Functional characterization of PGRP‐LC1 of Anopheles gambiae through deletion and RNA interference

Abstract Peptidoglycan recognition proteins (PGRP) play an important role in innate immunity in insects through the activation of the Imd pathway, which has been shown to be required in the antibacterial response in insects and in the limitation of the number of Plasmodium berghei oocysts developing in mosquito midgut. The LC1 gene of the PRGP family in Anopheles gambiae produces many products through alternative splicing. In this work, we demonstrate that PGRP‐LC1a alone is sufficient to activate the Imd pathway in the A. gambiae L3–5 cell line through a combination of terminal or internal deletions, and RNA interference against endogenous PGRP‐LC products. In the absence of endogenous PGRP‐LC proteins, the integrity of the cytoplasmic domain is necessary for LC1a function, while that of the extracellular domain is not. Moreover, the shorter the extracellular domain, the higher the activity for LC1a. However, the removal of either the cytoplasmic or the extracellular PGRP‐binding domain has little impact on the activity of LC1a in the presence of endogenous PGRP‐LC proteins.     

Functional characterization of the NF-κB transcription factor gene REL2 from Anopheles gambiae

The REL2 gene plays an important role in innate immunity against both Gram （＋） and Gram （-） bacteria and malaria parasites in Anopheles gambiae, the main vector of malaria in Africa. Through alternative splicing, REL2 produces two protein products, REL2F （with a Rel-homology domain as well as an inhibitory ankyrin repeat region） and REL2S （without the ankyrin repeats）. In the immune-competent cell line SualB from An. gambiae, REL2 has been shown to be a key regulator for cecropin A （or CEC1）. The high level expression of CEC1 in SualB was postulated to be the result of constitutive activation of REL2F. Here we showed that REL2F is indeed processed, albeit at a low level, in the SualB cell line. The primary cleavage requires residue 678 （an aspartic acid）. Proteolytic cleavage of REL2F can be enhanced by challenge with bacteria Escherichia coli and Bacillus subtilis, but not with fungus Beauveria bassiana. The inducible cleavage can be substantially reduced by RNA interference against PGRP-LC and CASPL1. Over-expression of REL2S or a constitutively active form of REL2F （REL2F380C or REL2F678） in An. gambiae cell line can further increase expression of CEC1 and other antimicrobial peptide genes. Over-expression of these constitutive active proteins in an immune naive cell line, MSQ43, from Anopheles stephensi, results in even more dramatic increased expression of antimicrobial peptides.     

一个调控抗菌肽表达的小菜蛾肽聚糖识别蛋白(PxPGRP-SA)

【目的】肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRPs)是昆虫免疫系统中一类重要的模式识别蛋白。本研究旨在阐明经苏云金芽孢杆菌Bacillus thuringiensis侵染后,小菜蛾Plutella xylostella PGRP-SA基因(命名为Px PGRP-SA)在体内的表达模式和对抗菌肽基因的表达调控。【方法】本研究利用实时荧光定量PCR(qRT-PCR)技术分析B.thuringiensis侵染小菜蛾幼虫后Px PGRP-SA的转录模式,通过RNAi技术结合抗血清封闭实验检测Px PGRP-SA对小菜蛾抗菌肽基因的表达调控作用。【结果】qRT-PCR检测表明,小菜蛾4龄幼虫在注射具有活性的B.thuringiensis 6 h后,Px PGRP-SA在脂肪体和血细胞中表达量迅速上升,其中脂肪体中的表达量在注射24 h后达到高峰,而在血细胞中的表达量在18 h后达到高峰。RNAi沉默小菜蛾4龄幼虫Px PGRP-SA的转录后,可显著降低小菜蛾脂肪体中cecropin,moricin-2,lysozyme和defensin 4个抗菌肽基因及Dorsal和Sptzle基因的mRNA转录水平;注射anti-Px PGRP-SA封闭小菜蛾体内Px PGRP-SA的活性后,也可降低小菜蛾脂肪体中4个抗菌肽基因的mRNA转录水平;Px PGRP-SA转录沉默后,同时导致添食B.thuringiensis的小菜蛾幼虫的存活率明显降低。【结论】Px PGRP-SA参与了小菜蛾体内抗菌肽cecropin,moricin-2,lysozyme和defensin基因的表达调控,并在免疫防御B.thuringiensis的侵染过程中起了重要的作用。     

