果蝇蛋白质激酶Pitslre通过调控抗菌肽表达参与天然免疫反应

为鉴定新的参与黑腹果蝇(Drosophila melanogaster)天然免疫信号通路调控的分子及作用机制,应用果蝇的Gal4/UAS系统敲低54个蛋白质激酶编码基因,分别利用革兰氏阳性菌（Enterococcus faecalis, E.faecalis）或革兰氏阴性菌（Erwinia carototovovora carototovovora 15, Ecc15）感染基因敲低果蝇,筛选参与果蝇天然免疫反应的蛋白质激酶。结果显示,全身性敲低蛋白质激酶Pitslre的果蝇感染E.faecalis或Ecc15 后,生存率降低,半致死时间LT50分别降低为对照组的66.7%和28.6%。相应的,Pitslre功能缺失导致革兰氏阳性菌和阴性菌分别感染后,Toll及IMD通路下游抗菌肽Drosomycin和Diptercin表达水平明显下降。在脂肪体和血淋巴细胞中特异性敲低Pitslre基因,导致革兰氏阳性菌及阴性菌感染后的果蝇半致死时间LT50分别缩短75%和90%,细菌载量分别升高约10倍。在果蝇S2细胞中,敲低Pitslre基因,导致细胞的抗菌肽Drosomycin、Attacin和Diptercin表达水平分别降低约50%。此外,通过免疫共沉淀实验检测Pitslre与预测存在相互作用的蛋白质TSC1、Rcd5和pbl之间的相互作用。综上所述,蛋白质激酶Pitslre参与果蝇天然免疫反应,在正向调控果蝇天然免疫Toll和IMD通路中发挥重要作用。     

果蝇蛋白质激酶Pitslre通过调控抗菌肽表达参与天然免疫反应

为鉴定新的参与黑腹果蝇(Drosophila melanogaster)天然免疫信号通路调控的分子及作用机制,应用果蝇的Gal4/UAS系统敲低54个蛋白质激酶编码基因,分别利用革兰氏阳性菌（Enterococcus faecalis, E.faecalis）或革兰氏阴性菌（Erwinia carototovovora carototovovora 15, Ecc15）感染基因敲低果蝇,筛选参与果蝇天然免疫反应的蛋白质激酶。结果显示,全身性敲低蛋白质激酶Pitslre的果蝇感染E.faecalis或Ecc15 后,生存率降低,半致死时间LT50分别降低为对照组的66.7%和28.6%。相应的,Pitslre功能缺失导致革兰氏阳性菌和阴性菌分别感染后,Toll及IMD通路下游抗菌肽Drosomycin和Diptercin表达水平明显下降。在脂肪体和血淋巴细胞中特异性敲低Pitslre基因,导致革兰氏阳性菌及阴性菌感染后的果蝇半致死时间LT50分别缩短75%和90%,细菌载量分别升高约10倍。在果蝇S2细胞中,敲低Pitslre基因,导致细胞的抗菌肽Drosomycin、Attacin和Diptercin表达水平分别降低约50%。此外,通过免疫共沉淀实验检测Pitslre与预测存在相互作用的蛋白质TSC1、Rcd5和pbl之间的相互作用。综上所述,蛋白质激酶Pitslre参与果蝇天然免疫反应,在正向调控果蝇天然免疫Toll和IMD通路中发挥重要作用。     

Alternative measures of response to Pseudomonas aeruginosa infection in Drosophila melanogaster

Studies of invertebrate immune defence often measure genetic variation either for the fitness cost of infection or for the ability of the host to clear the parasite. These studies assume that variation in measures of resistance is related to variation in fitness costs of infection. To test this assumption, we infected strains of the fruit fly, Drosophila melanogaster, with a pathogenic bacterium. We then measured the correlation between host bacterial load and the ability to survive infection. Despite the presence of genotypic variation for both traits, bacterial load and survival post-infection were not correlated. Our results support previous arguments that individual measures of immune function and the host's ability to survive infection may be decoupled. In light of these results, we suggest that the difference between tolerance and resistance to infection, a distinction commonly found in the plant literature, may also be of value in studies of invertebrate immunity.     

