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.     

Apolipophorin-III affects the activity of the haemocytes of Galleria mellonella larvae

Apolipophorin-III (apoLp-III) impaired the adhesion of plasmatocytes and a granular cell-subpopulation of larval Galleria mellonella to glass slides. The protein bound to haemocytes, limited the responses of the plasmatocytes to Bacillus subtilis and increased the percentage of a subgroup of granular cells with adhering bacteria. The total number of bacteria adhering to all the haemocytes on the slides declined. Injections of apoLp-III slowed bacterial removal from the haemolymph without affecting total haemocyte counts and impaired haemocyte attachment to glass slides. Purified apoLp-III bound to B. subtilis. ApoLp-III in serum bound to bacteria within 5 min, peaked at 15 min and was either shed or dissociated by 60 min. ApoLp-III bound to B. subtilis lowered the adhesion of the bacteria to the haemocytes and slowed the removal of the bacteria from the haemolymph.     

Choline and betaine ameliorate liver lipid accumulation induced by vitamin B6 deficiency in rats

We investigated the efficacy of supplementing the diet with choline or betaine in ameliorating lipid accumulation induced by vitamin B₆ (B₆) deficiency in rat liver. Male Wistar rats were fed a control, B₆-deficient, choline-supplemented (2, 4, or 6 g choline bitartrate/kg diet) B₆-deficient diet or betaine-supplemented (1, 2, or 4 g betaine anhydrous/kg diet) B₆-deficient diet for 35 d; all diets contained 9 g L-methionine (Met)/kg diet. Choline or betaine supplementation attenuated liver lipid deposition and restored plasma lipid profiles to control levels. These treatments restored the disruptions in Met metabolism and the phosphatidylcholine (PC)/phosphatidylethanolamine (PE) ratio induced by B₆ deficiency in liver microsomes. These results suggest that choline and betaine ameliorated liver lipid accumulation induced by B₆ deficiency via recovery of Met metabolism and very low-density lipoprotein secretion by restoring the supply of PC derived from PE.     

Factors affecting the reaggregation of rat brain microsomes solubilized with octyl glucoside and their relationship with the base-exchange activity of reaggregates

Rat brain microsomal membranes disaggregated by exposure to octyl glucoside were recovered by centrifugation after dialytic removal of the detergent. The composition of the dialysis medium (divalent cations, pH) was important to this effect; indeed, the reaggregation process which occurred during the dialytic step required the presence of either Ca²⁺ or Mg²⁺ and a slightly acidic pH. The lipid protein/ratio and choline and ethanolamine base-exchange of recovered particles depended on the conditions of dialysis although their lipid composition did not. The lipid composition of membranes was also varied by adding PE or PC to octyl glucoside-microsome suspensions. This treatment produced reaggregates possessing a low content of cholesterol and varying PC/PE ratios. Both choline and ethanolamine base-exchange activities were related to this parameter.     

Multiple phospholipid N-methyltransferases with distinct substrate specificities are encoded in Bradyrhizobium japonicum

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a few prokaryotes including members of the family Rhizobiaceae. In these bacteria, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum. At least two different phospholipid N-methyltransferases (PmtA and PmtX) have been postulated to convert phosphatidylethanolamine (PE) to PC in B. japonicum by three consecutive methylation reactions. However, apart from the known PmtA enzyme, we identified and characterized three additional pmt genes (pmtX1, pmtX3, and pmtX4), which can be functionally expressed in Escherichia coli, showing different substrate specificities. B. japonicum expressed only two of these pmt genes (pmtA and pmtX1) under all conditions tested. PmtA predominantly converts PE to monomethyl PE, whereas PmtX1 carries out both subsequent methylation steps. B. japonicum is the first bacterium known to use two functionally different Pmts. It also expresses a PC synthase, which produces PC via condensation of CDP-diacylglycerol and choline. Our study shows that PC biosynthesis in bacteria can be much more complex than previously anticipated.     

