Invertebrate Host Model Research Articles

A new killing assay with the Caenorhabditis elegans PX627 mutant to assess the virulence of uropathogenic Escherichia coli strains

The nematode Caenorhabditis elegans (C. elegans) is a prime invertebrate host model for studying uropathogenic Escherichia coli (UPEC) pathogenesis. The aim of this work was to develop a new C. elegans killing assay based on feeding bacteria by the nematode throughout its life from the egg. With this model, the lifespan of C. elegans rrf-3, temperature-sterile, mutant, and PX627, auxin-inducible infertile, mutant fed UPEC strains, was compared. The behavior of three clinical UPEC strains and the non-pathogenic Escherichia coli OP50 strain was analyzed. Survival curves were generated by the Kaplan–Meier method and compared by the log-rank test over 10 days of follow-up. There was no significant difference between the survival curves obtained with each of the two C. elegans mutants (PX627 and rrf-3) fed with each of the strains of E. coli (OP50, G1722, G1473 or ER41). The UPEC strains were classified according to their virulence in vivo in the C. elegans PX627 mutant. The most virulent strain was ER41 which harbored the virulence genes fimA, papC and hlyA, expressed hemolysis in vitro and showed no antibiotic resistance. The least virulent strain was G1722 which only harbored the two adhesion factor genes, was not hemolytic and was resistant to multiple antibiotics. The C. elegans PX627 mutant fed with UPEC bacteria from the egg stage is a simple and inexpensive invertebrate animal model for assessing the in vivo virulence of different strains. The early exposure of C. elegans to pathogenic bacteria at the egg stage, without the need to change the incubation temperature, is an advantage over previously described C. elegans killing assays.

Small Molecule Analysis of Extracellular Vesicles Produced by Cryptococcus gattii: Identification of a Tripeptide Controlling Cryptococcal Infection in an Invertebrate Host Model.

The small molecule (molecular mass <900 Daltons) composition of extracellular vesicles (EVs) produced by the pathogenic fungus Cryptococcus gattii is unknown, which limits the understanding of the functions of cryptococcal EVs. In this study, we analyzed the composition of small molecules in samples obtained from solid cultures of C. gattii by a combination of chromatographic and spectrometric approaches, and untargeted metabolomics. This analysis revealed previously unknown components of EVs, including small peptides with known biological functions in other models. The peptides found in C. gattii EVs had their chemical structure validated by chemical approaches and comparison with authentic standards, and their functions tested in a Galleria mellonella model of cryptococcal infection. One of the vesicular peptides (isoleucine-proline-isoleucine, Ile-Pro-Ile) improved the survival of G. mellonella lethally infected with C. gattii or C. neoformans. These results indicate that small molecules exported in EVs are biologically active in Cryptococcus. Our study is the first to characterize a fungal EV molecule inducing protection, pointing to an immunological potential of extracellular peptides produced by C. gattii.

Tobacco Hornworm (Manduca sexta) caterpillars as a novel host model for the study of fungal virulence and drug efficacy

ABSTRACT The two leading yeast pathogens of humans, Candida albicans and Cryptococcus neoformans, cause systemic infections in >1.4 million patients worldwide with mortality rates approaching 75%. It is thus imperative to study fungal virulence mechanisms, efficacy of antifungal drugs, and host response pathways. While this is commonly done in mammalian models, which are afflicted by ethical and practical concerns, invertebrate models, such as wax moth larvae and nematodes have been introduced over the last two decades. To complement existing invertebrate host models, we developed fifth instar caterpillars of the Tobacco Hornworm moth Manduca sexta as a novel host model. These caterpillars can be maintained at 37°C, are suitable for injections with defined amounts of yeast cells, and are susceptible to the most threatening yeast pathogens, including C. albicans, C. neoformans, C. auris, and C. glabrata. Importantly, fungal burden can be assessed daily throughout the course of infection in a single caterpillar’s feces and hemolymph. Infected caterpillars can be rescued by treatment with antifungal drugs. Notably, these animals are large enough for weight to provide a reliable and reproducible measure of fungal disease and to facilitate host tissue-specific expression analyses. M. sexta caterpillars combine a suite of parameters that make them suitable for the study of fungal virulence.

Type III secretion system confers enhanced virulence in clinical non-O1/non-O139 Vibrio cholerae

Vibrio cholerae O1 infections mainly are responsible for significant mortality and morbidity amongst children, however, non-O1/non-O139 V. cholerae have also been reported to cause mild to severe infections because of their virulence potential. The pathogenic mechanisms of non-O1, non-O139 isolates are not as clearly understood as for that of O1 and O139 isolates. Type three secretion system (TTSS) is also considered one of the important virulent factors and during the current study, we investigated the role of TTSS in association with non-O1/non-O139 clinical isolates. We report that the presence of TTSS in non-O1/non-O139 V. cholerae clinical isolate (D13) from a child confers more virulence compared to the one lacking it (D15) in another clinical case during the small cholera epidemic. Moreover, the antibiotic susceptibility profiles of D13 and D15 indicate that they are multiple drug resistance (MDR) isolates. The sequence analysis for TTSS cluster was carried out for D13 and compared with the TTSS positive reference Vibrio parahaemolyticus RIMD2210633 and V. cholerae AM19226 non-O1/non-O139. Furthermore, the pathogenic potential of D13 & D15 was also explored in simple and economical invertebrate host model, Galleria mellonella and the results revealed that TTSS+ve isolate (D13) was more virulent compared to TTSS-ve isolate (D15). We suggest that this distinct genetic difference, seen in natural variants D13 and D15, is also reflected by the clinical picture of the former in contributing towards the severity of disease symptoms and this finding was further validated by assessing virulence potential of both isolates using inexpensive G. mellonella infection model.

