APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2010, p. 310–317 Vol. 76, No. 10099-2240/10/$12.00 doi:10.1128/AEM.01301-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

Galleria mellonella as a Model System for StudyingListeria Pathogenesis�

Krishnendu Mukherjee,1 Boran Altincicek,1 Torsten Hain,2 Eugen Domann,2Andreas Vilcinskas,1* and Trinad Chakraborty2*

Institute of Phytopathology and Applied Zoology1 and Institute of Medical Microbiology,2 Justus Liebig University ofGiessen, Giessen, Germany

Received 4 June 2009/Accepted 30 October 2009

Essential aspects of the innate immune response to microbial infection are conserved between insects andmammals. This has generated interest in using insects as model organisms to study host-microbe interactions.We used the greater wax moth Galleria mellonella, which can be reared at 37°C, as a model host for examiningthe virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerialseptic infection, capable of clearly distinguishing between pathogenic and nonpathogenic Listeria strains andeven between virulent and attenuated Listeria monocytogenes strains. Virulence required listerial genes hithertoimplicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymphand hemocytes of infected larvae. Following Listeria infection, the expression of immune defense genes such asthose for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentiallyinduced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) signifi-cantly improved survival against subsequent L. monocytogenes challenge and strong antilisterial activity wasdetected in the hemolymph of LPS pretreated larvae. We conclude that the severity of septic infection with L.monocytogenes is modulated primarily by innate immune responses, and we suggest the use of Galleria as arelatively simple, nonmammalian model system that can be used to assess the virulence of strains of Listeriaspp. isolated from a wide variety of settings from both the clinic and the environment.

Listeriae are rod-shaped, motile, facultative, anaerobicGram-positive bacteria that are ubiquitously distributed in theenvironment (28). Of the six species that comprise the genusListeria, only L. monocytogenes and L. ivanovii are pathogenicand cause disease, while strains of the species L. innocua, L.welshimeri, L. seeligeri, and L. grayi are generally considered tobe nonpathogenic (26). L. monocytogenes is a major food-borne pathogen, and listeriosis is an invasive disease that in itsseverest form can lead to meningitis, meningoencephalitis, sep-ticemia, and abortions (38). Listeriosis occurs primarily inpregnant women, newborn infants, and the elderly as well as inimmunocompromised patients, with a mortality rate of about30% (22, 36). The virulence of L. monocytogenes has beenlinked to a 9.6-kb pathogenicity island designated vgc (viru-lence gene cluster) that comprises six genes encoding its majorvirulence determinants. These are (i) prfA, a master regulatorof many known listerial virulence genes; (ii) hly, encoding list-eriolysin, a hemolysin required for bacterial escape from thehost primary vacuole to the host cytoplasm; (iii) two phospho-lipase genes denoted plcA and plcB, for facilitating lysis of hostcell membranes; (iv) actA, encoding a surface bound proteinthat directs polymerization of host cell actin and is required for

intracellular motility; and (v) mpl, encoding a metalloprotein-ase which is thought to work together with the plcB product tofacilitate cell-to-cell spread (28). Presently, identification andcharacterization of novel virulence factors rely on assessingmutant bacteria for growth in the organs of infected mice.Nevertheless, the dependence on mouse infection models lim-its large-scale screening for additional mutants defective intheir ability to grow in the host intracellularly or for thoserequired to overcome host innate defenses (33).

The possibility of addressing many aspects of mammalianinnate immunity in invertebrates has opened a new arena fordeveloping invertebrate models to study human infections. Re-cently the use of invertebrate models, in particular the fruit flyDrosophila melanogaster, has been introduced for the study ofseptic listerial infections (37). Listeria mutants attenuated forvirulence in a mouse model exhibited lowered virulence in thismodel. The Drosophila model system has powerful genetictools available and has thus provided deeper insights into mo-lecular mechanisms of the interactions between Listeria andthe insect innate immune system (1, 8–10, 18, 24). However, arecent study has shown that even nonpathogenic L. innocuastrains cause lethal infections of Drosophila, limiting it use as adiscerning model for the study of virulence potential amongpathogenic L. monocytogenes isolates (32).

We have a longstanding interest in host-pathogen interac-tions of the greater wax moth, Galleria mellonella, in particularwith entomopathogenic microbes (55). Recently, Galleria hasalso emerged as a reliable model host to study the pathogen-esis of many human pathogens (7, 11, 12, 17, 21, 30, 31, 39–42,44, 46, 48–51). Among the advantages provided by the Galleriamodel host (e.g., low rearing costs, convenient injection feasi-

* Corresponding author. Mailing address for Trinad Chakraborty: In-stitute for Medical Microbiology, Justus Liebig University, FrankfurterStrasse 107, 35392 Giessen, Germany. Phone: 49 641 99-41251. Fax: 49641 99-41259. E-mail: trinad.chakraborty@mikrobio.med.uni-giessen.de.Mailing address for Andreas Vilcinskas: Institute of Phytopathology andApplied Zoology, Justus Liebig University, Heinrich-Buff-Ring 26-32,D-35392 Giessen, Germany. Phone: 49 641 99-37600. Fax: 49 641 99-37609. E-mail: Andreas.Vilcinskas@agrar.uni-giessen.de.

� Published ahead of print on 6 November 2009.

310

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.

bility, and status as an ethically acceptable animal model), it isof particular importance that Galleria has a growth optimum at37°C, to which human pathogens are adapted and which isessential for synthesis of many virulence/pathogenicity factors.Significantly, a correlation between the virulence of a pathogenin G. mellonella and that in mammalian models has been es-tablished (16, 25).

The innate immunity of Galleria is a complex, multicompo-nent response involving hemolymph coagulation, cellularphagocytosis, and phenol oxidase-based melanization. Impor-tantly, killing of pathogens is achieved similarly to that inmammals, i.e., by enzymes (e.g., lysozymes), reactive oxygenspecies, and antimicrobial peptides (e.g., defensins). Galleriaemploys recognition of nonself microbe-associated molecularpatterns by germ line-encoded receptors (e.g., Toll and pepti-doglycan recognition proteins) (52). Recently, we have foundthat Galleria also senses pathogens by danger signaling, bydetecting either nucleic acids released from damaged cells orpeptides resulting from proteolytic cleavage of self proteins bymatrix metalloproteinases (3–6).

