CHD1 Contributes to Intestinal Resistance against Infection by <em>P. aeruginosa</em> in <em>Drosophila melanogaster</em>

<div><p><em>Drosophila</em> SNF2-type ATPase CHD1 catalyzes the assembly and remodeling of nucleosomal arrays <em>in vitro</em> and is involved in H3.3 incorporation <em>in viin vivo</em> during early embryo development. Evidence for a role as transcriptional regulator comes from its colocalization with elongating RNA polymerase II as well as from studies of fly <em>Hsp70</em> transcription. Here we used microarray analysis to identify target genes of CHD1. We found a fraction of genes that were misregulated in <em>Chd1</em> mutants to be functionally linked to <em>Drosophila</em> immune and stress response. Infection experiments using different microbial species revealed defects in host defense in <em>Chd1</em>-deficient adults upon oral infection with <em>P. aeruginosa</em> but not upon septic injury, suggesting a so far unrecognized role for CHD1 in intestinal immunity. Further molecular analysis showed that gut-specific transcription of antimicrobial peptide genes was overactivated in the absence of infection in <em>Chd1</em> mutant flies. Moreover, microbial colonization of the intestine was elevated in <em>Chd1</em> mutants and oral infection resulted in strong enrichment of bacteria in the body cavity indicating increased microbial passage across intestinal epithelia. However, we did not detect enhanced epithelial damage or alterations of the intestinal stem cell population. Collectively, our data provide evidence that intestinal resistance against infection by <em>P. aeruginosa</em> in <em>Drosophila</em> is linked to maintaining proper balance of gut-microbe interactions and that the chromatin remodeler CHD1 is involved in regulating this aspect.</p> </div

Bacterial load is elevated in <i>Chd1</i>-mutant flies.

<p>(A) Bacterial load was analyzed in isolated guts and in whole flies from which intestines had been removed. qPCR was performed with primers targeting 16S rDNA in the absence of infection (−PA) as well as 3 days and 4 days after oral infection with <i>P. aeruginosa.</i> (B) 251659264<i>P. aeruginosa</i> titers are strongly increased in <i>Chd1^−/−</i> flies after infection. qPCR as in (A) with primers specific for <i>P. aeruginosa</i>. (C) Analysis of the gut-specific bacterium <i>Acetobacter</i> EW911. qPCR as in (A) with primers specific for <i>Acetobacter EW911</i>. Relative differences of bacterial genes and the fly <i>Rpl32</i> gene are expressed as 2^−ΔCT values. Values represent mean +/− SD of three independent experiments. Note that SD values are too small to show in the graph.</p

Deletion of <i>Chd1</i> does not cause aberrant numbers or distribution of ISCs in the fly intestine.

<p>(A, A’, A”) Co-staining of isolated guts of <i>Chd1^WT/WT</i> flies with antibodies against CHD1 and Delta revealed localization of CHD1 to ISCs. (B) CHD1 is present in the nuclei of large enterocytes. (C, C’) CHD1 colocalizes with mitotic, PH3-positive cells. CHD1, green; Dl, red; PH3, red; DNA was visualized by staining with DAPI (blue). (D) Guts of unchallenged and <i>P. aeruginosa</i> infected <i>Chd1^−/−</i> and <i>Chd1^WT/WT</i> flies were stained with anti-DI antibody. An area of the anterior midgut is shown. Images are presented with inverted colors to enhance clarity. Arrows indicate individual ISCs with cell membrane-associated Dl signal. No significant differences with respect to number or distribution of ISCs was observed in uninfected and infected wild-type and mutant flies.</p

CHD1 affects the expression of AMP genes in the gut.

<p>(A) Expression of several AMP genes is significantly upregulated in <i>Chd1^−/−</i> flies in the absence of infection. (B) The expression of several regulators of Imd pathway activity is not significantly altered in <i>Chd1^−/−</i> flies. RT-qPCR analysis of isolated guts of unchallenged (<i>−PA</i>) and <i>P. aeruginosa</i> infected (<i>PA</i>; 15 h) <i>Chd1^−/−</i> and <i>Chd1^WT/WT</i> flies was performed. Transcript levels of indicated genes were normalized against <i>Rpl32</i> and are expressed relative to those of the respective gene in <i>Chd1^WT/WT</i> guts. Values represent mean +/− SD of at least 3 independent experiments with 50 guts each (*P<0.05).</p

Whole genome expression profiling of <i>Chd1^−/−</i> larvae.

