A signaling protease required for melanization in Drosophila affects resistance and tolerance of infections - PubMed (original) (raw)
A signaling protease required for melanization in Drosophila affects resistance and tolerance of infections
Janelle S Ayres et al. PLoS Biol. 2008.
Abstract
Organisms evolve two routes to surviving infections-they can resist pathogen growth (resistance) and they can endure the pathogenesis of infection (tolerance). The sum of these two properties together defines the defensive capabilities of the host. Typically, studies of animal defenses focus on either understanding resistance or, to a lesser extent, tolerance mechanisms, thus providing little understanding of the relationship between these two mechanisms. We suggest there are nine possible pairwise permutations of these traits, assuming they can increase, decrease, or remain unchanged in an independent manner. Here we show that by making a single mutation in the gene encoding a protease, CG3066, active in the melanization cascade in Drosophila melanogaster, we observe the full spectrum of changes; these mutant flies show increases and decreases in their resistance and tolerance properties when challenged with a variety of pathogens. This result implicates melanization in fighting microbial infections and shows that an immune response can affect both resistance and tolerance to infections in microbe-dependent ways. The fly is often described as having an unsophisticated and stereotypical immune response where single mutations cause simple binary changes in immunity. We report a level of complexity in the fly's immune response that has strong ecological implications. We suggest that immune responses are highly tuned by evolution, since selection for defenses that alter resistance against one pathogen may change both resistance and tolerance to other pathogens.
Conflict of interest statement
Competing interests. The authors have declared that no competing interests exist.
Figures
Figure 1. Manipulation of Resistance and Tolerance Affects Host Health
We hypothesize that resistance and tolerance of a host can be manipulated in an independent manner, generating nine possible pairwise permutations to affect overall host health. Mutant phenotypes can be mapped onto a two-dimensional space where the axes are defined by health and pathogen load. We measure median survival time as a proxy for health and measure bacterial load directly in homogenized flies. The red dot represents the phenotype of a wild-type fly strain infected with a pathogen. Any given mutation could either have no effect or shift the phenotype to any of the eight red dots. Theoretically, phenotypic shifts can occur by altering either the resistance of the host, the tolerance, or both properties. The areas marked in blue show the area where shifts in resistance are expected to move the phenotypes; the pale yellow bar indicates the areas affected by shifts in tolerance, and the green shows areas that are caused by changes in both properties.
Figure 2. Disseminated Melanization in Infected Flies
Ventral, dorsal, and injection site views of the abdomen of L. monocytogenes– or medium-injected female w1118 and CG3066 mutants. Deposits of melanin at the injection site (green arrows) can be seen in both wild-type and mutant flies for both microbe and medium injections. No melanin is observed on either fly strain beyond what is seen at the injection site in the absence of infection. Large amounts of melanin are deposited on both the ventral and dorsal sides of the abdomen in wild-type flies when infected with L. monocytogenes, while no melanin is observed in L. monocytogenes-infected CG3066 mutants.
Figure 3. Quantification of Disseminated Melanization in Infected Flies
Male and female 5- to 7-d-old w1118 and CG3066 mutants were infected with the microbes listed in Table 1 or with medium and examined for disseminated melanization throughout the course of infections with L. monocytogenes, S. typhimurium, S. aureus, E. faecalis, S. pneumoniae, E. coli, and B. cepacia. Melanization can be observed in L. monocytogenes-infected flies approximately 4 d postinfection, while melanization in S. typhimurium and S. aureus-infected flies can be seen approximately 7 d postinfection. Values indicate the percentage of infected wild-type or CG3066 mutants that exhibit disseminated melanization, and data are represented as mean ± standard error of the mean. At least three groups of 20 flies were examined for each condition, and experiments were repeated three times and yielded similar results.
Figure 4. Survival of Infected CG3066 Mutants
Male 5- to 7-d-old flies were infected with bacteria or medium and survival was recorded daily for B. cepacia; E. coli; E. faecalis; L. monocytogenes infection in CG3066K02818 mutant (p < 0.0001); L. monocytogenes infection in UAS-CG3066 RNAi (p < 0.0001); S. aureus (p < 0.0001) and S. pneumoniae (p < 0.0001) with medium control; S. pneumoniae without medium control (p < 0.0001); S. typhimurium (p < 0.0001) and medium versus unmanipulated flies (p < 0.0001). Filled squares represent microbe-injected wild-type flies, filled circles indicate microbe-injected CG3066 mutant flies, open squares/dashed lines represent medium-injected wild-type flies, open circles/dashed lines indicate medium-injected CG3066 mutants, × indicates unmanipulated wild-type flies, and asterisks (*) indicate unmanipulated CG3066 mutants. For RNAi experiments expression was driven by the collagen-GAL4 driver, Cg-GAL4. Squares represent wild-type flies, circles represent Cg-GAL4/UAS-CG3066 RNAi, triangles represent Cg-GAL4/wild type, and diamonds represent UAS-CG3066 RNAi/wild type. Data are plotted as Kaplan-Meier plots and statistical significance was determined using log-rank analysis. Survival curves were repeated at least three times and yielded similar results each time.
Figure 5. Bacterial Growth in CG3066 Mutants
Bacterial growth was determined for B. cepacia; E. coli; E. faecelis; L. monocytogenes growth in CG3066K02818 mutants; L. monocytogenes growth in UAS-CG3066 RNAi; and E. coli, and S. pneumoniae. Male 5- to 7-d-old flies were infected with bacteria and collected at 0, 24, and 48 h postinfection. S. pneumoniae–infected flies were collected at 0, 2, 4, and 24 h postinfection. Flies were homogenized at each time point, serially diluted, and plated. For L. monocytogenes infection, flies were injected with 50 nl of 1 mg/ml gentamicin or water 3 h prior to plating. For B. cepacia, E. coli, S. pneumoniae, and S. typhimurium, solid black boxes indicate wild type, and solid white boxes indicate CG3066 mutant. For gentamicin chase of L. monocytogenes-infected CG3066 mutants, solid black boxes indicate water-treated wild-type flies, solid white boxes indicate water-treated CG3066 mutants, left diagonal hashed boxes indicate gentamicin-treated wild-type flies, and right diagonal hashed boxes indicate gentamicin-treated CG3066 mutants. For L. monocytogenes infection in RNAi flies, solid black boxes indicate wild-type flies, solid white boxes indicate Cg-GAL4/UAS-CG3066 RNAi, horizontal hashed boxes indicate Cg-GAL4/wild type, and vertical lined boxes indicate UAS-CG3066 RNAi/wild type. The _p_-value was determined with a nonparametric two-tailed _t_-test. Experiments for each microbe were repeated at least three times and gave similar results. * p < 0.01; ** p < 0.005.
Figure 6. Summary of Mutant Classes
We propose that resistance and tolerance can vary independently of each other, resulting in nine phenotypic classes. Three of these classes are clearly observable in CG3066 mutants when infected with E. faecalis, B. cepacia, or E. coli. Infections of CG3066 mutants fall into the class where resistance is increased but changes in tolerance cannot be easily measured here. Likewise, L. monocytogenes and S. typhimurium fall into the class where resistance is decreased but tolerance cannot be measured.
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