Microbiota-induced peritrophic matrix regulates midgut homeostasis and prevents systemic infection of malaria vector mosquitoes

<div><p>Manipulation of the mosquito gut microbiota can lay the foundations for novel methods for disease transmission control. Mosquito blood feeding triggers a significant, transient increase of the gut microbiota, but little is known about the mechanisms by which the mosquito controls this bacterial growth whilst limiting inflammation of the gut epithelium. Here, we investigate the gut epithelial response to the changing microbiota load upon blood feeding in the malaria vector <i>Anopheles coluzzii</i>. We show that the synthesis and integrity of the peritrophic matrix, which physically separates the gut epithelium from its luminal contents, is microbiota dependent. We reveal that the peritrophic matrix limits the growth and persistence of <i>Enterobacteriaceae</i> within the gut, whilst preventing seeding of a systemic infection. Our results demonstrate that the peritrophic matrix is a key regulator of mosquito gut homeostasis and establish functional analogies between this and the mucus layers of the mammalian gastrointestinal tract.</p></div

Effect of oral antibiotic treatment on the gene expression of the mosquito midgut.

<p><b>(A)</b> Bacterial load in the guts of 4 control and 4 antibiotic treated mosquito cohorts throughout a two blood meal (BM1 and BM2) time course assessed by qRT-PCR using universal 16S primers. Data were normalized within each biological replicate to the bacterial load in the control 96h sample. The mean plus/minus the standard error is shown. <b>(B)</b> Principal components analysis (PCA) plot of the 40 sequenced midgut samples after variance stabilizing transformation of count data. <b>(C)</b> Number of genes that were significantly upregulated (white bars) or downregulated (black bars) in each of the time points following antibiotic treatment. Genes showing an adjusted p-value <0.1 (Wald test with a Benjamini-Hochberg correction) were considered to be significantly regulated.</p

The peritrophic matrix regulates immune resistance to the microbiota.

<p><b>(A)</b> RNA-seq transcriptional profiles of <i>CEC1</i> and <i>GAM1</i> in the midgut of control (black lines) and antibiotic fed (grey lines) mosquitoes over a two blood meal (BM1 and BM2) time course. Dots indicate normalized counts in each of four biological replicates, with the line connecting the means. Statistical significance of a pairwise comparison of counts at each time point was assessed by a Wald test with a Benjamini-Hochberg correction. ‘*’ p<0.05; ‘**’ p<0.01; ‘***’ p<0.001 (<b>B)</b> <i>GAM1</i> expression, relative to AgS7, in the midgut 24h after feeding with blood supplemented with 100μM polyoxin D (PxD) or an equal volume of water (control), plus or minus antibiotic treatment, as determined by qRT-PCR. Mean plus/minus standard error is indicated. Statistical significance was assessed by an ANOVA on a linear mixed effect regression model. Each dot represents a pool of 8–10 guts, derived from 4–5 independent experiments. Ratios are normalized within biological replicates to the mean of the control pools (no polyoxin D, no antibiotics). <b>(C)</b> <i>Enterobacteriaceae</i> load, relative to AgS7, as determined by qRT-PCR using <i>Enterobacteriaceae</i> specific 16S primers. Normalization and statistical analysis were performed as described for (B). <b>(D)</b> Scatter plots of the load of specific bacteria families commonly found in the mosquito gut against <i>GAM1</i> expression in the midguts of control (top row) or polyoxin D-treated (bottom row) mosquitoes. Spearman’s rank correlation coefficient (r) and associated p-values (p) are indicated.</p

The peritrophic matrix prevents microbiota dissemination and systemic immune induction.

<p><b>(A-C)</b><i>CEC1</i> expression (A), <i>16S rRNA</i> quantification (B) and <i>Enterobacteriaceae 16S rRNA</i> quantification (C) in the carcass 72h after feeding with a blood meal supplemented with 100μM polyoxin D or water as a control, plus or minus antibiotic treatment, as determined by qRT-PCR. Each dot represents a pool of 8–10 carcasses, derived from 4 independent experiments. Data show mean and standard error. In A, ratios are normalized within biological replicates to the mean of the control pools (no polyoxin D, no antibiotics). In B-C, ratios are normalized within each biological replicate to the highest value across all conditions (‘100%’). In A-C, statistical significance was assessed by an ANOVA on a linear mixed effect regression model. <b>(D)</b> Scatter plots of relative <i>Enterobacteriaceae</i> load against <i>CEC1</i> expression in the carcass at 72h post blood feeding. Each dot represents a pool of 8–10 carcasses, derived from 4 independent experiments; data are normalized as in B and C. Spearman’s rank correlation coefficient and associated p-values are indicated. ‘*’ p<0.05.</p

The peritrophic matrix promotes restoration of gut homeostasis after blood feeding.