Innate immunity against malaria parasites in Anopheles gambiae

Malaria continues to exert a huge toll in the world today, causing approximately 400 million cases and killing between 1-2 million people annually. Most of the malaria burden is borne by countries in Africa. For this reason, the major vector for malaria in this continent, Anopheles gambiae, is under intense study. With the completion of the draft sequence of this important vector, efforts are underway to develop novel control strategies. One promising area is to harness the power of the innate immunity of this mosquito species to block the transmission of the malaria parasites. Recent studies have demonstrated that Toll and Imd signaling pathways and other immunity-related genes （encoding proteins possibly function in recognition or as effector molecules） play significant roles in two different arms of innate immunity： level of infection intensity and melanization of Plasmodium oocysts. The challenges in the future are to understand how the functions of these different genes are coordinated in defense against malaria parasites, and if different arms of innate immunity are cross-regulated or coordinated.     

Halocyntin and papillosin,two new antimicrobial peptides isolated from hemocytes of the solitary tunicate,Halocynthia papillosa

We report here the screening of five marine invertebrate species from two taxa (tunicates and echinoderms) for the presence of cationic antimicrobial peptides (AMP) in defence cells (hemocytes). Antimicrobial activities were detected only in the two tunicates Microcosmus sabatieri and Halocynthia papillosa. In addition, we report the isolation and characterization of two novel peptides from H. papillosa hemocytes. These molecules display antibacterial activity against Gram‐positive and Gram‐negative bacteria. Complete peptide characterization was obtained by a combination of Edman degradation and mass spectrometry. The mature molecules, named halocyntin and papillosin, comprise 26 and 34 amino acid residues, respectively. Their primary structure display no significant similarities with previously described AMP. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Crystal structure of human peptidoglycan recognition protein S (PGRP-S) at 1.70 A resolution

Peptidoglycan recognition proteins (PGRPs) are pattern recognition receptors of the innate immune system that bind peptidoglycans (PGNs) of bacterial cell walls. These molecules, which are highly conserved from insects to mammals, contribute to host defense against infections by both Gram-positive and Gram-negative bacteria. Here, we present the crystal structure of human PGRP-S at 1.70A resolution. The overall structure of PGRP-S, which participates in intracellular killing of Gram-positive bacteria, is similar to that of other PGRPs, including Drosophila PGRP-LB and PGRP-SA and human PGRP-Ialpha. However, comparison with these PGRPs reveals important differences in both the PGN-binding site and a groove formed by the PGRP-specific segment on the opposite face of the molecule. This groove, which may constitute a binding site for effector or signaling proteins, is less hydrophobic and deeper in PGRP-S than in PGRP-IalphaC, whose PGRP-specific segments vary considerably in amino acid sequence. By docking a PGN ligand into the PGN-binding cleft of PGRP-S based on the known structure of a PGRP-Ialpha-PGN complex, we identified potential PGN-binding residues in PGRP-S. Differences in PGN-contacting residues and interactions suggest that, although PGRPs may engage PGNs in a similar mode, structural differences exist that likely regulate the affinity and fine specificity of PGN recognition.     

The Plasmodium bottleneck: malaria parasite losses in the mosquito vector

Nearly one million people are killed every year by the malaria parasite Plasmodium. Although the disease-causing forms of the parasite exist only in the human blood, mosquitoes of the genus Anopheles are the obligate vector for transmission. Here, we review the parasite life cycle in the vector and highlight the human and mosquito contributions that limit malaria parasite development in the mosquito host. We address parasite killing in its mosquito host and bottlenecks in parasite numbers that might guide intervention strategies to prevent transmission.     

Therapeutic antimicrobial peptides may compromise natural immunity

Antimicrobial peptides (AMPs) have been proposed as a promising new class of antimicrobials despite warnings that therapeutic use could drive the evolution of pathogens resistant to our own immunity peptides. Using experimental evolution, we demonstrate that Staphylococcus aureus rapidly evolved resistance to pexiganan, a drug-candidate for diabetic leg ulcer infections. Evolved resistance was costly in terms of impaired growth rate, but costs-of-resistance were completely ameliorated by compensatory adaptation. Crucially, we show that, in some populations, experimentally evolved resistance to pexiganan provided S. aureus with cross-resistance to human-neutrophil-defensin-1, a key component of the innate immune response to infection. This unintended consequence of therapeutic use could drastically undermine our innate immune system's ability to control and clear microbial infections. Our results therefore highlight grave potential risks of AMP therapies, with implications for their development.     

