昆虫病原线虫共生菌Serratia nematodiphila DR186对果蝇致病机制的研究

Pathogenesis of entomopathogenic nematode symbiotic bacterial strain Serratia nematodiphila DR186 against fruit flies

[目的]通过昆虫病原线虫共生菌Serratia nematodiphila DR186侵染黑腹果蝇的野生型和免疫突变体,研究该共生菌侵染果蝇成虫的致病机制。[方法]利用饲喂和针刺方式将S.nematodiphila DR186接入野生型黑腹果蝇和其体液免疫途径突变体的肠道和血腔中引起侵染;通过统计2种侵染条件下各个突变体的存活率,血腔中共生菌的菌体数,结合荧光定量PCR检测两类抗菌肽Diptericin和Drosomycin的表达水平来研究S.nematodiphila DR186对果蝇的致病机制。[结果]S.nematodiphila DR186对野生型黑腹果蝇的肠道侵染存活率在7 d降至0,血腔侵染存活率在19 h降至0;S.nematodiphila DR186血腔侵染后,Imd途径的PGRP-LC、Dredd、Relish突变体相对Toll途径的Dif突变体和野生型更加敏感,在12 h内存活率低于50%,并在随后短时间内全部死亡。荧光定量PCR分析显示Diptericin和Drosomycin的相对表达水平在血腔侵染过程中均呈先上升再逐渐降低的趋势。Dredd突变体Drosomycin相对表达量在6 h达到88%,Dif突变体和野生型的Diptericin相对表达量在6 h分别达到75%和62%。S.nematodiphila DR186侵染肠道后对Imd途径和Toll途径突变体及野生型的存活率在7 d降至0。针刺法侵染的致病性高于饲喂法侵染,在19 h存活率降至0。[结论]S.nematodiphila DR186可能采用免疫逃逸机制从果蝇肠道成功侵染果蝇。果蝇抵抗S.nematodiphila DR186侵染的主要免疫途径是Imd体液免疫途径。

[ Objectives] In the present study,Serratia nematodiphila DR186 was introduced into wild-type and immune mutants of Drosophila melanogaster to study pathogenesis mechanism during the infection process. [ Methods] Feeding（ intestinal infection）and needling（ blood cavity infection）methods were used to inoculate S. nematodiphila DR186 into the gut and hemocoel of wild type and immune mutant flies of Imd and Toll immunological pathway. Survival rate of flies and bacterial proliferation curve within flies＇ hemocoel was employed to assess host susceptibility and virulence potential of pathogen. Expression level of Diptericin and Drosomycin,the antimicrobial peptides of Imd and Toll pathway was detected by qPCR. [ Results ] Survival rate of wild type flies which feed with bacterium DR186 decreased to 0 in 7 d,whereas survival rate using needing methods decreased to 0 in 19 h. After blood cavity infection of bacterium DR186, PGRP-LC mutants （ Imd）, Dredd mutants （Imd） and Relish mutants （Imd） were more sensitive than D/f mutants （Toll）and wildtype. Lower than 50% fly mutants of Imd pathway survival in 12 h and decreased to 0 survival rate in a short time after 12 h. Results of qPCR showed that expression of Diptericin and Drosomycin in the blood cavity infection had the same trend that up-regulated firstly and then gradually down-regulated. The relative expression level of Drosomycin in Dredd mutants reached 88% in 6 h,and those of Diptericin in D/f mutants and wild type respectively reached 75% and 62% in 6 h. Survival rate of wild type and mutants flies which feed with bacterium DR186 decreased to 0 in 7 d, while hemocoel infection need only 19 h to decreased to 0. [ Conclusions] In the process of intestinal infection, S. nematodiphila DR186 might have some immune escape mechanism to infect flies successfully. Imd pathway play a major role in flies＇ humoral immune systerm when facing with infection of S. nematodiphila DR186.