Immune response of Galleria mellonella after injection with non-lethal and lethal dosages of Candida albicans.

The immune response of Galleria mellonella to injection with non-lethal and lethal dosages of Candida albicans was compared. Larvae infected with the non-lethal dosage (2 × 104 cells/larva) did not show significant morphological changes, while those infected with the lethal dosage (2 × 105 cells/larva) showed inhibition of motility and cocoon formation and became darker around the area of injection after 24 h. While the administration of the lower dosage caused approx. 5- and 20-fold induction of genes for gallerimycin and galiomycin, respectively, the injection with the higher dosage induced approx. 25 and 120-fold expression of the respective genes. Similar differences were obtained for the insect metalloproteinase inhibitor (IMPI) and hemolin gene transcripts. The relatively low level of immune gene expression was confirmed by an assay of hemolymph antifungal activity, which was detected only in larvae infected with lethal dosage of C. albicans. Furthermore, greater amounts of immune-inducible peptides were detected in the hemolymph extracts in the same group of larvae. The stronger humoral immune response was not correlated with survival. Phenol oxidase (PO) activity was induced only in the hemolymph of larvae infected with the non-lethal dose; injection of the lethal dose resulted in strong inhibition of this enzyme after 24 h. We showed that PO is susceptible to regulation by immune priming with the non-lethal dose of C. albicans. The activity of this enzyme was enhanced in primed larvae at the time of re-injection. When both primed and non-primed larvae received 2 × 105 cells, the inhibition of PO was stronger in the primed group. G. mellonella infected with the lethal dose of C. albicans died despite the strong induction of humoral defence mechanisms. The priming-enhanced activity of PO was correlated with increased resistance to subsequent infection.

Host-pathogen interaction after infection of Galleria mellonella with the filamentous fungus Beauveria bassiana.

The filamentous fungus Beauveria bassiana is a natural pathogen of the greater wax moth Galleria mellonella. Infection with this fungus triggered systemic immune response in G. mellonella; nevertheless, the infection was lethal if spores entered the insect hemocel. We observed melanin deposition in the insect cuticle and walls of air bags, while the invading fungus interrupted tissue continuity. We have shown colonization of muscles, air bags, and finally colonization and complete destruction of the fat body-the main organ responsible for the synthesis of defense molecules in response to infection. This destruction was probably not caused by simple fungal growth, because the fat body was not destroyed during colonization with a human opportunistic pathogen Candida albicans. This may mean that the infecting fungus is able to destroy actively the insect's fat body as part of its virulence mechanism. Finally, we were unable to reduce the extremely high virulence of B. bassiana against G. mellonella by priming of larvae with thermally inactivated fungal spores.

Host-pathogen interactions upon the first and subsequent infection of Galleria mellonella with Candida albicans.

Insects are able to develop enhanced resistance in response to repeated infection. This phenomenon is called immune priming. In this work, so-called "primed" Galleria mellonella larvae were re-infected with a lethal dose of Candida albicans 48 h after injection of a non-lethal dose, while "non-primed" larvae were infected only with a lethal dose. The increased resistance of the primed larvae correlated with a slower rate of body colonisation by the fungus. Changes in the protein profiles were detected in the whole hemolymph of the primed insects. The analysis of low-molecular weight proteins and peptides obtained with the use of three different organic solvents and comparative quantitative HPLC analysis thereof showed that the primed larvae did not have higher amounts of any infection-inducible polypeptides than the non-primed larvae. Moreover, electrophoresis of low-molecular weight polypeptides revealed an even lower level of immune-induced peptides in the primed larvae than in the non-primed ones. Furthermore, the defence activity of larval hemolymph, i.e. the antifungal, antibacterial, and lysozyme-type activity, was up-regulated in the primed larvae at the time of re-infection and, consequently, at the early time points after the infection with the lethal dose. Twenty four hours after the infection, these parameters were equally high in the non-primed and primed larvae. Accordingly, at the time of the injection of the lethal dose, certain immune-inducible genes were up-regulated. However, 24 h after the infection with the lethal dose, their expression in both groups was incomparably higher than at the time of the infection and, in most cases, it was as high in the primed larvae as in the non-primed ones. We found that only anti yeast-like activity was enhanced 24 h after the re-infection. This correlated with results obtained by testing the priming effect in heterologous systems: the primed animals did not exhibit higher resistance to the other pathogens tested.

Humoral immune response of Galleria mellonella after repeated infection with Bacillus thuringiensis.