In this work we examined the Galleria model of septic in-fection for its ability to differentially distinguish between in-fections caused by strains with different virulence potentials inthe mouse infection model, as well as in avirulent strains ofListeria. We found that the Galleria model is highly discrimi-natory in assessing the pathogenic potential of Listeria spp.,and we observed a strong correlation with the virulence previ-ously determined in the mouse model of infection. Here, wepresent data indicating that the Galleria model also replicatesmany aspects of innate immune function, such as the consti-tutive expressions of potential antimicrobial factors followinginfection. Also, prior induction of immunity in Galleria canprotect larvae from septic infection with highly pathogenic L.monocytogenes.

MATERIALS AND METHODS

Insects, bacteria, and media. G. mellonella larvae were reared on an artificialdiet (22% maize meal, 22% wheat germ, 11% dry yeast, 17.5% bees wax, 11%honey, and 11% glycerin) at 32°C in darkness prior to use. Last-instar larvae,each weighing between 250 and 350 mg, were used in all experiments. Thedifferent Listeria species, serotypes, and mutants used in this experiment arelisted in Table 1. The wild-type strain Listeria monocytogenes EGD-e used in thisstudy belongs to serotype 1/2a (23). The bacterial cultures were grown aerobi-cally in brain heart infusion medium (BHI) (Difco, Franklin Lakes, NY) at 37°Cand on BHI agar plates. For long-term storage, Listeria strains were frozen inBHI with 30% glycerol at �80°C. For injection experiments, Listeria cultureswith a density of 109 CFU/ml in 10 ml of BHI broth growing in logarithmic phasewere used. Bacterial inoculums were washed and serially diluted using 0.9%NaCl to appropriate concentrations. Fifty microliters of each dilution was platedout on BHI agar plates and incubated at 37°C for 24 h, and the bacterial CFUwere used to calculate the inoculum injected. Cultures of L. innocua harboringthe pUvBBAC vector containing the vgc1 locus from L. monocytogenes EGD-ewere grown in the presence of 5 �g/ml erythromycin and 5 �g/ml kanamycin (27).The Escherichia coli host for plasmid constructions was INV�F�. Plasmid DNAwas transferred to INV�F� using the method of Hanahan (29). The electropo-ration protocol of Park and Stewart (43) was utilized for transformation of L.monocytogenes strains.

Deletion of the virulence gene cluster (vgc) comprising the genes prfA, plcA,hly, mpl, actA, and plcB in L. monocytogenes EGD-e. A PCR product of approx-imately 2,500 bp was generated with the forward primer 5�-TCTAATCGTGAACTAGCTG-3� and the reverse primer 5�-CGTAAGTGTTCGTGATGCAGCTTATG-3� using chromosomal DNA of the nonpathogenic strain L. innocuaNCTC 11289. The PCR product was cloned into plasmid pAUL-A and trans-formed into L. monocytogenes EGD-e, and the isogenic vgc mutant strain was

generated as described previously (47). A 12-kb fragment comprising the genesprfA, plcA, hly, mpl, actA, and plcB was replaced by the 2.5-kb genomic fragmentpresent between prs and ldh of L. innocua NCTC 11289. The loss of vgc wasconfirmed by sequencing and by immunoblotting with monoclonal antibodiesdirected against proteins PlcA, Hly, Mpl, ActA, and PlcB.

Construction of a chromosomal �uhpT deletion mutant of L. monocytogenesEGD-e. A �uhpT (or �hpt) mutant harboring only the first 22 amino acidresidues of UhpT was obtained as follows. Appropriate regions flanking the uhpTgene were PCR amplified with oligonucleotide primers uhpT-for1 (5�-AGAAACGGAGCTCGTGATTC-3�) and uhp-rev2 (5�-AAAGTGTTGGATCCATTGTTG-3�) or uhpT-for3 (5�-TAAGTTGGATCCAATGAGTG-3�) and uhpT-rev4(5�-GCTAAGTCGACTCAATCCG-3�), respectively. Both PCR products weredigested with BamHI and ligated to each other. The ligation product containingthe deletion was selectively amplified with oligonucleotide primers uhpT-for1and uhpT-rev4. The corresponding DNA fragment flanked by SacI and SalIrestriction sites was inserted into the temperature-sensitive shuttle vectorpAUL-A (36). The L. monocytogenes wild-type strain EGD-e was transformedwith this construct, and chromosomal integration of the plasmid and plasmidexcision and curing were carried out as previously described (35). Replacementof the wild-type allele by its truncated �uhpT derivative was confirmed bysequencing of the PCR product obtained with oligonucleotide primers uhpT-for1and uhpT-rev4.

G. mellonella injection and CFU count of L. monocytogenes. Bacterial inocu-lums were injected dorsolaterally into the hemocoel of last-instar larvae using1-ml disposable syringes and 0.4- by 20-mm needles mounted on a microappli-cator. After injection, larvae were incubated at 37°C. Caterpillars were consid-ered dead when they showed no movement in response to touch. No mortality ofGalleria larvae was recorded when they were injected with 0.9% NaCl. For CFUcounting, Galleria larvae were infected with L. monocytogenes (106 CFU/larva)and were homogenized in BHI medium with 1% Triton X-100. Homogenateswere plated onto Palcam Listeria selective agar plates (Heipha Diagnostika), andcolonies were counted after incubation at 37°C for 48 h. For each time point,homogenates of 10 larvae were plated individually for CFU count.

Preimmune activation of G. mellonella larvae and antibacterial activity assays.Last-instar larvae were injected independently with 10 mg/ml lipopolysaccharide(LPS) (purified Escherichia coli endotoxin 0111:B4) (catalog no. L2630; Sigma,Taufkirchen, Germany) or heat-killed L. monocytogenes to trigger strong im-mune responses. The heat-killed preparation of bacteria was obtained as follows.An exponential-phase bacterial culture was harvested, centrifuged, and washed

TABLE 1. Bacterial strains used in this study

Species and strain Serotype Reference

L. monocytogenesEGD-e 1/2a 23L99 4aL312 4bSLCC2376/ATCC 19116 4cATCC 19117 4dEGD-e �vgc Present studyEGD-e �uhpT Present studyEGD-e �prfA 14EGD-e �hly 25EGD-e �actA 13EGD-e �plcA 45EGD-e �plcB 25EGD-e �mpl 25EGD-e �inlAB 35

L. innocuaCLIP 11262 6aCLIP 11262 vgc1 27

L. welshimeri SLCC 5334/ATCC 35897 6b

L. grayi CLIP 12515

L. ivanovii PAM55 5

L. seeligeri SLCC 3954/ATCC 35967 1/2b

VOL. 76, 2010 LISTERIA PATHOGENESIS IN GALLERIA 311

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.

three times in 0.9% NaCl. The recovered bacteria were resuspended in NaCl andincubated at 85°C for 1 h. After two additional washes in NaCl, the wet weightof the bacterial pellet was adjusted to 10 mg/ml in 0.9% NaCl. Each 10 �l wasadministered directly into the hemolymph of the larvae to induce an immuneresponse. At 24 h after administration of LPS or heat-killed Listeria, 106 CFU ofL. monocytogenes strain EGD-e was injected into each larva for survival counts.To investigate the presence of antimicrobial activities in Galleria killing viableListeria, we used the inhibition zone assay. In brief, petri dishes (100 mm) werefilled with 7 ml BHI medium containing 0.7% high-purity agar-agar (Roth,Karlsruhe, Germany), and subsequently, 104 CFU of viable bacteria in logarith-mic growth phase was plated. Hemolymph samples from larvae were extracted at24 h following immune induction and inlaid into 4-mm-diameter wells previouslypunched into the agar. The diameters of clear zones were measured after 24 h ofincubation at 37°C.

