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Genetic, molecular and physiological basis of variation in Drosophila gut immunocompetence.

Bou Sleiman MS, Osman D, Massouras A, Hoffmann AA, Lemaitre B, Deplancke B - Nat Commun (2015)

Bottom Line: Gut immunocompetence involves immune, stress and regenerative processes.Using genome-wide association analysis, we identify several novel immune modulators.This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines.

View Article: PubMed Central - PubMed

Affiliation: 1] Global Health Institute, School of Life Sciences, Station 19, EPFL, 1015 Lausanne, Switzerland [2] Institute of Bioengineering, School of Life Sciences, Station 19, EPFL, 1015 Lausanne, Switzerland.

ABSTRACT
Gut immunocompetence involves immune, stress and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, we perform enteric infection of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila and observe extensive variation in survival. Using genome-wide association analysis, we identify several novel immune modulators. Transcriptional profiling further shows that the intestinal molecular state differs between resistant and susceptible lines, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study provides novel insights into the determinants underlying population-level variability in gut immunocompetence, revealing how relatively minor, but systematic genetic and transcriptional variation can mediate overt physiological differences that determine enteric infection susceptibility.

No MeSH data available.


Related in: MedlinePlus

Susceptibility to infection is highly variable among DGRP lines and multifactorial.(a) Bar graph showing for each of the 140 DGRP lines (x axis) the percentage of dead female flies (y axis) 3 days post-enteric infection with Pseudomonas entomophila (A 100). Data shown are averages from three biological replicates (±s.e. of the proportion; n>60 females per line). (b) A scatter plot of 78 DGRP lines revealing an absence of correlation in proportion death between enteric (by 3 days post-P. entomophila ingestion) and systemic (by 10 days post-septic injury with Ecc15) infection. DGRP line 25745 (red) is highly susceptible in both conditions and features a rare mutation in the dredd gene. (c) Quantification of P. entomophila-specific monalysin genomic DNA by qPCR reveals differences in P. entomophila clearance between four susceptible and four resistant DGRP lines over time (ANOVA P=0.00343 for the effect of susceptibility class; see Supplementary Methods for details on statistics). (d) Quantification of PH3-positive cells per female midgut dissected 8 h post enteric infection with P. entomophila reveals that infected resistant lines have more mitotically active stem cells than those of susceptible lines (n>30 guts per line; ANOVA P<0.00001 for difference between susceptibility classes). (e) Measurement of the incorporation of a methionine analogue, L-azidohomoalanine (green staining), in the R2 region56 of the anterior midgut shows that susceptible lines are not able to synthetize proteins after infection in contrast to resistant lines. Note that while the same midgut region was sampled, no gross morphological differences in the shape or regionalization of the gut can be observed between resistant and susceptible flies after infection. However, this does not rule out subtle differences at the cellular level. Scale bar 50 μm.
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f1: Susceptibility to infection is highly variable among DGRP lines and multifactorial.(a) Bar graph showing for each of the 140 DGRP lines (x axis) the percentage of dead female flies (y axis) 3 days post-enteric infection with Pseudomonas entomophila (A 100). Data shown are averages from three biological replicates (±s.e. of the proportion; n>60 females per line). (b) A scatter plot of 78 DGRP lines revealing an absence of correlation in proportion death between enteric (by 3 days post-P. entomophila ingestion) and systemic (by 10 days post-septic injury with Ecc15) infection. DGRP line 25745 (red) is highly susceptible in both conditions and features a rare mutation in the dredd gene. (c) Quantification of P. entomophila-specific monalysin genomic DNA by qPCR reveals differences in P. entomophila clearance between four susceptible and four resistant DGRP lines over time (ANOVA P=0.00343 for the effect of susceptibility class; see Supplementary Methods for details on statistics). (d) Quantification of PH3-positive cells per female midgut dissected 8 h post enteric infection with P. entomophila reveals that infected resistant lines have more mitotically active stem cells than those of susceptible lines (n>30 guts per line; ANOVA P<0.00001 for difference between susceptibility classes). (e) Measurement of the incorporation of a methionine analogue, L-azidohomoalanine (green staining), in the R2 region56 of the anterior midgut shows that susceptible lines are not able to synthetize proteins after infection in contrast to resistant lines. Note that while the same midgut region was sampled, no gross morphological differences in the shape or regionalization of the gut can be observed between resistant and susceptible flies after infection. However, this does not rule out subtle differences at the cellular level. Scale bar 50 μm.

