Biology Department, American University of Beirut, Beirut, Lebanon.
Entomology Department, Cornell Institute for Host-Microbe Interactions and Disease, Cornell University, Ithaca, New York, USA.
J Innate Immun. 2021;13(2):107-126. doi: 10.1159/000511401. Epub 2020 Nov 18.
Insect systemic immune responses to bacterial infections have been mainly studied using microinjections, whereby the microbe is directly injected into the hemocoel. While this methodology has been instrumental in defining immune signaling pathways and enzymatic cascades in the hemolymph, it remains unclear whether and to what extent the contribution of systemic immune defenses to host microbial resistance varies if bacteria invade the hemolymph after crossing the midgut epithelium subsequent to an oral infection. Here, we address this question using the pathogenic Serratia marcescens (Sm) DB11 strain to establish systemic infections of the malaria vector Anopheles gambiae, either by septic Sm injections or by midgut crossing after feeding on Sm. Using functional genetic studies by RNAi, we report that the two humoral immune factors, thioester-containing protein 1 and C-type lectin 4, which play key roles in defense against Gram-negative bacterial infections, are essential for defense against systemic Sm infections established through injection, but they become dispensable when Sm infects the hemolymph following oral infection. Similar results were observed for the mosquito Rel2 pathway. Surprisingly, blocking phagocytosis by cytochalasin D treatment did not affect mosquito susceptibility to Sm infections established through either route. Transcriptomic analysis of mosquito midguts and abdomens by RNA-seq revealed that the transcriptional response in these tissues is more pronounced in response to feeding on Sm. Functional classification of differentially expressed transcripts identified metabolic genes as the most represented class in response to both routes of infection, while immune genes were poorly regulated in both routes. We also report that Sm oral infections are associated with significant downregulation of several immune genes belonging to different families, specifically the clip-domain serine protease family. In sum, our findings reveal that the route of infection not only alters the contribution of key immunity genes to host antimicrobial defense but is also associated with different transcriptional responses in midguts and abdomens, possibly reflecting different adaptive strategies of the host.
昆虫对细菌感染的系统性免疫反应主要通过微注射来研究，即将微生物直接注入血腔。虽然这种方法对于定义血淋巴中的免疫信号通路和酶级联反应非常重要，但仍然不清楚如果细菌在经口感染后穿过中肠上皮进入血淋巴，系统性免疫防御对宿主微生物抗性的贡献是否以及在何种程度上发生变化。在这里，我们使用致病性的粘质沙雷氏菌（Sm）DB11 菌株来解决这个问题，通过败血症 Sm 注射或在 Sm 喂养后穿过中肠来建立疟疾病媒按蚊的系统性感染。通过 RNAi 的功能遗传研究，我们报告了两种体液免疫因子，硫酯蛋白 1 和 C 型凝集素 4，它们在防御革兰氏阴性细菌感染中起着关键作用，对于通过注射建立的针对 Sm 的系统性感染防御是必不可少的，但当 Sm 通过经口感染感染血淋巴时，它们变得可有可无。蚊 Rel2 途径也观察到了类似的结果。令人惊讶的是，用细胞松弛素 D 处理阻断吞噬作用并不影响蚊子对通过任何一种途径建立的 Sm 感染的易感性。通过 RNA-seq 对蚊中肠和腹部进行转录组分析表明，这两种组织对喂食 Sm 的反应更为明显。差异表达转录本的功能分类表明，代谢基因是对两种感染途径的最主要代表类群，而免疫基因在两种途径中都没有得到很好的调控。我们还报告说，Sm 的经口感染与属于不同家族的几个免疫基因的显著下调有关，特别是剪接结构域丝氨酸蛋白酶家族。总之，我们的发现表明，感染途径不仅改变了关键免疫基因对宿主抗菌防御的贡献，而且与中肠和腹部的不同转录反应相关联，这可能反映了宿主的不同适应策略。