Ex vivo infection of Galleria hemocytes. The hemocytes from Galleria weremaintained at 37°C in Schneider medium (BioWhittaker) supplemented with10% heat-inactivated fetal bovine serum (FBS) (Bio West). Intracellular growthof L. monocytogenes in primary Galleria hemocytes was monitored by using cellmonolayers on sterile coverslips for immune fluorescence microscopy observa-tion and on microtiter plates for estimation of bacterial CFU. Briefly, bacterialcultures logarithmically grown in BHI medium at 37°C were washed with 0.9%NaCl before infection. After 60 min of infection at 37°C, the hemocytes werecarefully washed three times with cell culture medium, followed by the additionof 1 ml of Schneider medium containing 50 �g/ml of gentamicin. To quantifybacterial intracellular growth, cell monolayers were lysed by sterile water con-taining 0.2% Triton X-100 for 4 h after L. monocytogenes infection, and CFUwere determined by plating dilutions of cell lysates on BHI plates followed byovernight incubation at 37°C. For microscopic analysis, cells were fixed by placinga drop of 3.7% paraformaldehyde on the coverslips and incubating at roomtemperature for 10 min. Coverslips were washed by dipping them into sterilephosphate-buffered saline (PBS), and then the hemocytes were permeabilizedwith 1 ml 0.2% Triton X-100 in PBS for 1 min and again washed in PBS. Thecoverslips were incubated with ActA N4 and ActA N81 (1:1) monoclonal anti-bodies (Helmholtz Zentrum for Infection Braunschweig; prepared by JurgenWehland) for 30 min at 33°C. After being washed three times with PBS, cover-slips were incubated with Cy3-labeled secondary anti-mouse antibody (1:100)(Dianova, Hamburg, Germany) and Alexa Fluor 488 conjugated to phalloidin(1:100) (Molecular Probes, Invitrogen, Carlsbad, CA) in PBS containing 1%bovine serum albumin for 30 min at 33°C. Subsequently, coverslips were washedthree times with PBS and mounted using Prolong Gold antifade reagent (In-vitrogen).

Quantitative real time RT-PCR. Three larvae per treatment for each timepoint were homogenized in 1 ml of Trizol reagent (Sigma), and whole animalRNA was extracted according to the manufacturer’s recommendations. RNAintegrity was confirmed by ethidium bromide gel staining, and quantities weredetermined spectrophotometrically. Quantitative real time reverse transcription-PCR (RT-PCR) was performed with the real-time PCR system Mx3000P (Strat-agene) using the FullVelocity SYBR green quantitative RT-PCR master mix(Stratagene) according to the protocols of the manufacturer. We used appropri-ate primers along with the 10 ng RNA per reaction to amplify the genes for 18SRNA (2), actin (3), IMPI (3), galiomycin (5), gallerimycin (3), and lysozyme (3).

Data analysis. All experiments were performed a minimum of three times.Significant differences between two values were compared with a paired Stu-dent’s t test. Values were considered significantly different when the P value wasless than 0.05.

RESULTS

Mortality in Listeria-infected Galleria larvae depends on thepathogen load. We examined the susceptibility of G. mellonellato a known pathogenic strain and a nonpathogenic strain ofListeria. Larvae were injected with 107, 106, 105, and 104 bac-teria of either L. monocytogenes strain EGD-e or L. innocua,and mortality was recorded up to 7 days postinjection. At 107

CFU we observed killing of Galleria irrespective of whether thepathogenic or nonpathogenic Listeria strain was used. At dosesbelow 106 CFU, clear differences in lethality between L. mono-cytogenes and L. innocua were observed (Fig. 1A and B). Dif-ferences in mortality were less apparent at lower doses (105

and below), and no deaths were recorded when larvae wereinjected with 0.9% saline alone. Thus, for subsequent experi-mental assays we used 106 CFU/larva as the inoculating dose tostudy septic infection by Listeria spp.

Listeria infection of G. mellonella resembles that seen withvertebrates. To examine whether cellular aspects of infectionare similar to those observed with vertebrate cells, we isolatedhemocytes from naive larvae and subjected them to infectionwith L. monocytogenes. Bacteria were incubated with hemo-cytes for 1 h to allow for invasion. Subsequently the superna-tant of cultures were replaced with fresh medium supple-mented with 50 �g/ml of gentamicin to kill extracellularListeria cells. Bacteria growing intracellularly were monitoredby immunofluorescence microscopy using an ActA-specificmonoclonal antibody. Actin-based motility of bacteria was de-tected by colocalization of intracellular bacteria with fluores-cence derived by actin-specific Alexa Fluor 488-conjugatedphalloidin. In infected cells, we detected intracellular bacteriaeither covered by actin “clouds” or undergoing rapid move-ment as judged by the lengths of their respective actin “comettails,” thus resembling the infection process seen previously invertebrate cells (Fig. 2A).

We also addressed the question of whether mortality ofGalleria is associated with the growth of L. monocytogenes in

FIG. 1. Dose-dependent survival of Galleria caterpillars after inoculation with L. monocytogenes and L. innocua. Bacteria were grown to logphase in BHI medium at 37°C. The time course of survival of the larvae when inoculated with pathogenic L. monocytogenes strain EGD-e (A)and/or apathogenic L. innocua (B) depended on the amount of CFU injected. Injection of 107, 106, 105, or 104 CFU/larvae resulted in highermortality with EGD-e than with L. innocua. Results represent means of at least three independent determinations � standard deviations for 10animals per treatment.

