Beneficial commensal bacteria promote Drosophila growth by downregulating the expression of peptidoglycan recognition proteins

Abstract

Commensal bacteria are known to promote host growth. Such effect partly relies on the capacity of microbes to regulate the host's transcriptional response. However, these evidences mainly come from comparing the transcriptional response caused by commensal bacteria with that of axenic animals, making it difficult to identify the animal genes that are specifically regulated by beneficial microbes. Here, we employ Drosophila melanogaster associated with Lactiplantibacillus plantarum to understand the host genetic pathways regulated by beneficial bacteria and leading to improved host growth. We show that microbial benefit to the host relies on the downregulation of peptidoglycan-recognition proteins. Specifically, we report that bacterial proliferation triggers the lower expression of PGRP-SC1 in larval midgut, which ultimately leads to improved host growth and development. Our study helps elucidate the mechanisms underlying the beneficial effect exerted by commensal bacteria, defining the role of immune effectors in the relationship between Drosophila and its gut microbes.

Keywords: immunology; microbiology; microbiome.

Graphical abstract

Figure 1

Lp^FlyG2.1.8 significantly alters Drosophila transcriptional response compared with Lp^NIZO2877 Bar chart of the genes with significantly differential expression (up- and downregulated) between Lp^FlyG2.1.8- and Lp^NIZO2877-associated larvae (p < 0.05 and −1.5- to +1.5-fold). Each color refers to the molecular function associated with each gene.

Figure 2

PGRP-SC1 downregulation by beneficial L. plantarum strains occurs in Drosophila gut and is regulated by L. plantarum ackA function Relative expression of PGRP-SC1 gene obtained by performing a qRT-PCR analysis on the transcriptome of yw Drosophila larvae (A) and dissected larval guts (B) mono-associated with the bacterial strains tested (x axis). Lines above each bar represent the mean with the standard deviation (SD) calculated by analyzing three biological replicates per condition. Relative expression was calculated as ΔC_T, using the housekeeping gene rp49 as reference gene. Statistical significance of the results with one-way ANOVA test analysis is included (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001). (C) Longitudinal size (mm) of Drosophila larvae measured after 7 days of incubation with the bacterial strain Lp^NIZO2877, Lp^FlyG2.1.8, Lp^DietG20.2.2, Lp^ΔackA, and Lp^NIZO2877 supplemented with N-acetyl glutamine (NAG), respectively. Each symbol refers to the larval size obtained from one out of the N ≥ 60 Drosophila larvae analyzed for each condition, with bars referring to the respective mean and standard deviation (SD). All conditions were compared with Lp^NIZO2877 by performing one-way ANOVA test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001).

Figure 3

PGRP-SC1 regulation and microbial load (A) Relative expression of PGRP-SC1 gene obtained by performing a qRT-PCR analysis on the transcriptome of Drosophila larvae mono-associated with L. plantarum strains (x axis). Lines above each bar represent the mean with the standard deviation (SD) calculated by analyzing three biological replicates per condition. Relative expression was calculated as ΔC_T, using the housekeeping gene rp49 as reference gene. Statistical significance of the results with one-way ANOVA test analysis is included:∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001. (B) Microbial load (CFU/mL) of the bacterial strains tested after 4 days of incubation in the fly diet and in the intestine of Drosophila larvae, respectively. Each symbol represents one out of the four replicates analyzed for each condition, with bars indicating the respective mean and standard deviation (SD). All conditions were compared with Lp^NIZO2877 by performing one-way ANOVA test (∗p ≤ 0.1, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001).

Figure 4

Lp^NIZO2877 recapitulates the same growth-promoting effect of Lp^FlyG2.1.8 in mutant PGRP-SC flies (A) Longitudinal size of Mex-Gal4>UAS-mCherry-RNAi larvae (control) and Mex-Gal4>UAS-PGRPSC-RNAi larvae (target) (A), Oregon-R wild-type (WT) population and larvae carrying a deletion of PGRP-SC gene cluster (PGRP-SC^Δ) (B) associated with the bacterial strains Lp^NIZO2877 and Lp^FlyG2.1.8 and measured 7 days after egg deposition on poor-nutrient diet. Each symbol refers to the larval size analyzed for each condition, with bars referring to the respective mean and standard deviation (SD). Each condition was compared with Lp^NIZO2877-associated larvae of the respective fly background by performing Mann-Whitney test (∗∗∗∗p ≤ 0.0001, ∗∗∗p ≤ 0.001, ns = not statistically significant). (C) Microbial load (CFU/mL) of bacteria retrieved from the diet of WT and PGRP-SC^Δ Drosophila larvae associated with the strains Lp^NIZO2877 and Lp^FlyG2.1.8, respectively. Each symbol represents the mean value for each technical replicate, whereas lines are the mean obtained from the values within the same condition with the respective standard deviation (SD). All conditions were compared with WT-Lp^NIZO2877 by performing one-way ANOVA test (∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001).