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

https://doi.org/10.1016/j.jip.2020.107327Get rights and content

Highlights

  • Defence activity of G. mellonella depended on the dosage of C. albicans.

  • Antifungal defence was higher after infection with a lethal dose of C. albicans.

  • G. mellonella phenol oxidase was inhibited by C. albicans.

  • Activity of phenol oxidase was modulated by priming.

Abstract

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.

Introduction

Elucidation of various aspects of insect defence mechanisms requires the use of both natural and opportunistic pathogens in infection experiments. The former allow understanding the immune response as a specific defence strategy directed against a natural pathogen, which has appeared as a result of host-pathogen co-evolution (Keebaugh and Schlenke, 2014). At the same time, studies of insect immune response to infection with opportunistic pathogens should not be ignored, as they can provide information about the general and universal properties of defence mechanisms. Insects possess only innate immunity, which shares many common features with the innate defence in vertebrates (Buchmann, 2014). These arthropods attract growing attention as convenient models for testing human pathogen virulence and drug efficiency in vivo (Cutuli et al., 2019). One of the best insect models to study the properties of innate defence is the greater wax moth Galleria mellonella (Lepidoptera: Pyralidae). Its ca. 6-week life cycle (at the optimal temperature) and the fact that one female can lay approx. 2000 eggs allow for efficient breeding. Also, the relatively large size of Galleria larvae facilitate injection and collection of hemolymph and other organs for further analysis (Wojda, 2017). G. mellonella live in beehives or, more often, in stored waxes, where the larvae feed on honey, pollen, and wax and cause galleriasis (Gulati and Kaushik, 2004, Kwadha et al., 2017, Williams, 1997). Larvae undergo seven moulting stages before pupation and are the stage most often used for injection experiments (Kwadha et al., 2017). Imagos live only ca. 12 d (females) or 21 d (males) and do not feed; their only role is reproduction. Galleria, like all insects, is protected by a chitin-containing integument. The internal organs of ectodermic origin such as the trachea, foregut, and hindgut, are covered by cuticle, protecting the insects from pathogens. If these barriers are broken, cellular and humoral immune responses can be triggered. Galleria plasmatocytes and granulocytes are able to engulf intruding bacteria, while larger foreign bodies and groups of microorganisms are entrapped in capsules and nodules; thus, isolating pathogens from the rest of the insect's body (Cytryńska et al., 2016). Molecular patterns of pathogens are recognised by receptors activating signalling pathways, mainly Toll and Imd. Imd functions exclusively in immunity, whereas the Toll pathway also regulates insect development (Viljakainen, 2015). The Imd pathway is triggered by most Gram-negative bacteria, and the Toll pathway is activated by Gram-positive bacteria, fungi, and danger signals (Ming et al., 2014). Gram-positive bacteria with lysine-type peptidoglycan are bound by short forms of Peptidoglycan Recognition Proteins: PGRP-SA and PGRP-SD, which leads to the activation of the Toll pathway. In turn, Gram-negative and some Gram-positive bacteria, e.g. Bacillus, containing DAP-type peptidoglycan (composed of diaminopimelic acid), are recognized by the so-called long forms of PGRPs: PGRP-LC and PGRP-LE (Charroux et al., 2009), which in turn activate the Imd pathway. Signalling through both pathways leads to the activation of transcription factors Dif and Relish, respectively, which are homologues of the human NF-κB factor (Lu et al., 2019a, Sheehan et al., 2018, Wang et al., 2019). As a result, effector molecules of humoral immunity, e.g. antimicrobial peptides (AMPs), appear in the hemolymph to kill the invading microorganisms mostly by destruction of their membranes (Wu et al., 2018). The main source of defence molecules is the fat body, an organ with very high metabolism and an analogue of the mammalian liver (Arrese and Soulages, 2010). In addition, the JAK/STAT pathway, which regulates many biological processes involving immunity, participates in hematopoiesis and cellular immunity, regulation of defence against viral infections, gut immunity, general stress response and wound healing (Bang, 2019, Wu et al., 2018). Synthesis of the dark pigment melanin is regulated by phenol oxidase (PO); the non-active form, prophenol oxidase (PPO), is released from oenocytoids after infection or injury. Melanin can be deposited either on the surface of invading microorganisms to facilitate recognition/killing of the pathogen or on the clot, which is thus hardened until the injured epidermis is restored (Eleftherianos and Revenis, 2011, Hillyer, 2016). The transcriptomes of infected and non-infected G. mellonella were compared by Vogel et al. (2011), revealing immune-regulated genes. Recently, the entire genome of the greater wax moth was published, which will contribute to identification of other molecules involved in the immunity of this animal (Lange et al., 2018).