312 MUKHERJEE ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.

infected larvae. Galleria larvae infected with EGD-e at 106

CFU/larva were homogenized in BHI medium containing 1%Triton X-100 and then plated onto Listeria-selective Palcamplates. L. monocytogenes colonies were counted following in-cubation at 37°C for 48 h. We observed a rapid decrease in theCFU count at 1 h postinfection, indicating that the constitutiveimmune defenses of Galleria were highly effective in reducingthe pathogen load (Fig. 2B). At 24 h and 48 h postinfection,successive increases in bacterial CFU were recorded. In Gal-leria larvae that had succumbed to infection at 96 h postinjec-tion, high numbers of bacteria were detected.

Listeria shows species-specific pathogenesis for Galleria. Weinvestigated the Galleria model for its ability to distinguish

between pathogenic and nonpathogenic strains comprising allknown Listeria species. There was a clear difference betweenpathogenic L. monocytogenes and the nonpathogenic L. in-nocua, L. seeligeri, L. welshimeri, and L. grayi strains. L. ivanoviiwas clearly less pathogenic in the Galleria model than L. mono-cytogenes but nevertheless still demonstrated a small but sig-nificant difference in mortality compared to the nonpathogenicL. innocua (Fig. 3A).

Serotype-specific virulence in Galleria. The ability to distin-guish between L. monocytogenes strains previously character-ized as being either highly virulent or attenuated in the mousemodel of infection was examined next. Among the differentserotypes tested, a serotype 4b strain was the most pathogenic,causing a significantly higher rate of killing than the serotype1/2a strain (EGD-e) (Fig. 3B). However strains of other sero-types of L. monocytogenes, such as 4a, 4c, and 4d, exhibitedsignificantly lower virulence, mirroring their reduced patho-genic potential previously seen in the mouse infection model(Fig. 3B).

Septic infection of Galleria is dependent on the vgc locus ofL. monocytogenes. The vgc locus encodes the major factorsrequired for virulence of L. monocytogenes (54). We generatedand used an EGD-e mutant with the vgc locus deleted, EGD-e�vgc (see Materials and Methods), and examined its ability tokill Galleria. The EGD-e �vgc strain was highly attenuated forkilling ability in comparison to the wild-type EGD-e in theGalleria model (P � 0.0005) (Fig. 4A). Isogenic strains lackingprfA, hly, actA, plcB, and mpl were highly attenuated for thekilling of infected larvae (Fig. 4B and C). Interestingly, dele-tion of plcA revealed no virulence attenuation. This was alsothe case with a mutant lacking internalins A and B.

We also assessed the contribution of an additional PrfA-regulated factor, the hexose phosphate transporter UhpT,which is required for efficient growth and survival of the bac-terium in infected vertebrate cells. The �uhpT mutant was alsofound to be attenuated for killing of infected larvae (Fig. 4C).

We used a recombinant L. innocua strain engineered toharbor the vgc locus and found that following infection it was

FIG. 2. Multiplication of L. monocytogenes in Galleria. (A) L.monocytogenes cells were stained using ActA antibodies (resulting inred fluorescence), and host actin of hemocytes was stained using Al-exa-phalloidin (resulting in green fluorescence). Note that Listeriaorganisms are spreading throughout the cytosol of the hemocyte, andactin tails at the poles of some of the bacteria are visible. (B) Todetermine rate of multiplication of L. monocytogenes in Galleria larvae,we determined the listerial load from infected larvae at several timepoints postinfection. For each time point, homogenates of 10 larvaewere plated individually for CFU count on Listeria selective Palcamagar plates. These results are shown as one dot, and resulting meanvalues of are shown in red. Surviving animals contained reduced liste-rial load, whereas dying larvae contained about 2 � 105 CFU, asindicated by a circle. The experiment was repeated three times withsimilar results.

FIG. 3. Time-dependent survival of Galleria larvae after inoculation with different Listeria species and L. monocytogenes serotypes. The timecourse of survival of the larvae varies with the type of Listeria species employed for inoculation. (A) Inoculation with 106 CFU/larva EGD-eresulted in a significantly higher rate of killing of larvae than inoculation with L. ivanovii, L. innocua, L. seeligeri, L. welshimeri, or L. grayi. OnlyL. ivanovii had a tendency for enhanced killing of Galleria with respect to L. innocua (P � 0.05). (B) Inoculation with different L. monocytogenesserotypes resulted in various rate of killing. Serotype 4b showed a significant high rate of killing of larvae, whereas the pathogeneses of 4a, 4c, and4d were strongly attenuated, with respect to the pathogenic serotype 1/2a strain EGD-e. Results represent means of at least three independentdeterminations � standard deviations for 10 animals per treatment.

VOL. 76, 2010 LISTERIA PATHOGENESIS IN GALLERIA 313

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.

significantly more pathogenic as observed by the higher ratesof killing of the larvae than with L. innocua alone (Fig. 5).Nevertheless, it was significantly less pathogenic than theEGD-e strain from which the vgc locus was derived (P � 0.05).

Expression patterns of genes encoding antimicrobial pep-tides. Galleria is capable of synthesizing a broad spectrum ofantimicrobial peptides in response to septic injury (43). Con-sequently, we were interested in examining whether antimicro-bial peptides are induced in Galleria upon septic Listeria in-fections. Larvae were infected with L. monocytogenes, andRNA was extracted at 1, 6, and 24 h postinfection. Transcrip-tional activation is represented as the fold change of expressionof immune-related genes in infected Galleria relative to themock-injected control larvae and normalized using the house-keeping 18S RNA gene (Fig. 6). Increased levels of lysozymeexpression were recorded throughout the whole period of L.monocytogenes infection. The amounts of immune-related gal-lerimycin and lysozyme mRNAs were found to be inducedabout 4.0-fold and 2.0-fold at 1 h postinfection. At 6 h postin-fection we observed increased galiomycin (11-fold), galleri-mycin (80-fold), and lysozyme (10-fold) mRNA levels. In-

duced expression of host actin (2.0-fold) was also found at 6 hfollowing L. monocytogenes infection. Interestingly, IMPImRNA levels were only induced at 24 h postinfection, whereasexpression levels of galiomycin, gallerimycin, and lysozymewere reduced at 6 h postinfection.

