Name: The Galleria mellonella Waxworm Infection Model for Disseminated Candidiasis (Video) | JoVE Journal
Uploaded: 2018-11-17T15:00:00.000+00:00
Duration: 7 min 33 s
Description: Learn the Galleria mellonella waxworm infection model for studying disseminated candidiasis using Candida albicans. This method enables high-throughput analysis of fungal virulence factors.

Candida species are common human commensals that are capable of emerging as opportunistic pathogens in severely immunocompromised and dysbiotic patients. Although many Candida species can cause disease, C. albicans is the most prevalent cause of disseminated candidiasis¹^,². Systemic disease results from C. albicans accessing the bloodstream through either direct penetration of previously restrictive host barriers or introduction at surgical sites and other breaches of the body³. Candida species utilize a range of pathogenic processes to cause systemic disease within the host including filamentation, biofilm formation, immune cell evasion and escape, and iron scavenging⁴. In vitro approaches exist to investigate individual pathogenic mechanisms, but animal models continue to provide the best option to investigate the entirety of disease outcome⁵^,⁶. Previous research has detailed many instances of promising in vitro investigations of virulence failing to reproduce in vivo⁷^,⁸. Thus, animal models are still required to assess virulence in vivo. Most disease models rely on mice to serve as a surrogate for human infections despite C. albicans inability to naturally colonize murine systems as a commensal⁹. Invertebrate models of disseminated candidiasis include the nematode Caenorhabditis elegans, the fruit fly Drosophila melanogaster, and the waxworm Galleria mellonella, although concerns about fundamental differences in basic physiology, host body temperatures, and routes of exposure have hampered their broad acceptance¹⁰^,¹¹.

Most recently, the G. mellonella waxworm infection model has been adopted to model pathogenicity of a wide range of bacterial and fungal pathogens¹²^,¹³^,¹⁴. Advantages of this model include its relatively low cost, increased throughput, ease of use, and reduced ethical concerns regarding animal beneficence compared to murine models. For researchers, this translates into increased ability to test multiple variables, stronger confidence intervals, more rapid experimentation, and bypass of animal protocols. G. mellonella has served as a platform to rapidly assess C. albicans virulence following perturbation of genes required for biofilm formation, filamentation, and gene regulation across clinical isolates¹¹^,¹⁵^,¹⁶. Recent studies have incorporated investigation of antifungal efficacy using G. mellonella to assess the pharmacokinetics of drug activity and resistance under in vivo settings, which are otherwise challenging and time consuming¹⁷^,¹⁸. Yet, studies of C. albicans virulence in G. mellonella have been complicated by reportedly high levels of variation within experiments and inconsistent protocols between research groups that produce differing virulence phenotypes between mice and waxworms¹¹^,¹³^,¹⁹^,²⁰^,²¹. Here, we outline a G. mellonella protocol to standardize C. albicans infections, increase reproducibility in virulence experiments, and demonstrate consistency with previously described studies of virulence in murine models.

Previous studies demonstrated that the C. albicans mating type-like (MTL) locus on chromosome 5 regulates cell identity and mating competence similar to Saccharomyces cerevisiae and other Ascomycete fungi²². The majority of C. albicans isolates are heterozygous at the MTL locus, encoding one of each of the MTLaand MTLα alleles (MTLa/α), and are consequently sterile¹⁵^,²³^,²⁴. Loss of one of the MTL alleles through loss of heterozygosity (LOH) or mutation leads to homozygous MTLa or MTLα strains that can undergo a phenotypic switch from the sterile 'white' state to the mating competent 'opaque' state²⁵. Previous work has highlighted that loss of MTL heterozygosity also reduces virulence in murine models of systemic infection across different strain backgrounds²⁶. Here, we detail the G. mellonella model for disseminated candidiasis using a genetically similar experimental set to depict the contribution of MTL heterozygosity to virulence in G. mellonella. We show that MTL configuration influenced C. albicans pathogenicity, where MTLα strains were less virulent with respect to both MTLa/α and MTLa cells, similar to findings within murine infection model²⁶.

The <em>Galleria mellonella</em> Waxworm Infection Model for Disseminated Candidiasis