One of the opportunistic organisms that can cause the death of G. mellonella larvae is the yeast-like fungus Candida albicans (Bergin et al., 2006, Marcos-Zambrano et al., 2019, Vertyporokh et al., 2019). C. albicans is often present as part of the natural microbiome of various organisms, including humans. In immune-compromised patients, it causes a disease called candidiasis. C. albicans has the ability to switch from the yeast-type to the filamentous type of growth, depending on ambient conditions. This feature contributes to the virulence of the fungus (Singkum et al., 2019). Many other virulence factors enabling C. albicans to colonise both vertebrate and invertebrate organisms have been identified. These include adhesins, invasins, and extracellular hydrolytic enzymes useful for invasion of host tissues (Schaller et al., 2005, Staniszewska et al., 2012, Wibawa, 2016). Although C. albicans is not a natural insect pathogen, a non-lethal dosage introduced into the hemocel of G. mellonella can induce enhanced resistance to repeated infection with the same fungus (Vertyporokh et al., 2019). This means that the insect immune system is able to “remember” a previous infection and protects the organism more efficiently when it encounters the pathogen for the second time. What is more, this so-called immune priming phenomenon was found to be specific; pre-infection did not prevent further infection with other organisms (Vertyporokh et al., 2019). Here, we show differences in immune response of G. mellonella to C. albicans depending on the dosage of fungal cells injected into the larval hemocel.

Section snippets

Insects, fungi, and injection

G. mellonella were reared on a natural diet, honeybee nest debris, at 28˚C in darkness. Final instar larvae, 3 d after the last moult and weighing about 200 mg, were used for testing. Candida albicans (strain ATCC 10231) was cultivated on YPD medium (1% yeast extract, 2% bactopepton, 2% glucose) at 30 °C with 120 rpm shaking. The fungi were centrifuged at 5500g and suspended in phosphate buffered saline (PBS, 140 mM NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, 1.76 mM KH2PO4 in pH 7.4). The density of

Symptoms of disease

The sensitivity of G. mellonella larvae to the intrahaemocelic injection of C. albicans cells strictly depended on the dose of the fungus. Only 5% of the larvae did not survive the dosage of 2 × 104 cells (LD5, non-lethal dosage). The ten-fold higher dosage caused the death of approx. 50% of the injected animals after 72 hpi, and, ultimately, approx. 95% at 120 h post injection (LD95; lethal dosage). The dosage of 2 × 106 caused very rapid death of all animals (LD100), mostly within the first

Discussion

C. albicans is an opportunistic pathogen that can kill G. mellonella when injected into the larval hemocel. Therefore, the greater wax moth is used as a non-vertebrate model to study pathogenicity, effectiveness of virulence factors, the ability to induce infection by different C. albicans mutants, and in in vivo tests of antifungal drugs (Brennan et al., 2002, Gu et al., 2018, Lu et al., 2019b, Marcos-Zambrano et al., 2019, Pereira et al., 2018, Trevijano-Contador and Zaragoza, 2018).

References (55)

  • C.H. Kim et al.

    Purification and cDNA cloning of a cecropin-like peptide from the great wax moth, Galleria mellonella

    Mol. Cell.

    (2004)
  • P. Mak et al.

    A different repertoire of Galleria mellonella antimicrobial peptides in larvae challenged with bacteria and fungi

    Dev. Comp. Immunol.

    (2010)
  • M. Ming et al.

    Persephone/Spätzle pathogen sensors mediate the activation of Toll receptor signaling in response to endogenous danger signals in apoptosis-deficient Drosophila

    J Biol Chem.

    (2014)
  • H. Schagger et al.

    Tricine-sodium dodecyl sulfatepolyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa

    Analyt. Biochem.

    (1987)
  • P. Taszłow et al.

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

    J. Invert. Pathol.

    (2017)
  • L. Vertyporokh et al.

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

    J. Insect Physiol.

    (2019)
  • Q. Wang et al.

    Peptidoglycan recognition proteins in insect immunity

    Mol. Immunol.

    (2019)
  • I. Wojda et al.

    Heat shock affects host-pathogen interaction in Galleria mellonella infected with Bacillus thuringiensis

    J. Insect Physiol.

    (2013)
  • E.L. Arrese et al.

    Insect fat body: energy, metabolism, and regulation

    Annu. Rev. Entomol.

    (2010)
  • I.S. Bang

    JAK STAT signaling in insect immunity

    Entomol. Res.

    (2019)
  • G. Bidla et al.

    Activation of insect phenoloxidase after injury: endogenous versus foreign elicitors

    J. Innate Immun.

    (2009)
  • M.R. Bolouri Moghaddam et al.

    The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides

    Biol. Chem.

    (2016)
  • K. Buchmann

    Evolution of innate immunity: clues from invertebrates via fish to mammals

    Front. Immunol.

    (2014)
  • M.A. Cutuli et al.

    Galleria mellonella as a consolidated in vivo model hosts: new developments in antibacterial strategies and novel drug testing

    Virulence

    (2019)
  • Cytryńska, M., Wojda, I., Jakubowicz, T., 2016. How insects combat infections. In: Ballarin L., Cammarata M. (red.),...
  • J. Dekkerová-Chupáčová et al.

    Up-Regulation of antimicrobial peptides gallerimycin and galiomicin in Galleria mellonella infected with Candida yeasts Displaying different virulence traits

    Mycopathologia

    (2018)
  • G.B. Dunphy et al.

    Virulence of Candida albicans mutants toward larval Galleria mellonella (Insecta, Lepidoptera, Galleridae)

    Can. J. Microbiol.

    (2003)
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