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. 2018 Feb 28;14(2):e1006899.
doi: 10.1371/journal.ppat.1006899. eCollection 2018 Feb.

PGRP-LD mediates A. stephensi vector competency by regulating homeostasis of microbiota-induced peritrophic matrix synthesis

Xiumei Song  1   2 , Mengfei Wang  1   2 , Li Dong  1   2 , Huaimin Zhu  3 , Jingwen Wang  1   2
Affiliations

PGRP-LD mediates A. stephensi vector competency by regulating homeostasis of microbiota-induced peritrophic matrix synthesis

Xiumei Song et al. PLoS Pathog. .

Abstract

Peptidoglycan recognition proteins (PGRPs) and commensal microbes mediate pathogen infection outcomes in insect disease vectors. Although PGRP-LD is retained in multiple vectors, its role in host defense remains elusive. Here we report that Anopheles stephensi PGRP-LD protects the vector from malaria parasite infection by regulating gut homeostasis. Specifically, knock down of PGRP-LD (dsLD) increased susceptibility to Plasmodium berghei infection, decreased the abundance of gut microbiota and changed their spatial distribution. This outcome resulted from a change in the structural integrity of the peritrophic matrix (PM), which is a chitinous and proteinaceous barrier that lines the midgut lumen. Reduction of microbiota in dsLD mosquitoes due to the upregulation of immune effectors led to dysregulation of PM genes and PM fragmentation. Elimination of gut microbiota in antibiotic treated mosquitoes (Abx) led to PM loss and increased vectorial competence. Recolonization of Abx mosquitoes with indigenous Enterobacter sp. restored PM integrity and decreased mosquito vectorial capacity. Silencing PGRP-LD in mosquitoes without PM didn't influence their vector competence. Our results indicate that PGPR-LD protects the gut microbiota by preventing hyper-immunity, which in turn promotes PM structurally integrity. The intact PM plays a key role in limiting P. berghei infection.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Influence of PGRP-D on P. berghei infection in A. stephensi.
(A) pgrp-ld silencing efficiency and specificity (B) in mosquitoes. Expression level of pgrp-ld was normalized to A. stephensi s7. Relative expression level of pgrp-ld in dsLD mosquitoes was normalized to the gene’s expression in dsGFP controls. (C) Oocyst number in dsRNA treated mosquitoes. Median oocysts number is indicated by the horizontal black bar. Each dot represents an individual mosquito. (D) Relative gene expression levels in dsRNA treated mosquitoes 26hr post parasite infection. (A, B and D) Error bars indicate standard error (n = 10). Significance was determined by Student’s-T test in (A), (B) and (D), by Mann-Whitney test in (C). *, P<0.05, **, P<0.01, ***, P<0.001. Results from one of three independent experiments are shown.
Fig 2
Fig 2. Influence of PGRP-LD on the gut microbiota.
(A) Relative expression levels of immunity-related genes in dsRNA treated, sugar fed mosquitoes. Error bars indicate standard error (n = 10). Culturable (B) and total gut microbiota (C) density was measured. Error bars indicate standard error (n = 10). Significance was determined by Student’s-T test. *, P<0.05, **, P<0.01, ***, P<0.001. Results from one of two independent experiments are shown. (D) Microbiota localization in midguts of dsGFP (D1-D3) and dsLD (D4-D6) were analyzed by FISH using a universal bacterial 16S rRNA gene probe (red). Nuclei were stained with DAPI (blue). D1 and D4 show DAPI staining. D2 and D5 show staining with 16S rRNA probe. D3 and D6 show merged images. Arrows denote gut microbiota. Images are representative of three independent experiments. Scale bars, 50 μm.
Fig 3
Fig 3. PM structure in dsRNA treated mosquitoes.
(A) PM structure was observed by H&E staining in dsGFP (A1 and A2) and dsLD (A3 and A4) mosquitoes at 100X (A1 and A3) and 200X (A2 and A4) magnification. Arrows denote the PM. Scale bars, 50 μm. (B) Dextran-feeding assay in dsRNA treated mosquitoes. The FITC signal is retained in the lumen of dsGFP control mosquitoes, which indicates that the dextran beads are contained within the PM (B1). The FITC signal is observed within gut epithelial cells (indicated by arrow) in dsLD mosquitoes, indicating that the beads can cross the PM (B2). Scale bars, 50 μm. Images are representative of at least two independent experiments. (C) PM gene expression in dsRNA treated mosquitoes. Relative gene expression level in dsRNA treated mosquitoes 24hr and 48 hr post blood meal. Error bars indicate standard error (n = 10).
Fig 4
Fig 4. PM structural integrity in normal and antibiotic treated mosquitoes.
PM structure was observed by H&E staining in normal (A and B) and antibiotic treated mosquitoes (C and D) at 100X (A and C) and 200X (B and D) magnification. Arrows denote the PM. Images are representative of three independent experiments. Scale bars, 50 μm. (E) Quantification of PM related gene expression in normal and antibiotic treated mosquitoes 24hr and 48 hr post blood meal. Error bars indicate standard error (n = 10).
Fig 5
Fig 5. The gut microbiota promotes PM formation.
(A) Culturable gut microbiota density in normal, antibiotic treated (Abx) and antibiotic treated mosquitoes recolonized with 1X105/ml (5), 1X106/ml (6) and 1X107/ml (7) Enterobacter sp. (B) Infection rate of P. berghei in normal, antibiotic treated (Abx) and antibiotic treated mosquitoes recolonized with 1X105/ml (5), 1X106/ml (6) and 1X107/ml (7) of Enterobacter sp. Median oocyst number is indicated by horizontal black bars. Each dot represents an individual mosquito. (C) PM structure was stained by H&E in normal (C1 and C4), antibiotic treated (C2 and C5) and antibiotic treated mosquitoes recolonized with 1X105/ml Enterobacter sp. (C3 and C6) with 100X (C1-3) and 200X (C4-6) magnification. Arrows denote the PM. Images are representative of two independent experiments. Scale bars, 100 μm.
Fig 6
Fig 6. Influence of PGRP-LD on parasitism in normal and antibiotic treated mosquitoes.
(A) Oocyst numbers in normal mosquitoes treated with dsRNA. (B) Oocyst numbers in antibiotic treated mosquitoes that received dsRNA. Median oocyst number is indicated by horizontal black bars. Each dot represents an individual mosquito. Significance was determined by Mann-Whitney test. ***, P<0.001. Results from one of three independent experiments are shown.
Fig 7
Fig 7. Model of influence of PGRP-LD on A. stephensi competence.
(A) PGRP-LD protects gut microbiota by negatively regulating immune responses. The gut microbes promote integrity of PM, which enhances vector resistance to parasite infection. (B) Knock down of pgrp-ld leads to the upregulation of immune effectors, which kills a significant number of gut microbes. Dysbiosis of the gut microbiota results in the presentation of a structurally compromised PM, which facilitates P. berghei infection.
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