The insect immune system relies on innate mechanisms only. However, there is an increasing number of data reporting that previous immune challenge with microbial elicitors or a low number of microorganisms can modulate susceptibility after subsequent lethal infection with the same or different pathogen. This phenomenon is called immune priming. Its biochemical and molecular mechanisms remain unravelled. Here we present that Galleria mellonella larvae that survived infection induced by intrahemocelic injection of a low dose of Bacillus thuringiensis were more resistant to re-injection of a lethal dose of the same bacteria but not other bacteria and fungi tested. This correlated with enhanced activity detected in full hemolymph as well as in separated hemolymph polypeptides. In addition, we observed differences in the hemolymph protein pattern between primed and non-primed larvae after infection with the lethal dose of B. thuringiensis. Expression of genes encoding inducible defence molecules was not enhanced in the primed larvae after the infection with the lethal dose of B. thuringiensis. It is likely that priming affects the turnover of immune related hemolymph proteins; hence, upon repeated contact, the immune response may be more ergonomic.

Expression of the insect metalloproteinase inhibitor IMPI in the fat body of Galleria mellonella exposed to infection with Beauveria bassiana.

The inducible metalloproteinase inhibitor (IMPI) discovered in Galleria mellonella is currently the only specific inhibitor of metalloproteinases found in animals. Its role is to inhibit the activity of metalloproteinases secreted by pathogenic organisms as virulence factors to degrade immune-relevant polypeptides of the infected host. This is a good example of an evolutionary arms race between the insect hosts and their natural pathogens. In this report, we analyze the expression of a gene encoding an inducible metalloproteinase inhibitor (IMPI) in fat bodies of the greater wax moth larvae Galleria mellonella infected with an entomopathogenic fungus Beauveria bassiana. We have used a natural infection, i.e. covering larval integument with fungal aerospores, as well as injection of fungal blastospores directly into the larval hemocel. We compare the expression of IMPI with the expression of genes encoding proteins with fungicidal activity, gallerimycin and galiomycin, whose expression reflects the stimulation of Galleria mellonella defense mechanisms. Also, gene expression is analyzed in the light of survival of animals after spore injection.

[Metalloproteases and their inhibitors: role in pathogenesis of selected examples]. / Metaloproteinazy i ich inhibitory: rola w patogenezie na wybranych przykladach.

Proteolytic enzymes and their inhibitors are crucial in host-pathogen interaction. Metalloproteases secreted by pathogenic microbes play an important role in destroying not only host tissues but also their immune proteins. Metalloproteinase inhibitors, in contrast, may serve as effective therapeutic agents, which is especially important because of the increasing number of microorganisms resistant to known antibiotics. The role of metalloproteases produced by the bacterium Pseudomonas aeruginosa in the colonization of the host organism is described. Attention has also been paid to the role of inhibitors of these enzymes in defense responses and underlined their potential role in inhibiting the development of infection.

[Different faces of phenoloxidase in animals]. / Rózne oblicza oksydazy fenolowej u zwierzat.

Phenoloxidases are oxidoreducting enzymes whose main function is the oxidation of phenols. The term phenoloxidase is often used interchangeably to describe three different enzymes: tyrosinase (EC 1.14.18.1), catechol oxidase, and laccase. Of these, only tyrosinase has two activities: (1) oxygenase activity to hydroxylate monophenols to ortho-diphenols and (2) oxidase activity responsible for further oxidation of ortho-diphenols to ortho-quinones. Tyrosinase is a key enzyme involved in the melanogenesis process, resulting in the formation of black-brown eumelanin and yellow-red feomelanin. In addition to the pigmentary role, human melanin protects against harmful ultraviolet radiation, while in invertebrate animals melanin is involved in the process of cuticle hardening, wound healing, clot formation, maintenance of intestinal homeostasis and defense reactions. In invertebrates, the tyrosinase is synthesized as a proenzyme that is activated by a serine proteases' cascade known as the phenoloxidase system. This system is considered as one of the innate immunity mechanisms.

Short-term heat shock affects the course of immune response in Galleria mellonella naturally infected with the entomopathogenic fungus Beauveria bassiana.

We aimed to investigate how exposition of infected insects to short-term heat shock affects the biochemical and molecular aspects of their immune response. Galleria mellonella larvae were exposed to 43°C for 15min, at the seventy second hour after natural infection with entomopathogenic fungus Beauveria bassiana. As a result, both qualitative and quantitative changes in hemolymph protein profiles, and among them infection-induced changes in the amount of apolipophorin III (apoLp-III), were observed. Heat shock differently affects the expression of the tested immune-related genes. It transiently inhibits expression of antifungal peptides gallerimycin and galiomicin in both the fat body and hemocytes of infected larvae. The same, although to a lesser extent, concerned apoLp-III gene expression and was observed directly after heat shock. Nevertheless, in larvae that had recovered from heat shock, apoLp-III expression was higher in comparison to unshocked larvae in the fat body but not in hemocytes, which was consistent with the higher amount of this protein detected in the hemolymph of the infected, shocked larvae. Furthermore, lysozyme-type activity was higher directly after heat shock, while antifungal activity was significantly higher also in larvae that had recovered from heat shock, in comparison to the respective values in their non-shocked, infected counterparts. These results show how changes in the external temperature modulate the immune response of G. mellonella suffering from infection with its natural pathogen B. bassiana.