Characterization of four Toll related genes during development and immune responses in Anopheles gambiae

Toll-like receptors (TLRs) are a group of evolutionary conserved proteins with diverse biological functions. In Drosophila melanogaster, Toll protein plays an important role in pattern formation in embryogenesis and in antimicrobial immunity in larvae and adults. In insects, Toll and two other related proteins, Tehao and 18-wheeler have been shown to participate in the activation of the innate immune responses to fungal and bacterial pathogens. In this paper we report the cloning and characterization of four TLR gene from malaria vector mosquito Anopheles gambiae, AgToll, AgToll6, AgTrex, and AgToll9, orthologues of DmToll, DmToll6, DmTollo (Toll8) and DmToll9 (CG5528) in Drosophila melanogaster. The expression profiles of these genes during development, in different adult tissues and after immune challenge were examined. As expected for the orthologue of Drosophila Toll, AgToll was found to be expressed highly in the ovary and may play a role in pattern formation during embryogenesis. AgToll9, surprisingly, was found to be highly expressed in the adult gut. The potential roles of these genes in development and immunity were discussed.     

Host ecology shapes geographical variation for resistance to bacterial infection in Drosophila melanogaster

1. Geographically distinct host populations often experience very different ecological conditions. These variable ecological conditions impact the strength of selection that these hosts experience from their parasites. 2. Numerous studies have characterized geographical patterns of resistance to infection among natural populations in the context of host-parasite local adaptation, but what other factors might contribute to these differences? 3. Here, we determined whether 20 naturally isolated populations of Drosophila melanogaster collected along the East Coast of the United States varied for survival after being inoculated with one of two species of bacteria--Lactococcus lactis and Pseudomonas aeruginosa. We then asked whether host environment accounted for the observed patterns of resistance. 4. Resistance to both types of infection varied spatially. The hosts' natural environment was predictive of the observed spatial variation in resistance to L. lactis, but not P. aeruginosa, infection. Specifically, hosts exposed to species-rich bacterial communities were more likely to survive the infection. 5. We conclude that biotic characteristics of the host environment, specifically the number of species of bacteria hosts encounter, shape host resistance to bacterial infection in nature. We discuss our results in the context of what is known about the evolutionary ecology of resistance in invertebrate systems.     

基于果蝇口腔感染的肠道训练免疫模型研究（英文）

目的 利用果蝇作为遗传工具从个体和分子层面研究果蝇的训练免疫效应,并为后续深入研究其分子机制提供依据。方法 首先构建无菌果蝇模型,在此基础上构建果蝇成虫及跨发育阶段训练免疫模型,用两种革兰氏阴性菌——胡萝卜软腐欧文氏菌（Erwinia carotovora carotovora 15）及铜绿假单胞菌（Pseudomonas aeruginosa）分别经口腔感染果蝇。在第一次感染完全消退后进行再次感染,然后通过比较果蝇在两个感染阶段的存活率和细菌量来衡量训练免疫的潜在效果。通过实时荧光定量PCR检测相应先天免疫相关基因的表达水平,研究革兰氏阴性菌对免疫缺陷（IMD）通路的诱导作用。结果 果蝇成虫及幼虫初次感染均可提高二次感染后的生存率、细菌清除效率及死亡时能承受的最高细菌负荷;二次感染的果蝇中,IMD通路中免疫反应基因的基础表达比未感染的高,这提供了获得感染抗性的分子基础;果蝇的免疫反应主要发生在中肠,二次免疫比初次免疫的效应更迅速且剧烈;二次免疫的果蝇中,肠道干细胞的数量显著多于初次感染。结论 果蝇肠道中强大的训练免疫可由同源或异源革兰氏阴性菌口腔感染引发,且免疫记忆可在整个发育阶段持...     

Universal primers for detection of common bacterial pathogens causing prosthetic joint infection

The diagnosis of low grade prosthetic joint infection is difficult and time consuming. Nested-PCR for universal bacterial DNA segments detection of "orthopaedic" bacteria was tested in a laboratory setting. This method is based on amplification of the 16S bacterial ribosomal RNA coding sequences. 11 species of the most frequent bacterial pathogens (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, Enterococcus faecium, Enterococcus faecalis, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, Serratia marcescens) involved in prosthetic joint infections were studied. All could be detected rapidly and sensitively by this method.     

The complexity of genetic variation in a simple immune system

Fruitflies derived from a wild population vary in their resistance to infection with the bacterial pathogen Serratia marcescens. A survey of nucleotide diversity in 21 genes involved in innate immunity concluded that 16 genes had polymorphisms associated with resistance to this specific pathogen. However, the effects of individual polymorphisms on the resistance phenotype were modest, and epistatic interactions appeared to be common. What might these findings tell us about genetic resistance to infection in humans?     

