport processes. Original studies of flight-related lipid transport that led to the discovery of apoLp-III have been followed by detailed studies of its structure and function relations, species distribution as well as its physiological roles beyond lipid transport. The non-exchangeable apoLp-I and -II, which are derived from a common precursor, are structural protein components of the multifunctional lipophorin particle. ApoLp-I/II have been identified as members of a broad lipid-binding protein family based on sequence similarities with their vertebrate counterparts. By contrast, apoLp-III can be found as a lipid-free hemolymph protein that associates with lipophorin during hormone-induced lipid mobilization. Based on structural characterization, apoLp-III belongs to a large family of exchangeable apolipoproteins characterized by segments of amphipathic alpha-helix. The remarkable structural adaptability of apoLp-III can be ascribed to its globular amphipathic alpha-helix bundle conformation wherein hydrophobic lipid-binding regions are stabilized in the absence of lipid by helix-helix interactions. Upon exposure to potential lipid surface-binding sites, the globular helix bundle opens to expose its hydrophobic interior permitting substitution of helix-helix contact in the bundle for helix-lipid interactions. Novel functions of apoLp-III beyond lipid transport have been identified recently. The expanding role of apoLp-III in innate immunity promises to offer exciting research opportunities in the future.     

Galleria mellonella apolipophorin III – an apolipoprotein with anti-Legionella pneumophila activity

The greater wax moth Galleria mellonella has been exploited worldwide as an alternative model host for studying pathogenicity and virulence factors of different pathogens, including Legionella pneumophila, a causative agent of a severe form of pneumonia called Legionnaires' disease. An important role in the insect immune response against invading pathogens is played by apolipophorin III (apoLp-III), a lipid- and pathogen associated molecular pattern-binding protein able to inhibit growth of some Gram-negative bacteria, including Legionella dumoffii. In the present study, anti-L. pneumophila activity of G. mellonella apoLp-III and the effects of the interaction of this protein with L. pneumophila cells are demonstrated. Alterations in the bacteria cell surface occurring upon apoLp-III treatment, revealed by Fourier transform infrared (FTIR) spectroscopy and atomic force microscopy, are also documented. ApoLp-III interactions with purified L. pneumophila LPS, an essential virulence factor of the bacteria, were analysed using electrophoresis and immunoblotting with anti-apoLp-III antibodies. Moreover, FTIR spectroscopy was used to gain detailed information on the type of conformational changes in L. pneumophila LPS and G. mellonella apoLp-III induced by their mutual interactions. The results indicate that apoLp-III binding to components of bacterial cell envelope, including LPS, may be responsible for anti-L. pneumophila activity of G. mellonella apoLp-III.     

Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.     

Adipokinetic response of a flightless grasshopper (Barytettix psolus): Functional components,defective response

The mature flightless grasshopper Barytettix psolus shows a very small adipokinetic response when injected with extracts of its own corpora cardiaca, although the fat body contains enough lipid for a strong response. When these extracts were injected into Melanoplus differentialis, a grasshopper capable of flight, or the moth Manduca sexta, much stronger adipokinetic responses were observed. Upon analysis of B. psolus extracts by HPLC, two components with adipokinetic activity were obtained. The major component appears to be identical to locust adipokinetic hormone (AKH) I. Extracts of B. psolus corpora cardiaca also activated fat body glycogen phosphorylase in B. psolus. This activation, however, did not result in an increase in hemolymph sugar, probably because of low levels of glycogen in the fat body. B. psolus hemolymph contains a high-density lipophorin (HDLp) consisting of the apolipophorins (apoLp) I and II and lipid. Both apoproteins are glycosylated. The hemolymph also contains apoLp-III, although this apoprotein apparently does not associate with HDLp to form a low-density lipophorin (LDLp) following AKH or corpora cardiaca extract injections. When B. psolus lipophorin and AKH were injected into Schistocerca americana, lipophorin took up lipids and combined with apoLp-III, forming LDLp. ApoLp-III from B. psolus injected into S. americana can also form LDLp, demonstrating that the components are functional. A lipid transfer particle isolated from M. sexta and injected into B. psolus does not improve the adipokinetic response. Thus, it appears that the adipokinetic response of B. psolus is not deficient because of the lack of AKH or functional lipophorins, but may be caused by the lack of a full response to AKH by fat body or the deficiency in hemolymph of some as yet unknown factor.     

The lipid composition of Legionella dumoffii membrane modulates the interaction with Galleria mellonella apolipophorin III

Apolipophorin III (apoLp-III), an insect homologue of human apolipoprotein E (apoE), is a widely used model protein in studies on protein–lipid interactions, and anti-Legionella activity of Galleria mellonella apoLp-III has been documented. Interestingly, exogenous choline-cultured Legionella dumoffii cells are considerably more susceptible to apoLp-III than non-supplemented bacteria. In order to explain these differences, we performed, for the first time, a detailed analysis of L. dumoffii lipids and a comparative lipidomic analysis of membranes of bacteria grown without and in the presence of exogenous choline. ³¹P NMR analysis of L. dumoffii phospholipids (PLs) revealed a considerable increase in the phosphatidylcholine (PC) content in bacteria cultured on choline medium and a decrease in the phosphatidylethanolamine (PE) content in approximately the same range. The interactions of G. mellonella apoLp-III with lipid bilayer membranes prepared from PLs extracted from non- and choline-supplemented L. dumoffii cells were examined in detail by means of attenuated total reflection- and linear dichroism-Fourier transform infrared spectroscopy. Furthermore, the kinetics of apoLp-III binding to liposomes formed from L. dumoffii PLs was analysed by fluorescence correlation spectroscopy and fluorescence lifetime imaging microscopy using fluorescently labelled G. mellonella apoLp-III. Our results indicated enhanced binding of apoLp-III to and deeper penetration into lipid membranes formed from PLs extracted from the choline-supplemented bacteria, i.e. characterized by an increased PC/PE ratio. This could explain, at least in part, the higher susceptibility of choline-cultured L. dumoffii to G. mellonella apoLp-III.