Gut microbiota is essential in PGRP-LA regulated immune protection against Plasmodium berghei infection

Abstract

Background: Malaria remains to be one of the deadliest infectious diseases and imposes substantial financial and social costs in the world. Mosquitoes rely on the immune system to control parasite infection. Peptidoglycan recognition proteins (PGRPs), a family of pattern-recognition receptors (PRR), are responsible for initiating and regulating immune signaling pathways. PGRP-LA is involved in the regulation of immune defense against the Plasmodium parasite, however, the underlying mechanism needs to be further elucidated.

Methods: The spatial and temporal expression patterns of pgrp-la in Anopheles stephensi were analyzed by qPCR. The function of PGRP-LA was examined using a dsRNA-based RNA interference strategy. Western blot and periodic acid schiff (PAS) staining were used to assess the structural integrity of peritrophic matrix (PM).

Results: The expression of pgrp-la in An. stephensi was induced in the midgut in response to the rapid proliferating gut microbiota post-blood meal. Knocking down of pgrp-la led to the downregulation of immune effectors that control gut microbiota growth. The decreased expression of these immune genes also facilitated P. berghei infection. However, such dsLA treatment did not influence the structural integrity of PM. When gut microbiota was removed by antibiotic treatment, the regulation of PGRP-LA on immune effectors was abolished and the knock down of pgrp-la failed to increase susceptibility of mosquitoes to parasite infection.

Conclusions: PGRP-LA regulates the immune responses by sensing the dynamics of gut microbiota. A mutual interaction between gut microbiota and PGRP-LA contributes to the immune defense against Plasmodium parasites in An. stephensi.

Keywords: Anopheles stephensi; Gut microbiota; Immune effectors; PGRP-LA; Peritrophic matrix; Plasmodium berghei.

Fig. 1

Gut microbiota is required for the expression of PGRP-LA. a Relative gene expression of pgrp-la in the midgut and carcass of mosquitoes before (− 24 h IB) and after (+ 24 h IB, + 48 h IB and + 72 h IB) feeding with infectious blood meal. b Relative gene expression of pgrp-la in midgut of sugar-fed (SF), 24 h post normal blood-fed (NB) and infectious blood-fed (IB) mosquitoes. c Relative gene expression of pgrp-la in normal and Abx midguts of sugar-fed (SF), 24 h post normal blood-fed (NB) and infectious blood-fed (IB) mosquitoes. Error bars indicate standard error of the mean (n = 8). Results from one of three independent experiments are shown. Significance was determined by Student’s t-test (for details, see Additional file 3: Text S1). *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant

Fig. 2

Influence of PGRP-LA on gut microbiota in An. stephensi. a PGRP-LA silencing efficiency and specificity. Relative expression level of pgrp-la in dsLA mosquitoes was normalized to the gene’s expression in dsGFP controls. Error bars indicate standard error of the mean (n = 10). Results from one of three independent experiments are shown. Significance was determined by Student’s t-test (t₍₈₎ = 3.431, P = 0.0089). b Median culturable gut microbiota in dsRNA-treated mosquitoes. Each dot represents an individual mosquito and horizontal lines represent the medians. Significance was determined by Mann–Whitney test (U = 15.00, P = 0.0074). c Relative expression level of 16S in dsRNA-treated mosquitoes. Each dot represents an individual mosquito and horizontal lines represent the medians. Significance was determined by Mann–Whitney test (U = 7.00, P = 0.0070). d Relative expression levels of immune-related genes in dsRNA-treated, sugar-fed mosquitoes normalized to the gene’s expression in dsGFP controls. Error bars indicate standard error of the mean (n = 8). Results from one of three independent experiments are shown. Significance was determined by Student’s t-test (for details, see Additional file 3: Text S1). *P < 0.05, **P < 0.01

Fig. 3

PGRP-LA is required for resistance to P. berghei. a Median oocyst number in dsRNA-treated mosquitoes. Each dot represents an individual mosquito and horizontal lines represent the medians. Results from one of three independent experiments are shown. Significance was determined by Mann–Whitney test (U = 132.00, P = 0.0003). b Relative expression levels of immune-related genes in dsRNA-treated mosquitoes 24 h post-infectious blood meal. Error bars indicate standard error of the mean (n = 8). Results from one of three independent experiments are shown. Significance was determined by Student’s t-test (for details, see Additional file 3: Text S1). *P < 0.05, **P < 0.01, ***P < 0.001. c Relative expression level of 16S in dsRNA-treated mosquitoes 24 h post-infectious blood meal. Each dot represents an individual mosquito and horizontal lines represent the medians. Significance was determined by Mann–Whitney test (U = 12.00, P = 0.0057)

Fig. 4

The influence of PGRP-LA on PM synthesis. a Relative expression levels of PM-related genes in dsRNA-treated mosquitoes 24 h, 36 h and 48 h post-infectious blood meal. Error bars indicate standard error of the mean (n = 8). Results from one of three independent experiments are shown. Significance was determined by Student’s t-test (for details, see Additional file 3: Text S1). *P < 0.05. b Western blot of Per1 in dsGFP-treated and dsLA-treated mosquitoes. Actin was used as a loading control. Results from one of three independent experiments are shown. c PAS staining of PM in dsGFP-treated and dsLA-treated mosquitoes at 100× and 200× magnification. Red arrowheads denote the PM. Results from one of three independent experiments are shown. Scale-bars: c, 100 μm

Fig. 5

Gut microbiota is necessary for anti-Plasmodium of PGRP-LA. a Fold change of immune-related genes in antibiotic-treated mosquitoes treated with dsRNA 24 h post-infectious blood meal. Error bars indicate standard error of the mean (n = 8). Results from one of three independent experiments are shown. Significance was determined by Student’s t-test (for details, see Additional file 3: Text S1). b Median oocyst number in antibiotics-treated mosquitoes treated with dsRNA. Each dot represents an individual mosquito and horizontal lines represent the medians. Significance was determined by Mann–Whitney test (U = 314.50, P = 0.6729). Abbreviation: ns, not significant

Fig. 6

Model of influence of PGRP-LA in An. stephensi. PGRP-LA is enriched in midgut in response to the proliferation of gut microbiota. It protects Anopheles from Plasmodium infection through initiating the synthesis of downstream immune effectors, including AMPs, NOS and TEP1. Such regulation requires the presence of gut microbiota