Mentions: To assess the extent of gut immunocompetence variation in genetically distinct individuals, we measured fly survival following enteric infection with the entomopathogenic bacterium Pseudomonas entomophila26 in 140 DGRP lines whose genomes have been comprehensively characterized for single nucleotide polymorphisms (SNPs) and non-SNP variants181927. We found striking and reproducible variation in the DGRP lines' survival (Fig. 1a; Supplementary Fig. 1a; Supplementary Table 1), comparable to previous observations regarding natural variation in systemic immunity in Drosophila23. While around 50% of the tested lines harbour the natural endosymbiont Wolbachia19, this had no effect on susceptibility (Supplementary Fig. 1b). To eliminate the possibility that the differential susceptibility of the lines is due to differences in commensal bacteria28, we infected five lines randomly chosen from each phenotypic class (resistant or susceptible) in germ-free conditions. The loss of commensals did not alter their relative susceptibility, indicating that the endogenous microbiota do not majorly impact on susceptibility class (Supplementary Fig. 1c). We also evaluated whether our results could be biased by differences in feeding behaviour between DGRP lines but found no consistent difference in food uptake between resistant and susceptible lines (Supplementary Fig. 1d). To determine if this variability in survival is specific to enteric infection, we assessed susceptibility of DGRP lines to systemic infection with Erwinia carotovora carotovora 15 (Ecc15) (Fig. 1b). We did not use P. entomophila since it leads to very fast lethality in this condition, which renders the scoring of a meaningful phenotype difficult. We found little correlation between the two infection conditions and pathogens (Pearson correlation, r=0.23, n=78, P=0.0395). This observation suggests that the determinants of gut immunocompetence are distinct from those that govern systemic immunity29. However, one line, 25745, was highly susceptible in both infection conditions (Fig. 1b). We found that this fly line contains a mutation in the dredd gene, a component of the immune deficiency (Imd) pathway required to resist Gram-negative bacterial infection730 (Supplementary Fig. 2a–d). Mutations with such a strong loss-of-function phenotype tend to be rare in a natural population and do not capture most of the underlying natural variation in gut immunocompetence20. For instance, the mutation we identified in dredd was found in only one of 205 genotyped DGRP lines18. Moreover, in a natural population, such a rare recessive allele would be mostly found in heterozygous form, which could explain why it has not been eliminated by purifying selection. We next examined whether the observed differences in survival is specific to P. entomophila by orally infecting DGRP lines with a clinical isolate of Pseudomonas aeruginosa (PA14). Specifically, using a similar infection protocol as for P. entomophila (Methods), we infected four randomly selected lines from the lower 10% in terms of survival to P. entomophila infection (that is, resistant) and four randomly from the upper 90% (that is, susceptible, excluding the dredd mutant line discussed above) and compared survival after 3 days (Supplementary Fig. 3). DGRP lines that were resistant to oral infection by P. entomophila were also resistant to PA14, while three of the four tested lines that were susceptible to P. entomophila were also susceptible to PA14. These results suggest that the DGRP phenotypes observed for P. entomophila infection may reflect a more general pattern in that they may be due to a common, likely bacterium-independent genetic and molecular mechanism that mediates oral infection susceptibility.


Genetic, molecular and physiological basis of variation in Drosophila gut immunocompetence.