The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids

Legionella micdadei is responsible for community- or nosocomial-acquired pneumonia as well as the influenza-like illness Pontiac fever. The aim of this study was to investigate the ability of L. micdadei to utilize extracellular choline for phosphatidylcholine (PC) synthesis and its consequences for the phospholipid composition of its membrane system and the interaction with the human LL-37 peptide. Comparative analysis of the PC content using isotopic labeling revealed that in presence of exogenous choline 98% of the total PC was synthesized via the Pcs pathway while the remaining 2% were generated via the PE-methylation (PmtA) pathway. PC species were to a greater extent defined by the Pcs pathway in the outer membrane than in the inner membrane. While no major changes in the bacterial lipid content were observed using ³¹P NMR, indication for utilization of longer acyl chains and slight increase of PG in response to choline addition was observed by a top-down lipidomics screen. The LL-37 peptide inhibited L. micdadei growth in a dose-dependent manner. Bacteria cultured with exogenous choline were more sensitive to the LL-37 peptide when compared to the standard culture condition. Our biophysical investigations show that the peptide perturbs bacterial-derived phospholipid monolayers and this interaction is dependent on the molar portion of PC. This interaction is responsible for the observed changes in the anti-L. micdadei activity of the LL-37 peptide.     

Apparent relative retention of the phosphatidylethanolamine molecular species 18:0–20:5(n−3), 16:0–22:6(n−3) and the sum 16:0–20:4(n−6) plus 16:0–20:3(n−9) in the liver microsomes of pig on an essential fatty acid deficient diet

Attempts at a better understanding of the cell membrane organization and functioning need to assess the physical properties which partly depend (i) on the positional distribution of the fatty acids in the membrane phospholipids (PLs) and (ii) on the way by which the PL molecular species are affected by exogenous fatty acids. To do that, the effects of essential (polyunsaturated) fatty acid (EFA) deficiency and enrichment were studied in the liver microsomes of piglets feeding on either an EFA-deficient diet or an EFA-enriched diet containing hydrogenated coconut oil or a mixture of soya + corn oils, respectively. After derivatization, the diacylated forms of choline and ethanolamine PLs were analyzed using a combination of Chromatographic techniques and fast-atom bombardmentmass spectrometry. The dinitrobenzoyl-diacylglycerol derivatives corresponding to the molecular species of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were identified. It appears that three factors brought about a marked apparent relative retention: the nature of (i) the base of the polar head, (ii) fatty acids at the sn−1 position and (iii) fatty acids at the sn−2 position. The highest apparent relative retentions were displayed by the 18:0–20:5(n−3)-PE and 16:0–22:6(n−3)-PE. It is noteworthy that the behavior of 20:3 n−9 — which is synthesized during the EFA-deficient diet by the same bioconversion system as 20:4 n−6 — was very similar to that of 20:4 n−6 during the formation of PC and PE molecular species and that the molecular species of PE containing 20:4(n−6) and 20:3(n−9), gathered together as metabolical homologues, were also apparently retained, particularly in association with 16:0. Present observations are consistent with some others showing retention or preferential distribution of EFA in PE and suggest that specific acyltransferase(s), ethanolamine phosphotransferase and methyltransferase would be mainly involved for PE and PC formation in liver endoplasmic reticulum. Fast-atom bombardment-mass spectrometry of intact phospholipids enables us to show that there is no very long chain dipolyunsaturated phospholipid in liver endoplasmic reticulum.     

Control of phosphatidylethanolamine metabolism in yeast: Diacylglycerol ethanolaminephosphotransferase and diacylglycerol cholinephosphotransferase are separate enzymes

Membrane preparations from Saccharomyces cerevisiae catalyze the transfer of phosphoethanolamine and phosphocholine from the cytidine dinucleotide derivatives to endogenous and exogenous 1,2-diacylglycerols. Utilizing CDP-[¹⁴C]ethanolamine and CDP-[¹⁴C]choline as isotopic substrates, diacylglycerol ethanolaminephosphotransferase (EPT) and diacylglycerol Cholinephosphotransferase (CPT) have been characterized in vitro. Both enzymes (i) require Mn²⁺; (ii) are stimulated by exogenous 1,2-diacylglycerols; and (iii) are inhibited by p-hydroxymercuribenzoate and CMP. Yeast EPT and CPT can be clearly distinguished on the basis of their different (i) pH optima; (ii) thermal sensitivities at 50 °C; (iii) concentration-dependent inhibition by CMP; and (iv) sensitivities to the hypolipidemic drug, DH-990. Reversibility experiments demonstrate that CDP-ethanolamine can be resynthesized by enzymatic reactions involving CMP and Phosphatidylethanolamine (PE) formed from the cytidine dinucleotide derivative or by the decarboxylation of phosphatidylserine (PS). Similarly, CDP-choline can be reformed by the reaction of CMP with PC synthesized from CDP-choline or by the sequential N-methylation of PE. A double-isotope experiment provides evidence that PE molecules synthesized via CDP-ethanolamine or by the decarboxylation of PS are converted to phosphatidylcholine (PC) by the methylation pathway at similar, if not identical, rates. The N-methylation of the metabolically specific pool of PE, synthesized from CDP-ethanolamine, is drastically reduced in membranes prepared from choline-grown cells. Neither EPT nor CPT appear to be induced by the addition of ethanolamine or choline, respectively, to the growth medium. However, the addition of 10 mm choline to the growth medium results in a 46% reduction in EPT activity. This change in EPT activity may be a regulatory response to lower rates of PE N-methylation in choline-grown cells.     