Activation of immunity in Galleria enhances the host de-fense against L. monocytogenes infection. Previous studies pro-vide evidence for the presence of inducible immune defensemolecules in Galleria that provide relatively long-lasting anti-microbial responses to repeated infections (12, 45). To exam-ine whether the prior induction of immune responses in Gal-leria would protect against subsequent infection by L.monocytogenes, we injected larvae with 100 �g LPS and thenchallenged them by injecting a dose of 106 CFU of L. mono-cytogenes 24 h later. LPS-mediated induction of immune re-sponses provided vigorous protection against subsequent in-fection by a lethal dose of L. monocytogenes (Fig. 7A). To

FIG. 4. Contributions of major virulence-related genes of L. monocytogenes in the mortality of Galleria. (A) The vgc locus is responsiblefor the pathogenicity of L. monocytogenes. L. monocytogenes with vgc deleted had a significant reduction of killing capacity in comparisonto EGD-e (P � 0.005) but still showed greater killing ability than L. innocua (P � 0.05). (B and C) Deletion of single virulence genes prfA,hly, actA, plcB, mpl, and uhpT (hexose-phosphate transporter gene) resulted in significantly reduced mortality in the Galleria model system.However deletion of vgc-associated plcA or inlA and intB caused no significant reduction in mortality rates. The P value for the mortalityrates between �uhpT and �inlAB was found to be 0.005, and that between �uhpT and �plcB was 0.05. Results represent means of at leastthree independent determinations � standard deviations for 10 animals per treatment.

FIG. 5. Insertion of EGD-e-derived vgc in L. innocua results ininduced virulence. Artificial introduction of the vgc1 locus into other-wise nonpathogenic L. innocua resulted in a significant increase ofvirulence with respect to that of wild-type L. innocua. Results repre-sent means of at least three independent determinations � standarddeviations. Each repetition contained 30 larvae per treatment.

FIG. 6. Transcriptional activation of actin and immune-responsivegenes following infection. The transcription levels of actin, galiomycin,gallerimycin, IMPI, and lysozyme were determined by quantitativereal-time RT-PCR analysis and are shown relative to the expressionlevels in mock-injected animals. Results were normalized to expressionof the housekeeping 18S RNA gene and represent means of threeindependent determinations � standard deviations.

314 MUKHERJEE ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.

examine the basis of reduced L. monocytogenes growth, weisolated hemolymphs from preimmunized and naive larvae andused them in an inhibition zone assay (see Materials and Meth-ods) that indicates the presence of antimicrobial activity. OnBHI agar plates plated with L. monocytogenes, we observedzones of inhibitory growth that were dependent on the con-centration of LPS used for preimmune activation, indicatinginducible antilisterial activity in hemolymph (Fig. 7B). Similarresults were observed when using heat-killed Listeria cells in-stead of LPS (data not shown).

DISCUSSION

Invertebrate infection models have been recently employedto investigate the pathogenesis of L. monocytogenes (18), but acomparative analysis of the pathogenic potentials of variousListeria species and serotypes obtained from various humanand environmental sources has not been previously addressedin these models. In this work we show that the Galleria modelwas able to clearly distinguish between pathogenic and non-pathogenic Listeria species and to discriminate between L.monocytogenes serotypes exhibiting attenuated virulence prop-erties. In addition, we report that mutants of L. monocytogeneslacking either single or multiple virulence factors are attenu-ated for pathogenicity in Galleria. Conversely, an avirulentstrain of L. innocua engineered to express the vgc locus of L.monocytogenes exhibited enhanced virulence in the Galleriamodel. Thus, the invertebrate host Galleria emulates manyaspects of Listeria infection seen in vertebrates.

Previous studies with Drosophila have revealed the relativecontribution of virulence factors of L. monocytogenes to septicinfection and demonstrated that the Drosophila S2 cell line canbe used to examine intracellular growth of Listeria (16). How-

ever, a recent study described some limitations of D. melano-gaster as a heterologous host for the study of pathogenesis ofseveral Gram-positive bacteria (26). Also, because Drosophilacannot be maintained at 37°C, it does allow experimental anal-ysis at a temperature to which mammalian pathogens areadapted. In another invertebrate infection model employingCaenorhabditis elegans, a Listeria mutant lacking actA wasfound to be lethal, thus also limiting the utility of C. elegans tostudy Listeria pathogenicity (53).

Listerial virulence in Galleria is influenced by the concen-tration of the inoculum injected. At high concentrations, i.e.,107 CFU/larva, even nonpathogenic Listeria species such as L.innocua induce septic death in Galleria. This is probably due toa threshold over which processes leading to larval death areinduced via the overwhelming activation of the innate immunesystem. At 106 CFU/larva, nonpathogenic Listeria species lack-ing virulence factors are probably engaged by cellular recep-tors recognizing bacterial pathogen-associated molecular pat-terns (PAMPs), such as peptidoglycan, leading to theactivation of the innate immune system and bacterial clear-ance. On the other hand, L. monocytogenes showed significantpathogenesis in Galleria. We show here that this can be attrib-uted to the expression of specific virulence factors responsiblefor the survival within the invertebrate host, e.g., through cy-totoxicity by listeriolysin or engaging cellular pathways thatutilize components of the host cytoskeleton by ActA, to affectthe course of infection. Hence, bacteria lacking the hexosephosphate transporter (uhpT) were significantly reduced forvirulence against infected Galleria larvae, implying that energy-rich phosphorylated derivatives of glucose are also importantsubstrates for bacterial growth in invertebrate cells.

However, some virulence factors, such as PlcA, appear to bedispensable for pathogenesis in Galleria. The lack of patho-genic potential has been previously observed for a plcA mutantin human umbilical vein endothelial cell (HUVEC) monolay-ers (20), suggesting that the listerial phosphatidylinositol phos-pholipase may have host- and cell-type-specific properties. Ashas also previously been observed, intravenous (i.v.) infectionof mice with either the inlA or inlB mutant did not reveal anypathogenic potential. This is not unexpected, as these are cell-tropic factors that are required for overcoming epithelial andendothelial barriers following oral infection (19).

The role of specific virulence factors of L. monocytogenes inGalleria infection was also illustrated by the increase in mor-tality caused by nonpathogenic L. innocua harboring the vgclocus from L. monocytogenes. We note, however, that there isa significant difference in the mortality rates of the pathogenicEGD-e strain and the virulent L. innocua vgc recombinantstrain, suggesting the presence of additional specific factorsencoded by the EGD-e genome that contribute to listerialpathogenesis in Galleria.

In conclusion, we note that while some listerial virulencegenes are generally needed for infection in mammals as well asin invertebrates, others have evolved for different hosts as wellas tissue-specific infections. Recently, chitinases capable ofhydrolyzing �-chitin from arthropods were found in some L.monocytogenes strains, which may be of importance for inver-tebrate infections (34).

