Galleria mellonella as a model system for studying Listeria pathogenesis

Abstract

Essential aspects of the innate immune response to microbial infection are conserved between insects and mammals. 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 degrees C, as a model host for examining the virulence potential of Listeria spp. Here we report that Galleria is an excellent surrogate model of listerial septic infection, capable of clearly distinguishing between pathogenic and nonpathogenic Listeria strains and even between virulent and attenuated Listeria monocytogenes strains. Virulence required listerial genes hitherto implicated in the mouse infection model and was linked to strong antimicrobial activities in both hemolymph and hemocytes of infected larvae. Following Listeria infection, the expression of immune defense genes such as those for lysozyme, galiomycin, gallerimycin, and insect metalloproteinase inhibitor (IMPI) was sequentially induced. Preinduction of antimicrobial activity by treatment of larvae with lipopolysaccharide (LPS) significantly improved survival against subsequent L. monocytogenes challenge and strong antilisterial activity was detected 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 a relatively simple, nonmammalian model system that can be used to assess the virulence of strains of Listeria spp. isolated from a wide variety of settings from both the clinic and the environment.

FIG. 1.

Dose-dependent survival of Galleria caterpillars after inoculation with L. monocytogenes and L. innocua. Bacteria were grown to log phase 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 higher mortality with EGD-e than with L. innocua. Results represent means of at least three independent determinations ± standard deviations for 10 animals per treatment.

FIG. 2.

Multiplication of L. monocytogenes in Galleria. (A) L. monocytogenes cells were stained using ActA antibodies (resulting in red fluorescence), and host actin of hemocytes was stained using Alexa-phalloidin (resulting in green fluorescence). Note that Listeria organisms are spreading throughout the cytosol of the hemocyte, and actin tails at the poles of some of the bacteria are visible. (B) To determine rate of multiplication of L. monocytogenes in Galleria larvae, we determined the listerial load from infected larvae at several time points postinfection. For each time point, homogenates of 10 larvae were plated individually for CFU count on Listeria selective Palcam agar plates. These results are shown as one dot, and resulting mean values of are shown in red. Surviving animals contained reduced listerial load, whereas dying larvae contained about 2 × 10⁵ CFU, as indicated by a circle. The experiment was repeated three times with similar results.

FIG. 3.

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

FIG. 4.

Contributions of major virulence-related genes of L. monocytogenes in the mortality of Galleria. (A) The vgc locus is responsible for the pathogenicity of L. monocytogenes. L. monocytogenes with vgc deleted had a significant reduction of killing capacity in comparison to 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 mortality rates between ΔuhpT and ΔinlAB was found to be 0.005, and that between ΔuhpT and ΔplcB was 0.05. Results represent means of at least three independent determinations ± standard deviations for 10 animals per treatment.

FIG. 5.

Insertion of EGD-e-derived vgc in L. innocua results in induced virulence. Artificial introduction of the vgc1 locus into otherwise nonpathogenic L. innocua resulted in a significant increase of virulence with respect to that of wild-type L. innocua. Results represent means of at least three independent determinations ± standard deviations. Each repetition contained 30 larvae per treatment.

FIG. 6.

Transcriptional activation of actin and immune-responsive genes following infection. The transcription levels of actin, galiomycin, gallerimycin, IMPI, and lysozyme were determined by quantitative real-time RT-PCR analysis and are shown relative to the expression levels in mock-injected animals. Results were normalized to expression of the housekeeping 18S RNA gene and represent means of three independent determinations ± standard deviations.

FIG. 7.

Effects of preimmune activation on subsequent challenge with L. monocytogenes. (A) Activation of the immune system by injecting 10 mg/ml of LPS 24 h prior Listeria infection resulted in a significant increase of survival of Galleria larvae (•) in comparison to untreated larvae (○). From totals of 10 mg/ml and 1 mg/ml of LPS stock solution 100 μg and 10 μg of LPS were injected into each larva for immune induction. (B) Hemolymph samples of the preimmune activated larvae produce antimicrobial effectors that inhibit the growth of L. monocytogenes. The size of the inhibition zone increased with the concentration of LPS used for preimmune activation. Similar results were obtained using heat-killed Listeria cells for immune activation prior to infection (data not shown). Results represent means of at least three independent determinations ± standard deviations. Each repetition contained 30 larvae per treatment. Statistically differences are indicated (*, P < 0.05; ***, P < 0.01; **, P < 0.005).