Bou Sleiman MS, Osman D, Massouras A, Hoffmann AA, Lemaitre B, Deplancke B - Nat Commun (2015)

Susceptibility to infection is highly variable among DGRP lines and multifactorial.(a) Bar graph showing for each of the 140 DGRP lines (x axis) the percentage of dead female flies (y axis) 3 days post-enteric infection with Pseudomonas entomophila (A 100). Data shown are averages from three biological replicates (±s.e. of the proportion; n>60 females per line). (b) A scatter plot of 78 DGRP lines revealing an absence of correlation in proportion death between enteric (by 3 days post-P. entomophila ingestion) and systemic (by 10 days post-septic injury with Ecc15) infection. DGRP line 25745 (red) is highly susceptible in both conditions and features a rare mutation in the dredd gene. (c) Quantification of P. entomophila-specific monalysin genomic DNA by qPCR reveals differences in P. entomophila clearance between four susceptible and four resistant DGRP lines over time (ANOVA P=0.00343 for the effect of susceptibility class; see Supplementary Methods for details on statistics). (d) Quantification of PH3-positive cells per female midgut dissected 8 h post enteric infection with P. entomophila reveals that infected resistant lines have more mitotically active stem cells than those of susceptible lines (n>30 guts per line; ANOVA P<0.00001 for difference between susceptibility classes). (e) Measurement of the incorporation of a methionine analogue, L-azidohomoalanine (green staining), in the R2 region56 of the anterior midgut shows that susceptible lines are not able to synthetize proteins after infection in contrast to resistant lines. Note that while the same midgut region was sampled, no gross morphological differences in the shape or regionalization of the gut can be observed between resistant and susceptible flies after infection. However, this does not rule out subtle differences at the cellular level. Scale bar 50 μm.
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Related In: Results  -  Collection