We differentiated numerous species and serotypes of Listeriabased on their ability to infect Galleria, showing that only the

FIG. 7. Effects of preimmune activation on subsequent challengewith L. monocytogenes. (A) Activation of the immune system by in-jecting 10 mg/ml of LPS 24 h prior Listeria infection resulted in asignificant increase of survival of Galleria larvae (F) in comparison tountreated larvae (E). From totals of 10 mg/ml and 1 mg/ml of LPSstock solution 100 �g and 10 �g of LPS were injected into each larvafor immune induction. (B) Hemolymph samples of the preimmuneactivated larvae produce antimicrobial effectors that inhibit the growthof L. monocytogenes. The size of the inhibition zone increased with theconcentration of LPS used for preimmune activation. Similar resultswere obtained using heat-killed Listeria cells for immune activationprior to infection (data not shown). Results represent means of at leastthree independent determinations � standard deviations. Each repe-tition contained 30 larvae per treatment. Statistically differences areindicated (*, P � 0.05; ***, P � 0.01; **, P � 0.005).

VOL. 76, 2010 LISTERIA PATHOGENESIS IN GALLERIA 315

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.

human pathogen species L. monocytogenes was lethal for Gal-leria. Among the L. monocytogenes serotypes, 4b is reported tobe the most invasive and pathogenic to mammals (15). Indeed,the heightened virulence of this serotype in Galleria underlinesthe discerning properties of this model system.

The ability of L. monocytogenes to overcome host immuneresponses and multiply within the host system was confirmedby monitoring bacterial CFU following infection. We observeda strong reduction of L. monocytogenes in larvae at very earlytimes, i.e., at 1 h postinfection, suggesting the presence ofeffective constitutively expressed components of innate im-mune responses in Galleria. Apart from the efficient constitu-tive immune system that Galleria employs to limit microbialgrowth, we show here that an induced response comprisingsequential and overlapping expression of antimicrobial pep-tides, lysozyme, and inhibitors of host and bacterial metallo-proteinases is required for complete elimination of bacteriacausing septic infections. This inducible immunity in Galleriaagainst Listeria infection seems to be nonspecific and can beinduced by products that are not part of the infecting patho-gen. Thus, as we show here, preactivation with LPS (which isnot present in Gram-positive bacteria) or heat-killed prepara-tions of L. monocytogenes can induce immune responses sim-ilar to those observed upon lethal challenge with pathogenicListeria.

Despite the clear utility of Galleria as a surrogate model toassess infections with L. monocytogenes, several limitations re-main. The relatively long time required to monitor killing oflarvae and the inability to assess oral infections are impedi-ments that need to be overcome. A further impediment is thelack of a genome sequence for Galleria and of a well-estab-lished method to generate mutants. In this study we have useddeath as an end point to monitor progress of infection. How-ever additional phenotype and cellular assays, such as signs ofmelanization, nodulation, inducibility of pupa formation, andclotting phenotypes, need to be incorporated to improve thediscerning power of the model system. The processes that arereproduced in mice and Galleria may represent ancient mech-anisms of cell-cell interactions. However, the enormous evolu-tionary distance between these models also makes it clear thatmany host-specific phenomena are likely to exist.

In conclusion, here we demonstrate that G. mellonella is asimple yet powerful model system for assessing virulence of L.monocytogenes. Our data indicate that following infection,pathogenic listeriae are able to overcome both constitutive andinducible components of invertebrate innate immunity. Bygenerating additional mutants, we can now further explore thismodel system to identify further bacterial factors that modu-late innate immunity to promote bacterial growth during in-fection.

ACKNOWLEDGMENTS

We thank Alexandra Amend, Nelli Schklarenko, and Meike Fischerfor excellent technical assistance.

This project was funded by the German Ministry of Educationand Research through ERANET program grant SPATELIS to T.H.and T.C. K.M. was supported by grants made available throughNGFN-2 to T.C.

We have no financial conflict of interest.

REFERENCES

1. Agaisse, H., L. S. Burrack, J. A. Philips, E. J. Rubin, N. Perrimon, and D. E.Higgins. 2005. Genome-wide RNAi screen for host factors required forintracellular bacterial infection. Science 309:1248–1251.

2. Altincicek, B., E. Knorr, and A. Vilcinskas. 2008. Beetle immunity: identifi-cation of immune-inducible genes from the model insect Tribolium casta-neum. Dev. Comp. Immunol. 32:585–595.

3. Altincicek, B., M. Linder, D. Linder, K. T. Preissner, and A. Vilcinskas.2007. Microbial metalloproteinases mediate sensing of invading pathogensand activate innate immune responses in the lepidopteran model host Gal-leria mellonella. Infect. Immun. 75:175–183.

4. Altincicek, B., S. Stotzel, M. Wygrecka, K. T. Preissner, and A. Vilcinskas.2008. Host-derived extracellular nucleic acids enhance innate immune re-sponses, induce coagulation, and prolong survival upon infection in insects.J. Immunol. 181:2705–2712.

5. Altincicek, B., and A. Vilcinskas. 2006. Metamorphosis and collagen-IV-fragments stimulate innate immune response in the greater wax moth, Gal-leria mellonella. Dev. Comp. Immunol. 30:1108–1118.

6. Altincicek, B., and A. Vilcinskas. 2008. Identification of a lepidopteranmatrix metalloproteinase with dual roles in metamorphosis and innate im-munity. Dev. Comp. Immunol. 32:400–409.

7. Aperis, G., B. B. Fuchs, C. A. Anderson, J. E. Warner, S. B. Calderwood, andE. Mylonakis. 2007. Galleria mellonella as a model host to study infection bythe Francisella tularensis live vaccine strain. Microbes Infect. 9:729–734.

8. Ayres, J. S., N. Freitag, and D. S. Schneider. 2008. Identification ofDrosophila mutants altering defense of and endurance to Listeria monocyto-genes infection. Genetics 178:1807–1815.

9. Ayres, J. S., and D. S. Schneider. 2006. Genomic dissection of microbialpathogenesis in cultured Drosophila cells. Trends Microbiol. 14:101–104.

10. Ayres, J. S., and D. S. Schneider. 2008. A signaling protease required formelanization in Drosophila affects resistance and tolerance of infections.PLoS Biol. 6:2764–2773.

11. Bergin, D., L. Murphy, J. Keenan, M. Clynes, and K. Kavanagh. 2006.Pre-exposure to yeast protects larvae of Galleria mellonella from a subse-quent lethal infection by Candida albicans and is mediated by the increasedexpression of antimicrobial peptides. Microbes Infect. 8:2105–2112.

12. Brennan, M., D. Y. Thomas, M. Whiteway, and K. Kavanagh. 2002. Corre-lation between virulence of Candida albicans mutants in mice and Galleriamellonella larvae. FEMS Immunol. Med. Microbiol. 34:153–157.