Responses of Anopheles gambiae complex mosquitoes to the use of untreated bednets in The Gambia

Population dynamics of the Anopheles gambiae complex of malaria vector mosquitoes were studied in four small hamlets in The Gambia. Bednets were used to reduce man/vector contact in two of the hamlets. High densities of An. gambiae, sensu lato, were present for only 3-8 weeks during the rainy season, depending on the position of the hamlet within the study area. The proportions of blood-fed mosquitoes caught indoors (83.0%) and existing from houses (11.6%) were lower in hamlets where bednets were used than in hamlets without (96.5% and 33.1% respectively). Fewer of the blood-fed mosquitoes had fed on man in houses where people slept under bednets (68.2%) than in those without (81.5%). However, the average number of infective bites received by children was still greater than one a year in hamlets where bednets were used. Consequently bednets are considered unlikely to be an effective malaria control measure so long as they are untreated with insecticide.     

Pyrethroid-treated bednet effects on mosquitoes of the Anopheles gambiae complex in The Gambia

The response of Anopheles gambiae complex mosquitoes to men sleeping under insecticide-impregnated or untreated bednets in six verandah trap huts was studied during the dry season in The Gambia. With this type of hut it was possible to collect live and dead indoor-resting mosquitoes and estimate the number of wild mosquitoes which entered, bloodfed on man, and exited each night. Bednets were treated with emulsions targetted to leave deposits of 25 mg/m2 lambda-cyhalothrin, or 5, 50 or 500 mg/m2 permethrin, diluted from emulsifiable concentrates (EC), or a blank formulation similar to the EC except that the permethrin was omitted; the sixth net was left untreated. Nets and sleepers were rotated between huts on different nights, the design being based on a series of Latin squares and conducted double-blind. Permethrin-impregnated bednets deterred mosquitoes from entering the huts. The degree of deterrency was proportional to the dosage of permethrin. This effect was also caused by the blank formulation and therefore attributed to other components of the formulation, rather than to the permethrin itself. The net impregnated with 500 mg permethrin per square metre gave the best individual protection, reducing mosquito bloodfeeding by 91% compared with untreated nets. However, lambda-cyhalothrin was proportionately more insecticidal than permethrin at doses of equivalent deterrency. At this stage of research, it remains conjectural whether chemical deterrency or killing of malaria vectors is better for community protection.     

Structure-function studies of chemokine-derived carboxy-terminal antimicrobial peptides

Recent reports which show that several chemokines can act as direct microbicidal agents have drawn renewed attention to these chemotactic signalling proteins. Here we present a structure-function analysis of peptides derived from the human chemokines macrophage inflammatory protein-3α (MIP-3α/CCL20), interleukin-8 (IL-8), neutrophil activating protein-2 (NAP-2) and thrombocidin-1 (TC-1). These peptides encompass the C-terminal α-helices of these chemokines, which have been suggested to be important for the direct antimicrobial activities. Far-UV CD spectroscopy showed that the peptides are unstructured in aqueous solution and that a membrane mimetic solvent is required to induce a helical secondary structure. A co-solvent mixture was used to determine solution structures of the peptides by two-dimensional ¹H-NMR spectroscopy. The highly cationic peptide, MIP-3α_51-70, had the most pronounced antimicrobial activity and displayed an amphipathic structure. A shorter version of this peptide, MIP-3α_59-70, remained antimicrobial but its structure and mechanism of action were unlike that of the former peptide. The NAP-2 and TC-1 proteins differ in their sequences only by the deletion of two C-terminal residues in TC-1, but intact TC-1 is a very potent antimicrobial while NAP-2 is inactive. The corresponding C-terminal peptides, NAP-2_50-70 and TC-1_50-68, had very limited and no bactericidal activity, respectively. This suggests that other regions of TC-1 contribute to its bactericidal activity. Altogether, this work provides a rational structural basis for the biological activities of these peptides and proteins and highlights the importance of experimental characterization of peptide fragments as distinct entities because their activities and structural properties may differ substantially from their parent proteins.     

Insect antimicrobial peptides show potentiating functional interactions against Gram-negative bacteria

Antimicrobial peptides (AMPs) and proteins are important components of innateimmunity against pathogens in insects. The production of AMPs is costly owing toresource-based trade-offs, and strategies maximizing the efficacy of AMPs at lowconcentrations are therefore likely to be advantageous. Here, we show thepotentiating functional interaction of co-occurring insect AMPs (the bumblebeelinear peptides hymenoptaecin and abaecin) resulting in more potentantimicrobial effects at low concentrations. Abaecin displayed no detectableactivity against Escherichia coli when tested alone atconcentrations of up to 200 μM, whereas hymenoptaecin affected bacterialcell growth and viability but only at concentrations greater than 2 μM.In combination, as little as 1.25 μM abaecin enhanced the bactericidaleffects of hymenoptaecin. To understand these potentiating functionalinteractions, we investigated their mechanisms of action using atomic forcemicroscopy and fluorescence resonance energy transfer-based quenching assays.Abaecin was found to reduce the minimal inhibitory concentration ofhymenoptaecin and to interact with the bacterial chaperone DnaK (anevolutionarily conserved central organizer of the bacterial chaperone network)when the membrane was compromised by hymenoptaecin. These naturally occurringpotentiating interactions suggest that combinations of AMPs could be usedtherapeutically against Gram-negative bacterial pathogens that have acquiredresistance to common antibiotics.     