Phosphatidylethanolamine synthesis is required for optimal virulence of Brucella abortus

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell.     

Methylglyoxal-mediated Gpd1 activation restores the mitochondrial defects in a yeast model of mitochondrial DNA depletion syndrome

Human MPV17, an evolutionarily conserved mitochondrial inner-membrane channel protein, accounts for the tissue-specific mitochondrial DNA depletion syndrome. However, the precise molecular function of the MPV17 protein is still elusive. Previous studies showed that the mitochondrial morphology and cristae organization are severely disrupted in the MPV17 knockout cells from yeast, zebrafish, and mammalian tissues. As mitochondrial cristae morphology is strictly regulated by the membrane phospholipids composition, we measured mitochondrial membrane phospholipids (PLs) levels in yeast Saccharomyces cerevisiae MPV17 ortholog, SYM1 (Stress-inducible Yeast MPV17) deleted cells. We found that Sym1 knockout decreases the mitochondrial membrane PL, phosphatidyl ethanolamine (PE), and inhibits respiratory growth at 37 ?C on rich media. Both the oxygen consumption rate and the steady state expressions of mitochondrial complex II and super-complexes are compromised. Apart from mitochondrial PE defect a significant depletion of mitochondrial phosphatidyl-choline (PC) was noticed in the sym1? cells grown on synthetic media at both 30 ?C and 37 ?C temperatures. Surprisingly, exogenous supplementation of methylglyoxal (MG), an intrinsic side product of glycolysis, rescues the respiratory growth of Sym1 deficient yeast cells. Using a combination of molecular biology and lipid biochemistry, we uncovered that MG simultaneously restores both the mitochondrial PE/PC levels and the respiration by enhancing cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase 1 (Gpd1) enzymatic activity. Further, MG is incapable to restore respiratory growth of the sym1?gpd1? double knockout cells. Thus, our work provides Gpd1 activation as a novel strategy for combating Sym1 deficiency and PC/PE defects.     

Unconventional membrane lipid biosynthesis in Xanthomonas campestris

All bacteria are surrounded by at least one bilayer membrane mainly composed of phospholipids (PLs). Biosynthesis of the most abundant PLs phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) is well understood in model bacteria such as Escherichia coli. It recently emerged, however, that the diversity of bacterial membrane lipids is huge and that not yet explored biosynthesis pathways exist, even for the common PLs. A good example is the plant pathogen Xanthomonas campestris pv. campestris. It contains PE, PG and CL as major lipids and small amounts of the N‐methylated PE derivatives monomethyl PE and phosphatidylcholine (PC = trimethylated PE). Xanthomonas campestris uses a repertoire of canonical and non‐canonical enzymes for the synthesis of its membrane lipids. In this minireview, we briefly recapitulate standard pathways and integrate three recently discovered pathways into the overall picture of bacterial membrane biosynthesis.     

Melittin-Induced Lipid Extraction Modulated by the Methylation Level of Phosphatidylcholine Headgroups

Protein- and peptide-induced lipid extraction from membranes is a critical process for many biological events, including reverse cholesterol transport and sperm capacitation. In this work, we examine whether such processes could display specificity for some lipid species. Melittin, the main component of dry bee venom, was used as a model amphipathic α-helical peptide. We specifically determined the modulation of melittin-induced lipid extraction from membranes by the change of the methylation level of phospholipid headgroups. Phosphatidylcholine (PC) bilayers were demethylated either by substitution with phosphatidylethanolamine (PE) or chemically by using mono- and dimethylated PE. It is shown that demethylation reduces the association of melittin with membranes, likely because of the resulting tighter chain packing of the phospholipids, which reduces the capacity of the membranes to accommodate inserted melittin. This reduced binding of the peptide is accompanied by an inhibition of the lipid extraction caused by melittin. We demonstrate that melittin selectively extracts PC from PC/PE membranes. This selectivity is proposed to be a consequence of a PE depletion in the surroundings of bound melittin to minimize disruption of the interphospholipid interactions. The resulting PC-enriched vicinity of melittin would be responsible for the observed formation of PC-enriched lipid/peptide particles resulting from the lipid efflux. These findings reveal that modulating the methylation level of phospholipid headgroups is a simple way to control the specificity of lipid extraction from membranes by peptides/proteins and thereby modulate the lipid composition of the membranes.     

