Scuola di Bioscienze e Biotecnologie, Università degli Studi di Camerino, Camerino, Italy.
Parasit Vectors. 2013 Jun 18;6(1):182. doi: 10.1186/1756-3305-6-182.
Malaria represents one of the most devastating infectious diseases. The lack of an effective vaccine and the emergence of drug resistance make necessary the development of new effective control methods. The recent identification of bacteria of the genus Asaia, associated with larvae and adults of malaria vectors, designates them as suitable candidates for malaria paratransgenic control.To better characterize the interactions between Asaia, Plasmodium and the mosquito immune system we performed an integrated experimental approach.
Quantitative PCR analysis of the amount of native Asaia was performed on individual Anopheles stephensi specimens. Mosquito infection was carried out with the strain PbGFPCON and the number of parasites in the midgut was counted by fluorescent microscopy.The colonisation of infected mosquitoes was achieved using GFP or DsRed tagged-Asaia strains.Reverse transcriptase-PCR analysis, growth and phagocytosis tests were performed using An. stephensi and Drosophila melanogaster haemocyte cultures and DsRed tagged-Asaia and Escherichia coli strains.
Using quantitative PCR we have quantified the relative amount of Asaia in infected and uninfected mosquitoes, showing that the parasite does not interfere with bacterial blooming. The correlation curves have confirmed the active replication of Asaia, while at the same time, the intense decrease of the parasite.The 'in vitro' immunological studies have shown that Asaia induces the expression of antimicrobial peptides, however, the growth curves in conditioned medium as well as a phagocytosis test, indicated that the bacterium is not an immune-target.Using fluorescent strains of Asaia and Plasmodium we defined their co-localisation in the mosquito midgut and salivary glands.
We have provided important information about the relationship of Asaia with both Plasmodium and Anopheles. First, physiological changes in the midgut following an infected or uninfected blood meal do not negatively affect the residing Asaia population that seems to benefit from this condition. Second, Asaia can act as an immune-modulator activating antimicrobial peptide expression and seems to be adapted to the host immune response. Last, the co-localization of Asaia and Plasmodium highlights the possibility of reducing vectorial competence using bacterial recombinant strains capable of releasing anti-parasite molecules.
疟疾是最具破坏性的传染病之一。缺乏有效的疫苗和药物耐药性的出现使得开发新的有效控制方法成为必要。最近发现的与疟疾传播媒介的幼虫和成虫有关的 Asaia 属细菌将其指定为疟疾转基因控制的合适候选者。为了更好地描述 Asaia、疟原虫和蚊子免疫系统之间的相互作用，我们进行了综合实验研究。
对个体按蚊标本中的天然 Asaia 数量进行定量 PCR 分析。用 PbGFPCON 菌株感染蚊子，并通过荧光显微镜计数中肠中的寄生虫数量。使用 GFP 或 DsRed 标记的 Asaia 菌株实现感染蚊子的定植。使用 An. stephensi 和 Drosophila melanogaster 血淋巴细胞培养物和 DsRed 标记的 Asaia 和 Escherichia coli 菌株进行逆转录 PCR 分析、生长和吞噬作用试验。
使用定量 PCR，我们量化了感染和未感染蚊子中 Asaia 的相对数量，表明寄生虫不会干扰细菌的繁殖。相关曲线证实了 Asaia 的积极复制，同时寄生虫数量急剧减少。“体外”免疫学研究表明，Asaia 诱导抗菌肽的表达，然而，在条件培养基中的生长曲线和吞噬作用试验表明，该细菌不是免疫靶标。使用 Asaia 和疟原虫的荧光菌株，我们定义了它们在蚊子中肠和唾液腺中的共定位。
我们提供了有关 Asaia 与疟原虫和按蚊关系的重要信息。首先，感染或未感染血餐引起的中肠生理变化不会对居住的 Asaia 种群产生负面影响，似乎受益于这种情况。其次，Asaia 可以作为免疫调节剂激活抗菌肽的表达，并且似乎适应宿主的免疫反应。最后，Asaia 和疟原虫的共定位突出了使用能够释放抗寄生虫分子的细菌重组菌株来降低媒介能力的可能性。