13. Chakraborty, T., F. Ebel, E. Domann, K. Niebuhr, B. Gerstel, S. Pistor, C. J.Temm-Grove, B. M. Jockusch, M. Reinhard, U. Walter, et al. 1995. A focaladhesion factor directly linking intracellularly motile Listeria monocytogenesand Listeria ivanovii to the actin-based cytoskeleton of mammalian cells.EMBO J. 14:1314–1321.

14. Chatterjee, S. S., H. Hossain, S. Otten, C. Kuenne, K. Kuchmina, S. Ma-chata, E. Domann, T. Chakraborty, and T. Hain. 2006. Intracellular geneexpression profile of Listeria monocytogenes. Infect. Immun. 74:1323–1338.

15. Chatterjee, S. S., S. Otten, T. Hain, A. Lingnau, U. D. Carl, J. Wehland, E.Domann, and T. Chakraborty. 2006. Invasiveness is a variable and hetero-geneous phenotype in Listeria monocytogenes serotype strains. Int. J. Med.Microbiol. 296:277–286.

16. Cheng, L. W., and D. A. Portnoy. 2003. Drosophila S2 cells: an alternativeinfection model for Listeria monocytogenes. Cell. Microbiol. 5:875–885.

17. Cowen, L. E., S. D. Singh, J. R. Kohler, C. Collins, A. K. Zaas, W. A. Schell,H. Aziz, E. Mylonakis, J. R. Perfect, L. Whitesell, and S. Lindquist. 2009.Harnessing Hsp90 function as a powerful, broadly effective therapeutic strat-egy for fungal infectious disease. Proc. Natl. Acad. Sci. U. S. A. 106:2818–2823.

18. Derre, I., M. Pypaert, A. Dautry-Varsat, and H. Agaisse. 2007. RNAi screenin Drosophila cells reveals the involvement of the Tom complex in Chlamydiainfection. PLoS Pathog. 3:1446–1458.

19. Disson, O., S. Grayo, E. Huillet, G. Nikitas, F. Langa-Vives, O. Dussurget,M. Ragon, A. Le Monnier, C. Babinet, P. Cossart, and M. Lecuit. 2008.Conjugated action of two species-specific invasion proteins for fetoplacentallisteriosis. Nature 455:1114–1118.

20. Drevets, D. A. 1998. Listeria monocytogenes virulence factors that stimulateendothelial cells. Infect. Immun. 66:232–238.

21. Fedhila, S., N. Daou, D. Lereclus, and C. Nielsen-LeRoux. 2006. Identifica-tion of Bacillus cereus internalin and other candidate virulence genes specif-ically induced during oral infection in insects. Mol. Microbiol. 62:339–355.

22. Fleming, D. W., S. L. Cochi, K. L. MacDonald, J. Brondum, P. S. Hayes,B. D. Plikaytis, M. B. Holmes, A. Audurier, C. V. Broome, and A. L. Rein-gold. 1985. Pasteurized milk as a vehicle of infection in an outbreak oflisteriosis. N. Engl. J. Med. 312:404–407.

23. Glaser, P., L. Frangeul, C. Buchrieser, C. Rusniok, A. Amend, F. Baquero,P. Berche, H. Bloecker, P. Brandt, T. Chakraborty, A. Charbit, F. Chetouani,E. Couve, A. de Daruvar, P. Dehoux, E. Domann, G. Dominguez-Bernal, E.Duchaud, L. Durant, O. Dussurget, K. D. Entian, H. Fsihi, F. Garcia-delPortillo, P. Garrido, L. Gautier, W. Goebel, N. Gomez-Lopez, T. Hain, J.Hauf, D. Jackson, L. M. Jones, U. Kaerst, J. Kreft, M. Kuhn, F. Kunst, G.Kurapkat, E. Madueno, A. Maitournam, J. M. Vicente, E. Ng, H. Nedjari, G.

316 MUKHERJEE ET AL. APPL. ENVIRON. MICROBIOL.

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.

Nordsiek, S. Novella, B. de Pablos, J. C. Perez-Diaz, R. Purcell, B. Remmel,M. Rose, T. Schlueter, N. Simoes, A. Tierrez, J. A. Vazquez-Boland, H. Voss,J. Wehland, and P. Cossart. 2001. Comparative genomics of Listeria species.Science 294:849–852.

24. Gordon, M. D., J. S. Ayres, D. S. Schneider, and R. Nusse. 2008. Pathogen-esis of listeria-infected Drosophila wntD mutants is associated with elevatedlevels of the novel immunity gene edin. PLoS Pathog. 4:e1000111.

25. Guzman, C. A., M. Rohde, T. Chakraborty, E. Domann, M. Hudel, J. Weh-land, and K. N. Timmis. 1995. Interaction of Listeria monocytogenes withmouse dendritic cells. Infect. Immun. 63:3665–3673.

26. Hain, T., S. S. Chatterjee, R. Ghai, C. T. Kuenne, A. Billion, C. Steinweg, E.Domann, U. Karst, L. Jansch, J. Wehland, W. Eisenreich, A. Bacher, B.Joseph, J. Schar, J. Kreft, J. Klumpp, M. J. Loessner, J. Dorscht, K. Neu-haus, T. M. Fuchs, S. Scherer, M. Doumith, C. Jacquet, P. Martin, P.Cossart, C. Rusniock, P. Glaser, C. Buchrieser, W. Goebel, and T.Chakraborty. 2007. Pathogenomics of Listeria spp. Int. J. Med. Microbiol.297:541–557.

27. Hain, T., S. Otten, U. von Both, S. S. Chatterjee, U. Technow, A. Billion, R.Ghai, W. Mohamed, E. Domann, and T. Chakraborty. 2008. Novel bacterialartificial chromosome vector pUvBBAC for use in studies of the functionalgenomics of Listeria spp. Appl. Environ. Microbiol. 74:1892–1901.

28. Hamon, M., H. Bierne, and P. Cossart. 2006. Listeria monocytogenes: amultifaceted model. Nat. Rev. Microbiol. 4:423–434.

29. Hanahan, D. 1983. Studies on transformation of Escherichia coli with plas-mids. J. Mol. Biol. 166:557–580.

30. Jackson, J. C., L. A. Higgins, and X. Lin. 2009. Conidiation color mutants ofAspergillus fumigatus are highly pathogenic to the heterologous insect hostGalleria mellonella. PLoS One 4:e4224.