Functional analysis of the gene cluster involved in production of the bacteriocin circularin A by Clostridium beijerinckii ATCC 25752

A region of 12 kb flanking the structural gene of the cyclic antibacterial peptide circularin A of Clostridium beijerinckii ATCC 25752 was sequenced, and the putative proteins involved in the production and secretion of circularin A were identified. The genes are tightly organized in overlapping open reading frames. Heterologous expression of circularin A in Enterococcus faecalis was achieved, and five genes were identified as minimally required for bacteriocin production and secretion. Two of the putative proteins, CirB and CirC, are predicted to contain membrane-spanning domains, while CirD contains a highly conserved ATP-binding domain. Together with CirB and CirC, this ATP-binding protein is involved in the production of circularin A. The fifth gene, cirE, confers immunity towards circularin A when expressed in either Lactococcus lactis or E. faecalis and is needed in order to allow the bacteria to produce bacteriocin. Additional resistance against circularin A is conferred by the activity of the putative transporter consisting of CirB and CirD.     

需氧菌阴道炎、细菌性阴道病阴道菌群分析

用阴道分泌物直接涂片、革兰染色、选择性培养分离和细菌预成酶谱分析等微生物学方法和生物化学方法,分析了正常女性、AV、BV患者的阴道菌群。正常女性阴道中以乳酸杆菌为主,产H2O2乳酸杆菌的检出率89%,AV的致病菌主要是革兰阳性率需氧菌、检出率可达80%,其中金黄色葡萄球菌,粪肠球菌和埃希氏大肠菌较为常见,B族链球菌的检出率较低;BV的致病菌主要是厌氧菌,以革兰阴性厌氧菌的检出率最高,加德纳菌的检出率不足40%。48例BV患者中有8例合并AV感染,31例AV患者中有6例合并BV感染。细菌谱分析发现,AV患者易感染消化链球菌、普雷沃菌等厌氧菌和加德纳菌,BV患者易感染粪肠球菌、大肠埃希菌等需氧菌。     

院内呼吸科下呼吸道感染病原学及耐药性结果分析

目的分析大连市第五人民医院2009年呼吸科送检1160例次痰标本细菌检查情况,为临床提供感染数据以及合理使用抗生素提供依据。方法按常规方法将临床送检的痰标本进行细菌培养、分离。使用BIO-FOSUN微生物鉴定药敏分析系统进行细菌鉴定及抗生素敏感试验。结果一年来共收痰标本1160例次,检出病原菌253例次,阳性率为21.8%。病原菌依次为肺炎克雷伯菌83株,铜绿假单胞菌57株,乙酸钙不动杆菌29株,阴沟肠杆菌14株,粘质沙雷菌13株,大肠埃希菌10株,,金黄色葡萄球菌5株等。大多数革兰阴性菌对亚胺培南较敏感,对头孢菌素类均有不同程度的耐药。革兰阳性球菌对万古霉素敏感,对其他抗生素有不同程度的耐药。结论该院呼吸科患者下呼吸道感染以革兰阴性杆菌为主,常见病原菌耐药性未呈现高水平、多重耐药情况。提示临床医生应与检验科密切配合,关注细菌感染情况,根据药敏试验结果合理选用抗生素。     

Natural variation in RPS2-mediated resistance among Arabidopsis accessions: correlation between gene expression profiles and phenotypic responses

Natural variation in gene expression (expression traits or e-traits) is increasingly used for the discovery of genes controlling traits. An important question is whether a particular e-trait is correlated with a phenotypic trait. Here, we examined the correlations between phenotypic traits and e-traits among 10 Arabidopsis thaliana accessions. We studied defense against Pseudomonas syringae pv tomato DC3000 (Pst), with a focus on resistance gene-mediated resistance triggered by the type III effector protein AvrRpt2. As phenotypic traits, we measured growth of the bacteria and extent of the hypersensitive response (HR) as measured by electrolyte leakage. Genetic variation among accessions affected growth of Pst both with (Pst avrRpt2) and without (Pst) the AvrRpt2 effector. Variation in HR was not correlated with variation in bacterial growth. We also collected gene expression profiles 6 h after mock and Pst avrRpt2 inoculation using a custom microarray. Clusters of genes whose expression levels are correlated with bacterial growth or electrolyte leakage were identified. Thus, we demonstrated that variation in gene expression profiles of Arabidopsis accessions collected at one time point under one experimental condition has the power to explain variation in phenotypic responses to pathogen attack.     