斯氏按蚊肽聚糖识别蛋白基因PGRP-LC1的克隆及功能分析

天生免疫系统是昆虫抵御外界病原入侵的主要方式。目前研究发现, Imd信号通路与按蚊感染柏氏疟原虫Plasmodium berghei的强度密切相关, 而PGRP-LC1是Imd信号通路最上游的受体之一。为了研究斯氏按蚊Anopheles stephensi肽聚糖识别蛋白PGRP-LC1, 采用RT-PCR并结合RACE技术克隆斯氏按蚊PGRP-LC1基因, 通过序列比较分析, 得到两条cDNA序列, 其开放阅读框分别为1 365 bp和1 290 bp, 3′非编码区为320 bp, 5′非编码区为240 bp。将两条cDNA分别命名为AsPGRP-LC1a（GenBank注册号 GU214232）和AsPGRP-LC1b（GenBank注册号GU214233）。AsPGRP-LC1a编码454个氨基酸, 分子量约为49.07 kDa;AsPGRP-LC1b编码429个氨基酸, 分子量约为46.3 kDa。AsPGRP-LC1b比AsPGRP-LC1a少一个长度为75 bp的外显子, 该外显子在冈比亚按蚊Anopheles gambiae PGRP-LC1基因的某些可变剪切形式中也有发现。分别将两个斯氏按蚊PGRP-LC1基因在冈比亚按蚊细胞系L3-5和斯氏按蚊细胞系MSQ43中过量表达, 通过双荧光素酶检测系统检测抗菌肽的表达情况, 结果显示克隆得到的PGRP-LC1基因在两种细胞系中均能够启动Imd信号通路, 为进一步研究斯氏按蚊的Imd信号通路提供了依据。     

Effects of antimicrobial peptides of neutrophils on tumor and normal host cells in culture

We carried out a study of the effects of two structurally different cationic antimicrobial peptides of cathelicidin family, porcine protegrin 1 (PG1) and caprine Bac5 on selected tumor and normal mammalian cells in vitro. Protegrins are amphiphilic β-hairpin molecules having broad-spectrum antimicrobial activity due to their marked membranolytic effects. Bac5 belongs to a group of proline-rich peptides, which adopt a polyproline type II extended helix and kill microorganisms rather by a nonlytic mechanism. We have shown that while PG1 exerted distinct and fast cytotoxic effects towards most of used tumor cells being in a lesser degree toxic for nontransformed host cells; the proline-rich peptide Bac5 possessed modest cytotoxic activity for all tested cells. The toxic effects of PG1 were partially declined in the presence of 10% fetal calf serum. It was revealed that PG1 was able to interact with proteins of serpin family (as was previously established for human defensins by Panyutich at al., 1995). Pre-incubation of PG1 with α1-antitrypsin caused the decrease of the cytotoxic activity of the peptide and, on the other hand, the antiprotease activity of α1-antitrypsin was reduced after the interaction of the serpin with PG1 (while Bac5 did not affect the antiprotease activity of α1-antitrypsin). We used BODIPY FL-tagged PG1 and Bac5 to study the internalization of the labeled peptides into target cells and their intracellular distribution by confocal microscopy. Bac5-BODIPY (at 5 μ M) was rapidly taken into the cells. PG1-BODIPY at non-toxic concentrations (1—3 μM) was also able to enter the cells without their damaging. By using flow cytometry we showed that lowering a temperature to 4°C caused a significant decrease in the uptake into K562 and U937 cells for both Bac5-BODIPY and PG1-BODIPY. A decline of target cells metabolism also diminished the process of both peptides internalization but for a lesser degree. In the presence of endocytosis inhibitors the penetration of Bac5-BODIPY and PG1-BODIPY into K562 cells was also reduced, but not completely abolished, suggesting that along with endocytosis process some direct penetration of the peptides across cell membranes takes place. The ability of the peptides to internalize into eukaryotic cells may contribute to the idea of participation of AMPs in varied intracellular events, occurring in normal or malignant host cells, for instance, in the modulation of intracellular serpins activity.     