31. Jander, G., L. G. Rahme, and F. M. Ausubel. 2000. Positive correlationbetween virulence of Pseudomonas aeruginosa mutants in mice and insects. J.Bacteriol. 182:3843–3845.

32. Jensen, R. L., K. S. Pedersen, V. Loeschcke, H. Ingmer, and J. J. Leisner.2007. Limitations in the use of Drosophila melanogaster as a model host forgram-positive bacterial infection. Lett. Appl. Microbiol. 44:218–223.

33. Lecuit, M. 2007. Human listeriosis and animal models. Microbes Infect.9:1216–1225.

34. Leisner, J. J., M. H. Larsen, R. L. Jorgensen, L. Brondsted, L. E. Thomsen,and H. Ingmer. 2008. Chitin hydrolysis by Listeria spp., including L. mono-cytogenes. Appl. Environ. Microbiol. 74:3823–3830.

35. Lingnau, A., E. Domann, M. Hudel, M. Bock, T. Nichterlein, J. Wehland,and T. Chakraborty. 1995. Expression of the Listeria monocytogenes EGDinlA and inlB genes, whose products mediate bacterial entry into tissueculture cell lines, by PrfA-dependent and -independent mechanisms. Infect.Immun. 63:3896–3903.

36. Linnan, M. J., L. Mascola, X. D. Lou, V. Goulet, S. May, C. Salminen, D. W.Hird, M. L. Yonekura, P. Hayes, R. Weaver, et al. 1988. Epidemic listeriosisassociated with Mexican-style cheese. N. Engl. J. Med. 319:823–828.

37. Mansfield, B. E., M. S. Dionne, D. S. Schneider, and N. E. Freitag. 2003.Exploration of host-pathogen interactions using Listeria monocytogenes andDrosophila melanogaster. Cell. Microbiol. 5:901–911.

38. Midelet-Bourdin, G., G. Leleu, S. Copin, S. M. Roche, P. Velge, and P. Malle.2006. Modification of a virulence-associated phenotype after growth of Lis-teria monocytogenes on food. J. Appl. Microbiol. 101:300–308.

39. Miyata, S., M. Casey, D. W. Frank, F. M. Ausubel, and E. Drenkard. 2003.Use of the Galleria mellonella caterpillar as a model host to study the role of

the type III secretion system in Pseudomonas aeruginosa pathogenesis. In-fect. Immun. 71:2404–2413.

40. Morton, D. B., G. B. Dunphy, and J. S. Chadwick. 1987. Reactions ofhemocytes of immune and non-immune Galleria mellonella larvae to Proteusmirabilis. Dev. Comp. Immunol. 11:47–55.

41. Mylonakis, E. 2008. Galleria mellonella and the study of fungal pathogenesis:making the case for another genetically tractable model host. Mycopatholo-gia 165:1–3.

42. Mylonakis, E., R. Moreno, J. B. El Khoury, A. Idnurm, J. Heitman, S. B.Calderwood, F. M. Ausubel, and A. Diener. 2005. Galleria mellonella as amodel system to study Cryptococcus neoformans pathogenesis. Infect. Im-mun. 73:3842–3850.

43. Park, S. F., and G. S. Stewart. 1990. High-efficiency transformation of Lis-teria monocytogenes by electroporation of penicillin-treated cells. Gene 94:129–132.

44. Park, S. Y., K. M. Kim, J. H. Lee, S. J. Seo, and I. H. Lee. 2007. Extracellulargelatinase of Enterococcus faecalis destroys a defense system in insect he-molymph and human serum. Infect. Immun. 75:1861–1869.

45. Paschen, A., K. E. Dittmar, R. Grenningloh, M. Rohde, D. Schadendorf, E.Domann, T. Chakraborty, and S. Weiss. 2000. Human dendritic cells in-fected by Listeria monocytogenes: induction of maturation, requirements forphagolysosomal escape and antigen presentation capacity. Eur. J. Immunol.30:3447–3456.

46. Peleg, A. Y., D. Monga, S. Pillai, E. Mylonakis, R. C. Moellering, Jr., andG. M. Eliopoulos. 2009. Reduced susceptibility to vancomycin influencespathogenicity in Staphylococcus aureus infection. J. Infect. Dis. 199:532–536.

47. Schaferkordt, S., and T. Chakraborty. 1995. Vector plasmid for insertionalmutagenesis and directional cloning in Listeria spp. Biotechniques 19:720–725.

48. Schell, M. A., L. Lipscomb, and D. DeShazer. 2008. Comparative genomicsand an insect model rapidly identify novel virulence genes of Burkholderiamallei. J. Bacteriol. 190:2306–2313.

49. Scully, L. R., and M. J. Bidochka. 2005. Serial passage of the opportunisticpathogen Aspergillus flavus through an insect host yields decreased saprobiccapacity. Can. J. Microbiol. 51:185–189.

50. Seed, K. D., and J. J. Dennis. 2008. Development of Galleria mellonella as analternative infection model for the Burkholderia cepacia complex. Infect.Immun. 76:1267–1275.

51. Seed, K. D., and J. J. Dennis. 2009. Experimental bacteriophage therapyincreases survival of Galleria mellonella larvae infected with clinically rele-vant strains of the Burkholderia cepacia complex. Antimicrob. Agents Che-mother. 53:2205–2208.

52. Seitz, V., A. Clermont, M. Wedde, M. Hummel, A. Vilcinskas, K. Schlatterer,and L. Podsiadlowski. 2003. Identification of immunorelevant genes fromgreater wax moth (Galleria mellonella) by a subtractive hybridization ap-proach. Dev. Comp. Immunol. 27:207–215.

53. Thomsen, L. E., S. S. Slutz, M. W. Tan, and H. Ingmer. 2006. Caenorhabditiselegans is a model host for Listeria monocytogenes. Appl. Environ. Microbiol.72:1700–1701.

54. Vazquez-Boland, J. A., M. Kuhn, P. Berche, T. Chakraborty, G. Dominguez-Bernal, W. Goebel, B. Gonzalez-Zorn, J. Wehland, and J. Kreft. 2001. Lis-teria pathogenesis and molecular virulence determinants. Clin. Microbiol.Rev. 14:584–640.

55. Vilcinskas, A., P. Gotz. 1999. Parasitic fungi and their interactions with theinsect immune system. Adv. Parasitol. 43:267–313.

VOL. 76, 2010 LISTERIA PATHOGENESIS IN GALLERIA 317

Dow

nloa

ded

from

http

s://j

ourn

als.

asm

.org

/jour

nal/a

em o

n 21

Nov

embe

r 20

21 b

y 45

.70.

1.18

9.