Monday, December 5, 2011

The fly, the amoeba, and the worm

The fly, the amoeba, and the worm
Robet Koch formulated in 1890 the Koch´s postulates as general guidelines that should be followed to identify pathogens causing diseases. One century later, Stanley Falkow established the molecular version of Koch's postulates to guide, this time, the identification of microbial genes encoding virulence factors. A key point of the molecular postulates is to test the virulence of the microorganism with the inactivated candidate virulence gene in an appropriate animal model. However, this is not always possible. Suitable animals models are lacking for many diseases such as brucellosis, typhoid and leprosy. And the models for tuberculosis and cholera do not reflect the biology of human infections. In addition, large scale analysis of virulence are costs prohibited due to the high number of animals that should be infected to get statistically significant results. And, last but not least, there are important ethical concerns on the use of vertebrate animals models (including mice and rats) for research. In the case of plant pathogens ethical concerns are not an issue, however the logistics behind a virulence experiment in plants represent a challenge (space, biosafety regulations, possibility of spreading in nature of genetically modified organisms,...).

To solve these issues, some years ago new models to test virulence were introduced: Drosophila melanogaster (the fruit-fly), Dyctiostelium discoideum (the social amoeba) and Caenorhabditis elegans (the soil nematode). With general skepticism, it was assumed that the same virulence factors important for virulence in humans/plants could play a role in the interplay with these surrogate hosts. Indeed, this has been case, and these three amigos (no offense for the actors, I like the movie) have made outstanding contributions to the microbial pathonegesis field.

Caenorhabditis elegans
Frederick Ausubel and co-wrorkers published pioneering studies analyzing the virulence of the human pathogen Pseudomonas aeruginosa in C. elegans, Arabidopsis (a plant model) and mice (Cell, 1999). Notably, they found an extensive overlap between the bacterial factors required to infect these different hosts. After the first papers describing the use of C.elegans as a suitable model to analyze the host-pathogen interface, the field has grown rapidly since and now more than 20 bacterial pathogens have been studied (for a summary see: Worm and pathogens).

Drosophila melanogaster
D. melanogaster has also been used as surrogate host to study bacterial virulence. The first pathogens tested were Pseudomonas and Serratia but later on Salmonella, Vibrio, Yersinia, and Staphylococcus have been shown to infect Drosophila. An important advantage of Drosophila as a model is that it possesses induce defense mechanisms similar to those of mammals with respect to both the regulatory pathways involved and the spectrum of their antimicrobial factors. Jules Hoffmann has received the 2011 Nobel Prize of Medicine by his discoveries in this field. 

Dyctiostelium discoideum
Bacteria are the main source of food of the free living amoebae D. discoideum. When the food supply is abundant the slime mold organisms live in unicellar form. Once the food becomes sparse they aggregate to form a multicellular fruiting body composed of two main cell types: stalk cells that support a spore-containing sorus. This social behaviour makes D. discoideium a good model to study cell development and motitly. Dictyostelium avidly engulf and kills most of the bacteria. It can be said that Dictyostelium is a primitive macrophage. This is why the organism is used as a host model for several intracellular pathogens including Legionella, Mycobacterium and Salmonella. Recently, and using D. discoideum as a tool, John Mekalanos and co-workers described a new system implicated in bacteria-bacteria defense and host-pathogen interactions (PNAS, 2006).

A very interesting feature common to the three models is the fact that they can be manipulated genetically. In other words, it is possible to run a 2-D virulence array to identify also host resistance genes in a systematic way. It should be noted that host resistance genes are poorly characterized and eventaully could be useful for therapeutic manipulation in the context of infections.

Galleria mellonella
A major drawback of the three models is the temperature at which the challenge is done, bellow 25C. This is an important limitation since many bacterial pathogens regulate the expression of many virulence factors in response to the host temperature, 37C. To circumvent this problem, recently it has been developed the wax moth larva, Galleria mellonella, as another alternative infection model. The infection challenge is done at 37C although unfortunately Galleria cannot be mutated and the genome is not yet available. Nevertheless, G. mellonella has both humoral and cellular immune response pathways mediated by antimicrobial peptides and phagocytic cells (hemocytes), respectively, enabling an assessment of host responses. Finally, and of great novelty, the G. mellonella infection model is amenable to antibiotic treatment, and thus, the efficacy of antimicrobial agents can be assessed. Galleria has been already utilized to study host-pathogen interactions in a range of organisms including P. aeruginosa, Burkholderia species, Proteus mirabilis, Listeria, Acinetobacter, and several pathogenic fungi.

Model host systems are powerful tools for studying virulence because they have the capacity to highlight similarities, contrast differences, and provide important insights. However, they are not substitute hosts, since no one host can fully replicate another. Model hosts will undoubtedly continue to find new uses and applications.

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