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f1: Susceptibility to infection is highly variable among DGRP lines and multifactorial.(a) Bar graph showing for each of the 140 DGRP lines (x axis) the percentage of dead female flies (y axis) 3 days post-enteric infection with Pseudomonas entomophila (A 100). Data shown are averages from three biological replicates (±s.e. of the proportion; n>60 females per line). (b) A scatter plot of 78 DGRP lines revealing an absence of correlation in proportion death between enteric (by 3 days post-P. entomophila ingestion) and systemic (by 10 days post-septic injury with Ecc15) infection. DGRP line 25745 (red) is highly susceptible in both conditions and features a rare mutation in the dredd gene. (c) Quantification of P. entomophila-specific monalysin genomic DNA by qPCR reveals differences in P. entomophila clearance between four susceptible and four resistant DGRP lines over time (ANOVA P=0.00343 for the effect of susceptibility class; see Supplementary Methods for details on statistics). (d) Quantification of PH3-positive cells per female midgut dissected 8 h post enteric infection with P. entomophila reveals that infected resistant lines have more mitotically active stem cells than those of susceptible lines (n>30 guts per line; ANOVA P<0.00001 for difference between susceptibility classes). (e) Measurement of the incorporation of a methionine analogue, L-azidohomoalanine (green staining), in the R2 region56 of the anterior midgut shows that susceptible lines are not able to synthetize proteins after infection in contrast to resistant lines. Note that while the same midgut region was sampled, no gross morphological differences in the shape or regionalization of the gut can be observed between resistant and susceptible flies after infection. However, this does not rule out subtle differences at the cellular level. Scale bar 50 μm.
Mentions: To assess the extent of gut immunocompetence variation in genetically distinct individuals, we measured fly survival following enteric infection with the entomopathogenic bacterium Pseudomonas entomophila26 in 140 DGRP lines whose genomes have been comprehensively characterized for single nucleotide polymorphisms (SNPs) and non-SNP variants181927. We found striking and reproducible variation in the DGRP lines' survival (Fig. 1a; Supplementary Fig. 1a; Supplementary Table 1), comparable to previous observations regarding natural variation in systemic immunity in Drosophila23. While around 50% of the tested lines harbour the natural endosymbiont Wolbachia19, this had no effect on susceptibility (Supplementary Fig. 1b). To eliminate the possibility that the differential susceptibility of the lines is due to differences in commensal bacteria28, we infected five lines randomly chosen from each phenotypic class (resistant or susceptible) in germ-free conditions. The loss of commensals did not alter their relative susceptibility, indicating that the endogenous microbiota do not majorly impact on susceptibility class (Supplementary Fig. 1c). We also evaluated whether our results could be biased by differences in feeding behaviour between DGRP lines but found no consistent difference in food uptake between resistant and susceptible lines (Supplementary Fig. 1d). To determine if this variability in survival is specific to enteric infection, we assessed susceptibility of DGRP lines to systemic infection with Erwinia carotovora carotovora 15 (Ecc15) (Fig. 1b). We did not use P. entomophila since it leads to very fast lethality in this condition, which renders the scoring of a meaningful phenotype difficult. We found little correlation between the two infection conditions and pathogens (Pearson correlation, r=0.23, n=78, P=0.0395). This observation suggests that the determinants of gut immunocompetence are distinct from those that govern systemic immunity29. However, one line, 25745, was highly susceptible in both infection conditions (Fig. 1b). We found that this fly line contains a mutation in the dredd gene, a component of the immune deficiency (Imd) pathway required to resist Gram-negative bacterial infection730 (Supplementary Fig. 2a–d). Mutations with such a strong loss-of-function phenotype tend to be rare in a natural population and do not capture most of the underlying natural variation in gut immunocompetence20. For instance, the mutation we identified in dredd was found in only one of 205 genotyped DGRP lines18. Moreover, in a natural population, such a rare recessive allele would be mostly found in heterozygous form, which could explain why it has not been eliminated by purifying selection. We next examined whether the observed differences in survival is specific to P. entomophila by orally infecting DGRP lines with a clinical isolate of Pseudomonas aeruginosa (PA14). Specifically, using a similar infection protocol as for P. entomophila (Methods), we infected four randomly selected lines from the lower 10% in terms of survival to P. entomophila infection (that is, resistant) and four randomly from the upper 90% (that is, susceptible, excluding the dredd mutant line discussed above) and compared survival after 3 days (Supplementary Fig. 3). DGRP lines that were resistant to oral infection by P. entomophila were also resistant to PA14, while three of the four tested lines that were susceptible to P. entomophila were also susceptible to PA14. These results suggest that the DGRP phenotypes observed for P. entomophila infection may reflect a more general pattern in that they may be due to a common, likely bacterium-independent genetic and molecular mechanism that mediates oral infection susceptibility.

Bottom Line: Gut immunocompetence involves immune, stress and regenerative processes.Using genome-wide association analysis, we identify several novel immune modulators.This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines.

View Article: PubMed Central - PubMed

Affiliation: 1] Global Health Institute, School of Life Sciences, Station 19, EPFL, 1015 Lausanne, Switzerland [2] Institute of Bioengineering, School of Life Sciences, Station 19, EPFL, 1015 Lausanne, Switzerland.

ABSTRACT
Gut immunocompetence involves immune, stress and regenerative processes. To investigate the determinants underlying inter-individual variation in gut immunocompetence, we perform enteric infection of 140 Drosophila lines with the entomopathogenic bacterium Pseudomonas entomophila and observe extensive variation in survival. Using genome-wide association analysis, we identify several novel immune modulators. Transcriptional profiling further shows that the intestinal molecular state differs between resistant and susceptible lines, already before infection, with one transcriptional module involving genes linked to reactive oxygen species (ROS) metabolism contributing to this difference. This genetic and molecular variation is physiologically manifested in lower ROS activity, lower susceptibility to ROS-inducing agent, faster pathogen clearance and higher stem cell activity in resistant versus susceptible lines. This study provides novel insights into the determinants underlying population-level variability in gut immunocompetence, revealing how relatively minor, but systematic genetic and transcriptional variation can mediate overt physiological differences that determine enteric infection susceptibility.

No MeSH data available.


Related in: MedlinePlus