New Perspectives on Galleria mellonella Larvae as a Host Model Using Riemerella anatipestifer as a Proof of Concept

Abstract

Galleria mellonella larvae have been used as a host model to study interactions between pathogens and hosts for several years. However, whether the model is useful to interrogate Riemerella anatipestifer infection biology remained unknown. This study aimed to exploit the potential of G. mellonella larvae and reveal their limitations as a host model for R. anatipestifer infection. G. mellonella larvae were shown to be effective for virulence evaluations of different R. anatipestifer strains. Furthermore, the virulent strain R. anatipestifer CH-1 had a stronger ability to proliferate than the attenuated strain R. anatipestifer ATCC 11845 in both G. mellonella larvae and ducklings. Unconventionally it was shown that G. mellonella larvae cannot be used to evaluate the efficacy of antimicrobials and their combinations. Additionally, it was shown that certain virulence factors, such as OmpA (B739_0861), B739_1208, B739_1343, and Wza (B739_1124), were specific only for ducklings, suggesting that G. mellonella larvae must be cautiously used to identify virulence factors of R. anatipestifer. Evaluation of heme uptake-related virulence genes, such as tonB1 and tonB2, required preincubating the strains with hemoglobin before infection of G. mellonella larvae since R. anatipestifer cannot obtain a heme source from G. mellonella larvae. In conclusion, this study revealed the applicability and limitations of G. mellonella as a model with which to study the pathogen-host interaction, particularly in the context of R. anatipestifer infection.A fter Robert Koch's work, Stanley Falkow established the molecular version of Koch's postulates, which have guided the study of the microbial virulence determinants in infectious diseases since the late 1980s (1). One of the key purposes of the molecular version of Koch's postulates is to test whether a microorganism is attenuated when a candidate virulence gene is inactivated. Thus, the use of animal models to identify the virulence factors of pathogens is indispensable. However, animal models are accompanied by ethical problems and cannot be used in large numbers for statistical analysis. Recently, nonmammalian models, including Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, and larvae of the greater wax moth, Galleria mellonella, have been used for biological research (2-4). These organisms share many advantages over mammalian models, including being easy to obtain, cost-effective, and more ethically acceptable. However, the optimal temperature for C. elegans, D. melanogaster, and Danio rerio is below 28°C (5, 6). These models are defective since the optimum temperature for most mammal pathogens is 37°C. In contrast, the G. mellonella larva model is able to survive at 37°C. Moreover, the immune system of G. mellonella larvae

Innate Immune Responses of Galleria mellonella to Mycobacterium bovis BCG Challenge Identified Using Proteomic and Molecular Approaches

“…Induction of expression was highest at 4 h pi, and thereafter decreased over the time course despite the presence of a persistent infection. Similar observations were made in an RT-qPCR study of G. mellonella innate immune responses to infection with Riemerella anatipestifer , a Gram-negative bacterium, where genes involved in innate immunity (e.g., encoding AMPs and opsonin), were highest at 4 h pi, and declined over time ( Liu et al., 2019 ). In this study, the trend between the presence of protein and the corresponding mRNA improved over the course of infection, but the abundance of protein was largely inconsistent with gene expression signals, with only 20% in agreement between 48-168 h pi.…”

Functional characterization of Fur in iron metabolism, oxidative stress resistance and virulence of Riemerella anatipestifer

“…The above results showed that the absence of fur led to a potentially increase in intracellular iron content, indicating that Fur is involved in the regulation of iron transport. Therefore, to verify whether R. anatipestifer CH-1 Fur can regulate the transcription of putative iron uptake-related genes, we detected the mRNA levels of TonB-dependent receptor genes B739_0103 and B739_0173 , which were up-regulated in an iron-limited environment and considered iron uptake-related genes [ 31 , 33 ]. The results showed that the transcription of B739_0103 and B739_0173 was markedly increased in R. anatipestifer CH-1 ∆fur compared to that in the wild-type strain, and their transcript levels were not affected when 100 µM EDDHA was added to the GCB medium (Figure 5 ).…”

“…In our previous study, we demonstrated that a fur deletion strain of R. anatipestifer showed reduced virulence in the Galleria mellonella model [ 33 ]. To investigate if Fur plays a role in the pathogenesis of R. anatipestifer CH-1 in poultry, we used a duckling model [ 29 , 30 ] to determine the LD 50 of the fur mutant.…”

“…Then, samples of the mixture were spread onto plates supplemented with chloramphenicol and incubated overnight at 37 °C. The correct clone was identified as described in a previous study [ 33 ]. The mutant R. anatipestifer CH-1Δ recA Δ fur was constructed by the same method on the basis of R. anatipestifer CH-1 Δ fur , which was constructed in a previous study [ 33 ].…”

“…Real-time PCR was performed with SYBR Green master mix (Q111-03, Vazyme) using a CFX Connect real-time PCR detection system (Bio-Rad Laboratories, Hercules, CA, USA). Then, the transcription levels of TonB-dependent receptor genes B739_0103 and B739_0173 were detected in each sample using specific primers [ 33 ]. Relative fold changes were calculated as described previously with the threshold cycle (ΔΔCT) method, considering the efficiency of the PCR for each target [ 37 ].…”