Variable fitness effects of Wolbachia infection in Drosophila melanogaster

Maternally inherited Wolbachia bacteria are extremely widespread among insects and their presence is usually associated with parasitic modifications of host fitness. Wolbachia pipientis infects Drosophila melanogaster populations from all continents, but their persistence in this species occurs despite any strong parasitic effects. Here, we have investigated the symbiosis between Wolbachia and D. melanogaster and found that Wolbachia infection can have significant survival and fecundity effects. Relative to uninfected flies, infected females from three fly strains showed enhanced survival or fecundity associated with Wolbachia infection, one strain showed both and one strain responded positively to Wolbachia removal. We found no difference in egg hatch rates (cytoplasmic incompatibility) for crosses between infected males and uninfected females, although there were fecundity differences. Females from this cross consistently produced fewer eggs than infected females and these fecundity differences could promote the spread of infection just like cytoplasmic incompatibility. More surprising, we found that infected females often had the greatest fecundity when mated to uninfected males. This could also promote the spread of Wolbachia infection, though here the fitness benefits would also help to spread infection when Wolbachia are rare. We suggest that variable fitness effects, in both sexes, and which interact strongly with the genetic background of the host, could increase cytoplasmic drive rates in some genotypes and help explain the widespread persistence of Wolbachia bacteria in D. melanogaster populations. These interactions may further explain why many D. melanogaster populations are polymorphic for Wolbachia infection. We discuss our results in the context of host-symbiont co-evolution.     

Functional analysis of immune response genes in Drosophila identifies JNK pathway as a regulator of antimicrobial peptide gene expression in S2 cells

The templates of innate immunity have ancient origins. Thus, such model animals as the fruit fly, Drosophila melanogaster, can be used to identify gene products that also play a key role in the innate immunity in mammals. We have used oligonucleotide microarrays to identify genes that are responsive to gram-negative bacteria in Drosophila macrophage-like S2 cells. In total, 53 genes were induced by greater than threefold in response to Escherichia coli. The induction of all these genes was peptidoglycan recognition protein LC (PGRP-LC) dependent. Twenty-two genes including 10 of the most strongly induced genes are also known to be up-regulated by septic injury in vivo. Importantly, we identified 31 genes that are not known to respond to bacterial challenge. We carried out targeted dsRNA treatments to assess the functional importance of these gene products for microbial recognition, phagocytosis and antimicrobial peptide release in Drosophila S2 cells in vitro. RNAi targeting three of these genes, CG7097, CG15678 and beta-Tubulin 60D, caused altered antimicrobial peptide release in vitro. Our results indicate that the JNK pathway is essential for normal antimicrobial peptide release in Drosophila in vitro.     

Phage abortive infection in lactococci: variations on a theme

Abortive infection (Abi) systems, also called phage exclusion, block phage multiplication and cause premature bacterial cell death upon phage infection. This decreases the number of progeny particles and limits their spread to other cells allowing the bacterial population to survive. Twenty Abi systems have been isolated in Lactococcus lactis, a bacterium used in cheese-making fermentation processes, where phage attacks are of economical importance. Recent insights in their expression and mode of action indicate that, behind diverse phenotypic and molecular effects, lactococcal Abis share common traits with the well-studied Escherichia coli systems Lit and Prr. Abis are widespread in bacteria, and recent analysis indicates that Abis might have additional roles other than conferring phage resistance.     

Genotype-by-environment interactions and adaptation to local temperature affect immunity and fecundity in Drosophila melanogaster

Natural populations of most organisms harbor substantial genetic variation for resistance to infection. The continued existence of such variation is unexpected under simple evolutionary models that either posit direct and continuous natural selection on the immune system or an evolved life history "balance" between immunity and other fitness traits in a constant environment. However, both local adaptation to heterogeneous environments and genotype-by-environment interactions can maintain genetic variation in a species. In this study, we test Drosophila melanogaster genotypes sampled from tropical Africa, temperate northeastern North America, and semi-tropical southeastern North America for resistance to bacterial infection and fecundity at three different environmental temperatures. Environmental temperature had absolute effects on all traits, but there were also marked genotype-by-environment interactions that may limit the global efficiency of natural selection on both traits. African flies performed more poorly than North American flies in both immunity and fecundity at the lowest temperature, but not at the higher temperatures, suggesting that the African population is maladapted to low temperature. In contrast, there was no evidence for clinal variation driven by thermal adaptation within North America for either trait. Resistance to infection and reproductive success were generally uncorrelated across genotypes, so this study finds no evidence for a fitness tradeoff between immunity and fecundity under the conditions tested. Both local adaptation to geographically heterogeneous environments and genotype-by-environment interactions may explain the persistence of genetic variation for resistance to infection in natural populations.