<p><b>(A)</b> Midgut bacterial load at 24h and 48h after a blood meal, as determined by qRT-PCR. At 48h, midguts were pooled according to whether (+) or not (-) the blood bolus was present. Each dot represents a pool of 5 guts, derived from two independent experiments. Ratios are normalized within biological replicates to the mean of the 24h pools. Mean plus/minus standard error is indicated. Statistical significance was assessed by an ANOVA on a linear mixed effect regression model. ‘*’ p<0.05; ‘**’ p<0.01; ‘***’ p<0.001. <b>(B)</b> <i>Enterobacteriaceae</i> and <i>Acetobacteraceae</i> load in midguts 72h after feeding on blood supplemented with 100μM polyoxin D (PxD) or an equal volume of water (control), as determined by qRT-PCR. Each dot represents a pool of 8–10 guts, derived from 4 independent experiments. Ratios are normalized within biological replicates to the mean of the control pools. Mean plus/minus standard error is indicated. Statistical significance was assessed and is presented as described above. <b>(C)</b> Thin abdominal section 24h post blood meal stained with anti-LPS antibody. White arrowheads indicate LPS staining. <b>(D)</b> Gram-stained thin abdominal sections 24h post blood meal, with or without polyoxin D supplementation. Bacteria are stained light purple. “Lu” lumen; “Ep” epithelium.</p

Multiple aligned OTU sequences associated with midgut microbiota of Anopheles mosquitoes sampled from Ghana

Data file included the following information: Operational Taxonomic Unit (OTU) name;Bacterial taxon assigned to OTU;Mosquito species associated with the OTU;Status of collection site from with the mosquito samples were collected;Season during which mosquito samples were collected

Description of <i>PGRP-LA</i> genomic locus and isoforms.

<p><b>A.</b> Scheme of the locus containing <i>PGRP-LA</i>, <i>PGRP-LC</i> and <i>PGRP-LF</i>. Each gene contains at least one PGRP domain (orange) and PGRP-LA and LC contain a transmembrane domain (TM, green) and a RHIM motif (blue). No signal peptide has been predicted in the <i>PGRP-LA</i> sequence, and the C-terminal sequence (purple) contains 2 Cys residues. <i>PGRP-LA</i> encodes three isoforms, depicted under the gene: boxes represent the exons, of which the coding sequence is colored in red. <i>PGRP-LA^2A</i> (<i>LA^2A</i>) deletion was performed by imprecise excision of the <i>P</i>-element G14937 (KAIST library) and <i>PGRP-[LA,LC]^Δ (LALC^Δ)</i> by FRT mediated deletion of the region between the P-elements 1930 and 4396. <i>PGRP-LC^E12</i> (<i>LC^E12</i>) deletion has already been published <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069742#pone.0069742-Gottar1" target="_blank">[22]</a>. In PGRP-LA_C, the hatched box represents a sequence between a start and a stop codon, but which is not predicted to be the coding sequence (Flybase). Fp, Rg: localization of the primers used for RT-qPCR. B. Alignment of the proteic sequences of the PGRP domains of PGRP-LA in <i>Drosophila</i> (<i>Dm</i>), <i>Anopheles gambiae</i> (<i>Ag</i>) and <i>Culex quinquefasciatus</i> (<i>Cq</i>) and of <i>Drosophila</i> PGRP-LCx, of which the crystal structure has already been solved. Blue boxes contain conserved amino acids (identities and similarities are highlighted and written in red respectively). The residues that are directly in contact with TCT in the structure of the complex with PGRP-LCx <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069742#pone.0069742-Chang2" target="_blank">[37]</a> are marked with yellow triangles. The numbering corresponds to PGRP-LCx. The two residues insertion in the β2-α1 loop and the four residues deletion in the β4–β5 loop are denoted with green and red stars, respectively.</p

<i>PGRP-LA_D</i> over-expression leads to induction of the Imd pathway.

<p><b>A.</b> Measurement of <i>Diptericin</i> (<i>Dpt</i>) by RT-qPCR in whole males over-expressing each isoform of <i>PGRP-LA</i> under the control of the ubiquitous <i>da-Gal4</i> driver, using <i>UAS-PGRP-LA_C</i> (<i>LA_C</i>), <i>UAS-PGRP-LA_D</i> (<i>LA_D</i>) and <i>UAS-PGRPLA_F</i> (<i>LA_F</i>) transgenes. <b>B.</b> Measurement of <i>Dpt</i> by RT-qPCR in <i>PGRP-LC</i>, <i>Dredd</i>, or <i>Tak1</i>-deficient whole males over-expressing <i>PGRP-LA_D</i> under the control of the ubiquitous <i>da-Gal4</i> driver. Results are shown as fold change of <i>Dpt</i> expression versus wild-type (+) unchallenged controls. Data are expressed as a percentage of <i>Dpt/RpL32</i> 6 h after septic injury (SI) and are the mean of three experiments; error bars indicate standard errors. In <b>A</b>, <b>B</b>, data were analyzed by ANOVA1 followed by Dunnett's multiple comparison test using wt (i.e. da-Gal4 x w) (<b>A</b>) and wt (SI) (<b>B</b>) as references (a and b groups are statistically different, p<0.01 (<b>A</b>) and p<0.05 (<b>B</b>)).</p

Shannon-Weaver indices compared among samples collected in dry and rainy season.

<p>Crosses in the box indicate the mean, and black lines are medians.</p

Microarray characterization of the tracheal immune response in wild-type larvae.

<p><b>A.</b> Distribution of regulated genes, based on their up or down-regulation and their fold change in the microarray. Black and white bar portions represent the genes whose expression is affected or not affected in <i>Rel^E20</i> respectively. <b>B.</b> Comparison of the distribution of genes up-regulated in the tracheae upon <i>Ecc15</i> bacterial infection to that of genes induced in the gut upon <i>Ecc15</i> ingestion and in whole flies upon septic injury with <i>Ecc15 </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069742#pone.0069742-Buchon1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069742#pone.0069742-DeGregorio2" target="_blank">[47]</a>. * indicates that the gene expression is affected in <i>Rel^E20</i>. The number of genes induced in each tissue is indicated in brackets. <b>C.</b> Repartition of induced (left) and repressed (right) genes in defined categories of gene ontology.</p