Gao et al. Parasites Vectors (2020) 13:3 https://doi.org/10.1186/s13071-019-3876-y Parasites & Vectors RESEARCH Open Access Gut microbiota is essential in PGRP-LA regulated immune protection against Plasmodium berghei infection Li Gao1,2, Xiumei Song1,2 and Jingwen Wang1,2* Abstract Background: Malaria remains to be one of the deadliest infectious diseases and imposes substantial fnancial and social costs in the world. Mosquitoes rely on the immune system to control parasite infection. Peptidoglycan recog- nition 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 para- site, 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 schif (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 proliferat- ing gut microbiota post-blood meal. Knocking down of pgrp-la led to the downregulation of immune efectors that control gut microbiota growth. The decreased expression of these immune genes also facilitated P. berghei infection. However, such dsLA treatment did not infuence the structural integrity of PM. When gut microbiota was removed by antibiotic treatment, the regulation of PGRP-LA on immune efectors 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 inter- action between gut microbiota and PGRP-LA contributes to the immune defense against Plasmodium parasites in An. stephensi. Keywords: PGRP-LA, Gut microbiota, Peritrophic matrix, Immune efectors, Plasmodium berghei, Anopheles stephensi Background Anopheles-targeted intervention strategies [2]. Con- Malaria, a mosquito-borne disease (MBD), is caused sequently, a further understanding of the interaction by parasites of the genus Plasmodium. It remains to between the Anopheles mosquito and Plasmodium is be a high concern of the World Health Organization extremely urgent. due to the continued emergence and spread of drug- Te main bottleneck for Plasmodium infection in the resistant parasites and insecticide-resistant mosquitoes mosquito is the traverse of ookinetes across the mid- [1]. Malaria prevention and control primarily relies on gut [3, 4]. During this process, two physical barriers are encountered by Plasmodium. Te frst barrier, peri- trophic matrix (PM), composed of chitin, proteoglycans *Correspondence: [email protected] and proteins, is produced by intestinal epithelial cells 1 State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai 200438, People’s Republic of China in response to blood-feeding [5, 6]. PM poses an indis- Full list of author information is available at the end of the article pensable role in the defense of Plasmodium because its © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Gao et al. Parasites Vectors (2020) 13:3 Page 2 of 9 maturation time coincides with the ookinete invasion mosquitoes has no infuence on the outcome of infection time [7]. When artifcially increasing the thickness of with P. berghei. Tese data elucidate that the anti-Plas- PM by feeding mosquitoes with latex particles and ani- modium efect of PGRP-LA depends on the homeostasis mal blood, the number of P. gallinaceum oocysts signif- of gut microbiota. cantly decreases in Aedes aegypti [8]. Midgut epithelium is the second barrier that inhibits Plasmodium infection Methods [9]. When ookinetes start to traverse the midgut epithe- Mosquito rearing and antibiotic treatment lium, epithelial nitration will be activated, promoting Te An. stephensi mosquito (the Hor strain) was reared in thioester-containing protein 1 (TEP1)-mediated lysis of the insectary at a temperature of 28 °C, relative humidity Plasmodium [10, 11]. Once inside the cell cytoplasm, the of 80% and a 12:12 h light/dark photocycle. Adults were invaded intestinal epithelial cells tend to undergo apop- fed on 10% sucrose solution and mouse blood. For antibi- tosis that extrudes ookinetes from the epithelium [7, 12]. otic treatment experiment, newly emerged adult mosqui- Besides, epithelial cells are also immune competent cells, toes were orally administrated with 10% sucrose solution involved in the production of nitric oxide (NO), anti- containing 10 U/ml penicillin, 10 μg/ml streptomycin microbial peptides (AMPs) and reactive oxygen species and 15 μg/ml gentamycin daily for 3 days [20]. Ten the (ROS) to limit Plasmodium survival [13, 14]. antibiotic-treated mosquitoes and untreated mosquitoes Mosquito gut microbiota is another important factor were collected and surface sterilized. Te homogenates that can infuence the outcome of Plasmodium infection were plated onto LB-agar to test the efcacy of antibiotic [15–19]. Oral administration of live or heat-inactivated treatment. bacteria isolated from mosquito midgut signifcantly decreases the infection rate of P. falciparum [20]. Wick- Plasmodium berghei infection erhamomyces anomalus, a yeast involved in symbiotic Six to eight-week-old BALB/c mice were injected intra- relationships with Anopheles, is able to directly elimi- peritoneally (ip) with 106 infected RBCs with GFP-tagged nate Plasmodium through secreting killer toxin [21, P. berghei (ANKA). To evaluate parasitemia, tail smears 22]. Another stably inherited gut commensal bacteria, were taken and stained with Giemsa (Baso Diagnostics Asaia, can be genetically modifed and directly inhib- Inc, Zhuhai, China), the number of parasites per 3000 its pathogen development by secreting antiplasmodial RBCs were counted [33]. When the parasitemia reached efectors [23]. In addition, these microbes inhibit para- 4–6%, the infected mice were used to feed mosquitoes sites indirectly through promoting the formation of PM that had been starved overnight; the mosquitoes were and stimulating the oxidative defense system and NF-κB then maintained at 21 °C. Unengorged mosquitoes were dependent immune responses [24–27]. removed 24 h post-blood meal. Midguts were dissected Mosquito immune response plays a pivotal role in pro- and infection intensity (oocyst number) were determined tecting the host against pathogen infection. Peptidoglycan microscopically at day 8 post-infection. recognition proteins (PGRPs) are a family of important pattern-recognition receptors (PRR) that initiate immune Knockdown by RNA interference signaling pathways [28, 29]. In Anopheles gambiae, PGRP- PCR amplifcation from 606 to 1082 bp of pgrp-la LC is the receptor of NF-κB transcription factor REL2- (ASTE016413) was performed using T7-tagged prim- mediated signaling pathway. It plays an important role ers. Te plasmid eGFP (BD Biosciences, San Jose, USA) in maintaining the homeostasis of gut microbiota [27]. was used as a template for control dsRNA amplifcation. PGRP-LD protects the mosquito from Plasmodium infec- Te double-stranded RNA (dsRNA) was synthesized tion through regulating gut microbiota-mediated PM using the MEGAscript RNA kit (Ambio, Invitrogen, formation in Anopheles stephensi [30]. PGRP-LB serves Shanghai, China). Two to three-day-old mosquitoes were as a negative regulator of immune pathways in Aedes and injected with 69 nl dsRNA (4 μg/μl) intra-thoracically Anopheles mosquitoes [31, 32]. PGRP-LA also partici- using a nanoject II microinjector (Drummond, Philadel- pates in antiparasitic immune defenses, but the underlin- phia, USA). Mosquitoes treated with an equal volume of ing mechanism needs to be further elucidated [31]. dsGFP and distilled water were used as controls. For the In this study, we demonstrate that the expression of antibiotic treatment experiment, dsRNAs were injected pgrp-la is induced in the midgut in response to a blood three days after antibiotic treatment. Te midguts of meal. Such induction is due to the rapid proliferation dsRNA-treated mosquitoes were dissected two days of gut microbiota post-feeding. Once gut microbiota post-treatment and knock down efcacy was examined is removed by antibiotic treatment, PGRP-LA fails to by qPCR as previously described [30]. For