Galleria Mellonella as a Model Invertebrate Host for the Study of Muriform cells of Dematiaceous Fungi

To study the pathogenesis of chromoblastomycosis using the alternative model host Galleria mellonella. We analyzed the virulence of different dematiaceous fungal strains and the host immune responses (hemocytes density and morphological changes) to Fonsecaea monophora by the alternative infection model. Then detected the development of the pathogenic muriform cells within larvae under microscope. Increasing inocula resulted in greater larval mortality and Cladophialophora carrionii was the most virulent. Low inocula activated the humoral immune response significantly. Moreover, the conidia underwent morphological transition to muriform cells within larvae. We developed an invertebrate host model that can be used to evaluate the virulence of dematiaceous fungi, which may provide further insights into overcoming current limitations in studying chromoblastomycosis in vivo.

Genome Sequence of Galleria mellonella (Greater Wax Moth).

ABSTRACTThe larvae of the greater wax moth, Galleria mellonella, are pests of active beehives. In infection biology, these larvae are playing a more and more attractive role as an invertebrate host model. Here, we report on the first genome sequence of Galleria mellonella.

Quorum sensing-dependent metalloprotease VvpE is important in the virulence of Vibrio vulnificus to invertebrates

Vibrio vulnificus, a Gram-negative bacterium, is an opportunistic human pathogen responsible for fatal septicemia caused by contaminated sea foods in eastern Asia. Quorum sensing (QS) is a cell-density dependent gene regulation mechanism that controls the expression of many virulence genes in various bacteria and V. vulnificus has been also suggested to express their virulence genes through the QS system. In this study, we investigated the role of QS system and QS-regulated exoproteases in the virulence of V. vulnificus using several invertebrate host models, Tenebrio molitor, an insect, Caenorhabditis elegans, a nematode, and brine shrimp (Artemia), an aquatic crustacean. When the culture supernatant of smcR (major QS regulator of V. vulnificus) mutant was injected to T. molitor larvae, it failed to induce the melanization of T. molitor larvae, while the culture supernatant of luxO (upstream negative regulator of smcR) mutant more strongly induced the melanization than wild type. These results demonstrated that QS system of V. vulnificus is crucial for virulence to T. molitor larvae. Among several QS-dependently expressed exoproteases of V. vulnificus, vvpE encoding a metalloprotease was mainly responsible for the melanization of T. molitor larvae, in that the culture supernatant of vvpE mutant failed to induce the melanization. This result was confirmed using the C. elegans and Artemia salina model systems, in which the vvpE mutant strains were attenuated in killing C. elegans and A. salina, compared with wild type, indicating that VvpE is important in the infection of V. vulnificus. In conclusion, we suggest that QS system and a QS-dependent exoprotease, VvpE are crucial for the V. vulnificus virulence to invertebrates.

Enterococcus infection biology: lessons from invertebrate host models.

The enterococci are commensals of the gastrointestinal tract of many metazoans, from insects to humans. While they normally do not cause disease in the intestine, they can become pathogenic when they infect sites outside of the gut. Recently, the enterococci have become important nosocomial pathogens, with the majority of human enterococcal infections caused by two species, Enterococcus faecalis and Enterococcus faecium. Studies using invertebrate infection models have revealed insights into the biology of enterococcal infections, as well as general principles underlying host innate immune defense. This review highlights recent findings on Enterococcus infection biology from two invertebrate infection models, the greater wax moth Galleria mellonella and the free-living bacteriovorous nematode Caenorhabditis elegans.

Antifungal resistance does not necessarily affect Candida glabrata fitness

Although there has been an overall good coverage of Candida glabrata infections by the echinocandins, emergence of antifungal resistance during therapy has been reported. We investigated, by using an invertebrate host model, the fitness of sequential C. glabrata isolates with different echinocandins susceptibility patterns. The studied strains were isolated from a case of recurrent fungemia with a fatal outcome due to C. glabrata that developed cross-resistance to echinocandins during caspofungin therapy. The sequential strains isolated post-therapy showed a S663P mutation in the Fks2p hot spot 1. In vivo study in the invertebrate host Galleria mellonella did not suggest a fitness cost related to the acquired antifungal resistance, the three isolates displayed a similar rate of killing (P = 0·54). We observed a clear correlation between emergence of antifungal resistance and persistence of the causal agent, probably aided by the unchanged fitness and unresponsiveness in vivo to the adopted therapy.