Differences in the pattern of attack of acidic,neutral, and basic phospholipases A2 of A. halys blomhofii on human erythrocyte membranes: Problems in interpretation of phospholipid location

Purified acidic (pI 4.9), neutral (pI 6.9), and basic (pI 8.7) phospholipase A₂ from Agkistrodon halys blomhofii showed characteristically different patterns of hemolysis and phospholipid hydrolysis of intact human erthyrocytes. Acidic and neutral enzymes were nonlytic in the early periods of incubations with intact erythrocytes whereas the basic enzyme caused immediate hemolysis (5–8%). Under nonlytic conditions acidic and neutral enzymes hydrolyzed only phosphatidyl choline (PC) (20 and 50%, respectively), whereas basic enzyme hydrolyzed not only PC (60%) but nearly 15% of the phosphatidylethanolamine (PE). Both PC and PE were hydrolyzed significantly when the three phospholipases A₂ were incubated individually with erythrocyte lysate or hypotonic ghosts (sealed or unsealed). The order of substrate preference for acidic and neutral enzymes was always PC > PE. On the contrary basic enzyme exhibited the property of substrate specificity reversal. It hydrolyzed PC faster than PE when the membranes were sealed whereas PE hydrolysis was faster than PC hydrolysis in unsealed membranes. Interestingly only the basic enzyme showed activity in the absence of Ca²⁺ and in the presence of 0.5 mm EDTA. Phospholipase C (Bacillus cereus or Clostridium perfringens) did not show the property of substrate specificity reversal although their ability to hydrolyze PC and PE was different. In general this study demonstrates the unique activity patterns of three physically different pure phospholipases A₂ on human erythrocyte membranes which could be of value in selectively modifying membrane phospholipids. In addition it also throws an important light on the fact that results obtained with phospholipases should be interpreted with caution particularly as regards the localization of phospholipids in membranes.     

Feedback regulation of CTP:phosphocholine cytidylyltransferase translocation between cytosol and endoplasmic reticulum by phosphatidylcholine

The mechanism for the increased association of CTP:phosphocholine cytidylyltransferase (CT) with membranes of hepatocytes derived from choline-deficient, compared with choline-supplemented rats, has been investigated. The cells were maintained in culture for 4 h in a choline- and methionine-deficient medium. (Methionine is required for synthesis of phosphatidylcholine (PC) via methylation of phosphatidylethanolamine.) Afterward, the cells were incubated +/- choline for various times up to 4 h. In the presence, but not in the absence, of choline there was a translocation of CT activity from membranes to cytosol. During this time period there was no change in the amounts of unesterified fatty acids or diacylglycerol recovered from the hepatocytes. In addition, there was no evidence for a difference in the incorporation of 32P into CT or other cytosolic proteins isolated from hepatocytes +/- choline. In contrast, there was a highly significant correlation between the concentration of PC in the membranes and the increased activity of CT in the cytosol (R = 0.98) and the decreased activity in the membranes (R = 0.93). The concentration of PC could alternatively be altered by incubation of the choline-deficient hepatocytes with methionine or lyso-PC. With either of these supplementations highly significant correlation coefficients were observed between the concentration of PC in membranes and decreased activity of CT in membranes or increased activity in cytosol. The concentration of PC was reduced in the endoplasmic reticulum, but not the Golgi membranes, isolated from choline-deficient compared with choline-supplemented livers. The data suggest that the amount of PC in the endoplasmic reticulum feedback regulates the amount of CT associated with this membrane.     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Intracellular localization of phosphatidylcholine and phosphatidylethanolamine synthesis in cotyledons of cotton seedlings