<p>(A) Gene ontology classification of genes that display at least 2-fold up (left) or downregulation (right). Immunity-linked genes account for a considerable fraction of all misregulated genes. (B) Assignment of immunity-linked misregulated genes in <i>Chd1^−/−</i> larvae to several functional subcategories. Color bars denote the magnitude of aberrant regulation.</p

Loss of CHD1 renders flies more susceptible to oral but not to systemic infection by <i>P. aeruginosa</i>.

<p>(A) Kaplan-Meier plots displaying survival rates of female <i>Chd1^−/−</i>, <i>Chd1^WT/WT</i>, <i>Tl^rv1/Tl^r3</i> and <i>Dredd^EP1412</i> flies after feeding with 5% sucrose solution containing either <i>S. aureus</i>, <i>P. aeruginosa</i> (both 10⁹–10¹⁰ cfu/ml), <i>R. oryzae</i> (5×10⁸ spores/ml) or no microbes for 15 h. <i>Chd1-</i>deficient flies are significantly more susceptible to oral infections with <i>S. aureus</i> and <i>P. aeruginosa</i> (*P<0.05; n = 80) than <i>Chd1^WT/WT</i> flies. (B) Survival rates of female <i>Chd1^−/−</i>, <i>Chd1^WT/WT</i>, <i>Tl^rv1/Tl^r3</i> and <i>Dredd^EP1412</i> flies following septic injury with different microbe solutions or NaCl as above (10⁶–10⁷ cfu or spores/ml). No significant differences of survival of infected <i>Chd1^−/−</i> (n = 200) and <i>Chd1^WT/WT</i> (n = 320) flies were observed.</p

Chao1 and Shannon indices of <i>Chd1</i> mutant and wild-type sample replicates.

<p>Three sets of filtering parameters were used to analyse the data.</p

Distribution of bacterial families within the gut microbiome of <i>Chd1</i> mutant and wild-type flies.

<p>Three sets of filtering parameters were used to analyse the data.</p

Impact of the Chromatin Remodeling Factor CHD1 on Gut Microbiome Composition of <i>Drosophila melanogaster</i>

<div><p>The composition of the intestinal microbiota of <i>Drosophila</i> has been studied in some detail in recent years. Environmental, developmental and host-specific genetic factors influence microbiome composition in the fly. Our previous work has indicated that intestinal bacterial load can be affected by chromatin-targeted regulatory mechanisms. Here we studied a potential role of the conserved chromatin assembly and remodeling factor CHD1 in the shaping of the gut microbiome in <i>Drosophila melanogaster</i>. Using high-throughput sequencing of 16S rRNA gene amplicons, we found that <i>Chd1</i> deletion mutant flies exhibit significantly reduced microbial diversity compared to rescued control strains. Specifically, although <i>Acetobacteraceae</i> dominated the microbiota of both <i>Chd1</i> wild-type and mutant guts, <i>Chd1</i> mutants were virtually monoassociated with this bacterial family, whereas in control flies other bacterial taxa constituted ~20% of the microbiome. We further show age-linked differences in microbial load and microbiota composition between <i>Chd1</i> mutant and control flies. Finally, diet supplementation experiments with <i>Lactobacillus plantarum</i> revealed that, in contrast to wild-type flies, <i>Chd1</i> mutant flies were unable to maintain higher <i>L</i>. <i>plantarum</i> titres over time. Collectively, these data provide evidence that loss of the chromatin remodeler CHD1 has a major impact on the gut microbiome of <i>Drosophila melanogaster</i>.</p></div

Loss of CHD1 causes decreased species diversity in the gut microbiome.

<p>(A) Relative abundance of bacterial families (97% similarity threshold) determined by 16S rDNA sequencing in <i>Chd1</i>^<i>WT/WT</i> and <i>Chd1</i>^<i>-/-</i> samples. Families present at levels less than 1.5% were summarized as “others“. (B) Heatmap of the 25 most abundant 97% identity OTUs within <i>Chd1</i>^<i>WT/WT</i> and <i>Chd1</i>^<i>-/-</i> guts. OTU classification down to the lowest level possible is shown. Color bars denote the relative abundance (log10 values) of each OTU within the respective sample. OTUs are clustered according to their average relative abundance. (C) Heatmap showing the abundance of <i>Acetobacter</i> and <i>Lactobacillus</i> species in <i>Chd1</i>^<i>WT/WT</i> and <i>Chd1</i>^<i>-/-</i> samples identified by alignment of sequencing reads to all respective sequences in the SILVA database at an identity threshold of 99%. All OTUs with fewer than 10 counts, were excluded. Color bars denote the relative abundance (log10 values) of each species within the respective sample.</p