Photodynamic and Antibiotic Therapy Impair the Pathogenesis of Enterococcus faecium in a Whole Animal Insect Model

Enterococcus faecium has emerged as one of the most important pathogens in healthcare-associated infections worldwide due to its intrinsic and acquired resistance to many antibiotics, including vancomycin. Antimicrobial photodynamic therapy (aPDT) is an alternative therapeutic platform that is currently under investigation for the control and treatment of infections. PDT is based on the use of photoactive dye molecules, widely known as photosensitizer (PS). PS, upon irradiation with visible light, produces reactive oxygen species that can destroy lipids and proteins causing cell death. We employed Galleria mellonella (the greater wax moth) caterpillar fatally infected with E. faecium to develop an invertebrate host model system that can be used to study the antimicrobial PDT (alone or combined with antibiotics). In the establishment of infection by E. faecium in G. mellonella, we found that the G. mellonella death rate was dependent on the number of bacterial cells injected into the insect hemocoel and all E. faecium strains tested were capable of infecting and killing G. mellonella. Antibiotic treatment with ampicillin, gentamicin or the combination of ampicillin and gentamicin prolonged caterpillar survival infected by E. faecium (P = 0.0003, P = 0.0001 and P = 0.0001, respectively). In the study of antimicrobial PDT, we verified that methylene blue (MB) injected into the insect followed by whole body illumination prolonged the caterpillar survival (P = 0.0192). Interestingly, combination therapy of larvae infected with vancomycin-resistant E. faecium, with antimicrobial PDT followed by vancomycin, significantly prolonged the survival of the caterpillars when compared to either antimicrobial PDT (P = 0.0095) or vancomycin treatment alone (P = 0.0025), suggesting that the aPDT made the vancomycin resistant E. faecium strain more susceptible to vancomycin action. In summary, G. mellonella provides an invertebrate model host to study the antimicrobial PDT and to explore combinatorial aPDT-based treatments.

Assessment of virulence diversity of methicillin-resistant Staphylococcus aureus strains with a Drosophila melanogaster infection model

BackgroundStaphylococcus aureus strains with distinct genetic backgrounds have shown different virulence in animal models as well as associations with different clinical outcomes, such as causing infection in the hospital or the community. With S. aureus strains carrying diverse genetic backgrounds that have been demonstrated by gene typing and genomic sequences, it is difficult to compare these strains using mammalian models. Invertebrate host models provide a useful alternative approach for studying bacterial pathogenesis in mammals since they have conserved innate immune systems of biological defense. Here, we employed Drosophila melanogaster as a host model for studying the virulence of S. aureus strains.ResultsCommunity-associated methicillin-resistant S. aureus (CA-MRSA) strains USA300, USA400 and CMRSA2 were more virulent than a hospital-associated (HA)-MRSA strain (CMRSA6) and a colonization strain (M92) in the D. melanogaster model. These results correlate with bacterial virulence in the Caenorhabditis elegans host model as well as human clinical data. Moreover, MRSA killing activities in the D. melanogaster model are associated with bacterial replication within the flies. Different MRSA strains induced similar host responses in D. melanogaster, but demonstrated differential expression of common bacterial virulence factors, which may account for the different killing activities in the model. In addition, hemolysin α, an important virulence factor produced by S. aureus in human infections is postulated to play a role in the fly killing.ConclusionsOur results demonstrate that the D. melanogaster model is potentially useful for studying S. aureus pathogenicity. Different MRSA strains demonstrated diverse virulence in the D. melanogaster model, which may be the result of differing expression of bacterial virulence factors in vivo.

Pesticide exposure strongly enhances parasite virulence in an invertebrate host model

Parasites are a common and constant threat to organisms at all levels of phyla. The virulence of a parasite, defined as the impact on survival and reproduction of its host, depends on the specific host–parasite combination and can also be influenced by environmental conditions. Environmental pollution might be an additional factor influencing host–parasite interactions. We here aimed to test whether the combined stress of pollutant exposure and parasite challenge results in stronger impacts on host organisms than expected from the single stressors applied alone. We used the water flea Daphnia magna and two of its endoparasites, the bacterium Pasteuria ramosa and the microsporidium Flabelliforma magnivora, as invertebrate host–parasite models. For each parasite, we tested in a full‐factorial design for interactions between parasitism and pollution using the neurotoxic pesticide carbaryl as a model substance. Sublethal concentrations of the pesticide synergistically enhanced the virulence of both parasites by increasing host mortality. Furthermore, host castration induced by P. ramosa was accelerated by carbaryl exposure. These effects likely reflect decreased host resistance due to direct or indirect immunosuppressive activity of carbaryl. The present study provides experimental evidence that the in vivo development of infectious diseases can be influenced by a pesticide at environmentally realistic concentrations. This implies that host–parasite interactions and subsequently co‐evolution might be influenced by environmental pollution at toxicant concentrations being sublethal to parasite‐free hosts. Standard toxicity testing as employed in the current way of conducting ecological risk assessments for anthropogenic substances does not consider natural antagonists such as infectious diseases, and thereby likely underestimates the impact these substances may pose to natural populations in the environment.

Galleria mellonella as a Model System To Study Cryptococcus neoformans Pathogenesis

Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37 degrees C and 30 degrees C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.