家蚕肽聚糖识别蛋白L1参与Imd信号通路

【目的】肽聚糖识别蛋白(peptidoglycan recognition proteins,PGRP)作为一个重要的模式识别受体,在家蚕Bombyx mori的先天免疫中发挥重要的作用。本研究主要探讨家蚕PGRP-L1基因的功能及其所参与的免疫信号通路。【方法】本实验通过RT-PCR扩增获得家蚕PGRP-L1基因。利用微生物诱导实验对5龄起蚕分别注射大肠杆菌Escherichia coli、酿酒酵母Saccharomyces cerevisiae、枯草芽孢杆菌Bacillus subtilis和PBS,12 h后取体壁和头部提取RNA,然后采用RT-PCR和凝胶电泳技术测定BmPGRP-L1基因的表达水平;利用RNA干涉实验向5龄起蚕注射PGRP-L1 dsRNA或PBS,6 h后再分别注射3种灭活的微生物或PBS,然后检测家蚕体壁及头部的免疫相关转录因子(Rel,Cactus和Relish)以及家蚕多个抗菌肽(antimicrobial peptides,AMPs)基因(攻击素基因Attacin,Moricin和葛佬素基因Gloverin)的表达情况。【结果】微生物诱导实验显示,注射大肠杆菌的实验组比注射PBS的对照组Bm PGRP-L1基因转录水平显著上调,而注射酿酒酵母和枯草芽孢杆菌的实验组Bm PGRP-L1转录水平没有变化。利用RNAi技术成功敲低Bm PGRP-1表达,对Bm PGRP-L1敲低的5龄起蚕注射灭活的微生物,敲低实验组relish因子表达低于正常对照组,相应地抗菌肽基因的表达也有不同程度的下调。【结论】结果提示,BmPGRP-L1基因参与家蚕对革兰氏阴性菌大肠杆菌的免疫响应;Bm PGRP-L1基因在家蚕的体壁和头中参与Imd信号通路。     

Movement of Anopheles gambiae s.l. malaria vectors between villages in The Gambia

ovement of mosquitoes belonging to the Anopheles gambiae complex (mixed wild populations of An.arabiensis, An.gambiae and An.melas ) between three neighbouring rural villages in The Gambia was investigated by mark-release-recapture. A total of 12,872 mosquitoes were collected in bednets, marked with a magenta fluorescent powder and released over a 15-day period in one of the villages. A further 15,507 mosquitoes were collected in exit traps, marked with a yellow powder and released over the same period. Mosquitoes were captured daily in all three villages using pyrethrum spray catches, as well as bednet and exit trap catches. The catching period extended for 6 days after the last day of release.
Of the mosquitoes released, 372 (1.3%) were recaptured 2–21 days later. Of these recaptures, 272 were caught in the release village, and 98 were caught in other villages situated 1–1.4 km away. The 'movement index' between villages was calculated as 17.2% (12.2–22.4% confidence limits) for mosquitoes released after feeding and 20.1% (14.7–25.3%) for those released unfed.
These results suggest that movement of mosquitoes between neighbouring villages in The Gambia seriously affects the entomological evaluation of pyrethroid-impregnated bednet programmes in areas where treated and untreated villages are interspersed.     

Impact of permethrin-treated bednets on malaria transmission by the Anopheles gambiae complex in The Gambia

Malaria vector mosquitoes belonging to the Anopheles gambiae complex were studied in four hamlets in The Gambia. All inhabitants were given bednets treated either with a placebo (milk) in two hamlets or with the pyrethroid insecticide permethrin (500 mg/m2) in two other hamlets. Malaria transmission occurred mainly during a few weeks of the rainy season, in September and October 1987. The indoor resting densities of mosquitoes in permethrin-treated hamlets were reduced, and we estimated over 90% reduction in biting on man by An. gambiae Giles sensu stricto in these hamlets. No mosquitoes were found under permethrin-treated bednets compared with eighty-one recovered from placebo-treated bednets. Mosquitoes exited more readily from rooms where permethrin-treated bednets were used than from rooms with placebo-treated nets. The annual mean probability that a child would receive an infective bite was estimated to be 0.09 in hamlets with insecticide-treated bednets, compared with 1.9 where placebo-treated bednets were used. Permethrin-treated bednets are therefore recommended as a means of effectively reducing the risk of exposure to malaria transmission, particularly in areas of low seasonal transmission.