|
 The
outcome of the interaction between a pathogen
and a host depends on the interplay between virulence
factors of the microorganism and host responses
to the infection. Some virulence factors are induced
only in the host and may therefore require specialized
techniques to be identified, based on detection
of these genes in vivo or the survival of mutagenized
strains within specific host environments. Previous
studies have shown that non-vertebrate hosts can
be used to study established and identify novel
virulence determinants in a variety of microbial
animal pathogens. The hypothesis is that selection
for genes involved in interactions with both mammalian
and non-mammalian hosts may specifically identify
genes of fundamental relevance to pathogenesis
independent of the model system used (read
more).
Therefore, investigators have increasingly turned
to invertebrates as facile and inexpensive hosts
to model a variety of human pathogens. If killing
of invertebrate hosts by pathogens mimics key
features of mammalian pathogenesis, then it should
be possible to use invertebrates as facile and
inexpensive hosts for high throughput study of
microbial pathogenesis.
We developed three invertebrate systems for the
study of the fungal pathogens. These non-mammalian
hosts are:
- Caenorhabditis elegans
- Drosophila
melanogaster and
- Galleria mellonella.
Each of these systems provides some unique
advantages. We are using non-mammalian hosts
and especially Caenorhabditis
elegans to study host-pathogen interactions. C.
elegans is a facile model (read
more)
and has enabled us to study virulence factors
of the model fungal pathogen Cryptococcus
neoformans.
Using this system we have ascertained a number
of C.
neoformans mutants that are hypovirulent
in C. elegans (read
more).
The current approach in antimicrobial studies is to analyze the “host”, the “pathogen”, or the “anti-microbial compound”. This artificial separation is dictated by the lack of model systems in which all approaches can be used simultaneously (read more). This is a major hindrance to developing novel antimicrobial agents and groundbreaking therapies. For example, the currently accepted "gold standard" approach of using an in vitro assay to determine the minimal inhibitory concentration (MIC) of compounds does not allow the simultaneous evaluation of toxicity, the identification of compounds that affect virulence traits that are expressed only in vivo, or compounds that have an immuno-modulatory effect that augments the host response to infection. To achieve a real paradigm shift in antimicrobial discovery, we are developing whole animal C. elegans-based high throughput assays for the identification and characterization of antifungal compounds. We found that Candida albicans as well as other Candida species are ingested by C. elegans and establish a persistent lethal infection in the C. elegans intestinal track. Importantly, key components of Candida pathogenesis in mammals, such as biofilm and filament formation, are also involved in nematode killing. The assay is performed in liquid media using standard 96-well plate technology. A screen of 1,266 compounds with known pharmaceutical activities identified 15 (~1.2%) that prolonged survival of C. albicans-infected nematodes and inhibited in vivo filamentation of C. albicans. We have tested three of these compounds in the murine model of candidiasis and two of these compounds identified, caffeic acid phenethyl ester (CAPE), a major active component of honeybee propolis, and the fluoroquinolone agent enoxacin, exhibited anti-fungal activity in mice. (read more) These surrogate hosts fill an important niche in microbial pathogenesis research and, along with established mammalian models provide us with a unique opportunity to identify and study basic, evolutionarily conserved aspects of microbial virulence and host response (http://www.asm.org/microbe/index.asp?bid=55040).
 Finally, prokaryote-eukaryote interactions are ubiquitous, and have important medical and environmental significance. Despite this, a paucity of data exists on the mechanisms and pathogenic consequences of bacterial-fungal encounters within a living host. We utilized the nematode C. elegans as a substitute host to study the interactions between two ecologically related and clinically troublesome pathogens, the prokaryote, Acinetobacter baumannii, and the eukaryote, C. albicans http://www.pnas.org/content/105/38/14585 After co-infecting C. elegans with these organisms, we observed that A. baumannii inhibits filamentation, a key virulence determinant of C. albicans. This antagonistic, cross-kingdom interaction led to attenuated virulence of C. albicans, as determined by improved nematode survival when infected with both pathogens. In vitro co-infection assays in planktonic and biofilm environments supported the inhibitory effects of A. baumannii toward C. albicans, further showing a predilection of A. baumannii for C. albicans filaments. Interestingly, we demonstrate a likely evolutionary defense by C. albicans against A. baumannii, whereby C. albicans inhibits A. baumannii growth once a quorum develops. This counter-offensive is at least partly mediated by the C. albicans quorum-sensing molecule farnesol. We utilized the C. elegans co-infection A. baumannii-C. albicans model to evaluate an A. baumannii mutant library, leading to the identification of several mutants attenuated in their inhibitory activity toward C. albicans. These findings present an extension to the current paradigm of studying mono-microbial pathogenesis in C. elegans, and by use of genetic manipulation, provides a whole animal model system to investigate the complex dynamics of a polymicrobial infection.
Overall, as part of our efforts we have demonstrated that: 1. There are extensive similarities between fungal infections in mammals and non-vertebrate models including nematode and insect hosts. 2. The model microscopic nematode C. elegans grown on non-pathogenic yeasts has a life span similar to or longer than nematodes fed on the usual laboratory food source, but human pathogenic yeasts kill C. elegans. 3. A number of virulence traits that are involved in mammalian infection from a variety of fungal pathogens are associated with C. elegans disease. 4. C. elegans assays can be used to identify novel virulence factors that are associated with mammalian infection and these virulence factors provide important insights in fungal biology. 5. C. elegans assays can be used to identify and study compounds with antifungal efficacy and multidrug resistance (MDR) mechanisms. 6. During polymicrobial infection, prokaryote-eukaryote interactions modulate fungal virulence. 7. There are evolutionarily conserved pathways for the innate sensing of fungal pathogens. We identified C. elegans receptors that (similar to and their mammalian orthologues) mediate host defense against C. neoformans.
Also, the laboratory remains deeply committed in the education of students and in assisting younger scientists and physicians to reach their full potential. We actively participate in the programs organized by the MGH Multicultural Affairs Office and the Harvard Initiative for Global Health, while through collaborations with scientists in other countries. Eleftherios Mylonakis, M.D.
Division of Infectious Diseases
55 Fruit Street
Gray Jackson 5, Room GRJ-504
Boston, Massachusetts 02114-2696
(617) 726-3812
E-mail: emylonakis@partners.org |