Subfractionation of clarified cotyledon homogenates of cotton (Gossypium hirsutum L.) seedlings on sucrose gradients revealed a single coincident peak of cholinephosphotransferase (EC 2.7.8.2) (CPT) and ethanolaminephosphotransferase (EC 2.7.8.1) (EPT) activities, which equilibrated with the main peak of Antimycin A-insensitive NADH:cytochrome c reductase (CCR) activity. The small percentage of CPT and EPT activities (less than 5% of the total) in glyoxysome-enriched pellets equilibrated with cytochrome c oxidase activity, not with catalase activity. Preincubation of microsomes (containing 83% of total CPT and EPT activities) in 0.2 millimolar MgCl₂ followed by subfractionation on sucrose gradients resulted in peak CPT and EPT activities equilibrating with peak CCR activity at 24% (w/w) sucrose. Preincubation of microsomes with ¹⁴C-CDPcholine (or ¹⁴C-CDPethanolamine) resulted in synthesis and incorporation of ¹⁴C-phosphatidylcholine (PC) (or ¹⁴C-phosphatidylethanolamine, PE) into membranes at the same density. Increasing the Mg²⁺ concentration to 2.0 millimolar facilitated binding of ribosomes and caused a concomitant shift in density (to 34% w/w sucrose) of peak CPT, EPT, and CCR activities. Under these conditions, newly synthesized and incorporated ¹⁴C-PC (or PE) was recovered in these membranes. Transmission electron microscopy of this fraction confirmed binding of ribosomes to membranes. Radiolabeling in vivo of cotyledons with [methyl-¹⁴C] choline chloride or [1,2 ethanolamine-¹⁴C] ethanolamine hydrochloride resulted in a linear incorporation of radiolabel into PC or PE in a time dependent manner. Subfractionation of homogenates of radiolabeled cotyledons on sucrose gradients showed that membranes sedimenting at 24% (w/w) sucrose (ER) contained the majority of radiolabeled PC and PE with a minor peak at 40% (w/w) sucrose (mitochondria), but no radioactive PC or PE was recovered in glyoxysomes. These results indicate that ER in cotyledons of germinated cotton seedlings is the primary subcellular site of PC and PE synthesis. This is similar to the situation in endosperm tissue but distinctly different from root and hypocotyl tissue where Golgi are a major subcellular site of PC and PE synthesis.     

Dietary phosphatidylcholine supplementation reduces atherosclerosis in Ldlr−/− male mice2

Choline is an essential nutrient required for various biological processes. Eggs, dairy, and meat are rich in phosphatidylcholine (PC), whereas cereal and legumes are rich in free choline. Excess dietary choline leads to increase plasma trimethylamine N-oxide (TMAO). Epidemiological studies suggest that plasma TMAO is a biomarker for atherosclerosis and it has been suggested that a lower intake of eggs and meat would reduce choline consumption and thus reduce atherosclerosis development. To investigate whether the form of dietary choline influences atherosclerosis development in Ldlr^−/−, we randomly fed Ldlr^−/−male mice (aged 8 – 10 wk) one of the three 40% (calories) high fat diets (with 0.5% w/w of cholesterol): Control (0.1% w/w free-choline, CON), choline-supplemented (0.4% free-choline, CS), or PC-supplemented (0.1% free-choline and 0.3% choline from PC, PCS). After 12-wk of dietary intervention, the animals were euthanized and tissues and blood collected. Aortic atherosclerotic plaque area, plasma choline, lipid metabolites, and spleen and peripheral blood cell phenotypes were quantified. Surprisingly, the PCS group had significantly lower atherosclerotic lesions while having 2-fold higher plasma TMAO levels compared with both CON and CS groups (P<0.05). In the fasting state, we found that PCS decreased plasma very low-density lipoprotein-cholesterol (VLDL-C) and apolipoprotein B48 (APOB48), and increased plasma high-density lipoprotein-cholesterol (HDL-C). However, very low-density lipoprotein (VLDL) secretion was not affected by dietary treatment. We observed lower levels of circulating pro-atherogenic chemokines in the PCS group. Our study suggests that increased dietary PC intake does not induce a pro-atherogenic phenotype.     

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

The role of Pseudomonas aeruginosa elastase B in activation of the humoral immune response in Galleria mellonella larvae was investigated. The results of our study showed that elastase B injected at a sublethal concentration was responsible for eliciting the humoral immune response in G. mellonella larvae. The insects exhibited increased antibacterial activity, namely, we observed appearance of antimicrobial peptides and a higher level of lysozyme in cell-free hemolymph. Elastase B seems to be a more potent elicitor than thermolysin because similar maximal antibacterial activity levels were observed at a 5-fold lower concentration. We also demonstrated that there were differences in the kinetics of induction of antimicrobial activity between thermolysin and elastase B. The maximum level was observed 18 h post challenge of thermolysin and 38 h after injection of elastase B. It was also shown that, 24 h after elastase injection, the relative levels of apoLp-III in the hemolymph significantly increased in comparison with control G. mellonella larvae. The activation of immune responses in metalloproteinase-challenged larvae involved synthesis of metalloproteinase inhibitors which increased the survival rates of insects both against the lethal dose of thermolysin as well as against viable pathogenic bacterial cells of P. aeruginosa.