摘要
志贺菌(Shigella)为革兰阴性菌,是细菌性痢疾的病原体,主要通过粪一口途径传播,侵入结肠粘膜。在我国,细菌性痢疾是发病率仅次于病毒性肝炎和肺结核的第三大传染病,血清型以福氏2a型为主。近年来,由于抗生素的不规范使用以及耐药基因的水平传播,志贺菌在临床上呈现多重耐药,对青霉素类、磺胺类、四环素类抗生素耐药率极高,甚至对喹诺酮类、头孢菌素类等目前临床常用药物的耐药率也不断上升,因此需要研究新的治疗策略加以应对。以抑制细菌毒力为思路可为新治疗策略的研究提供理论依据。志贺菌侵袭宿主细胞、在细胞间扩散并释放毒素,是其致病过程的关键环节,其毒力与它的黏附力、侵袭力、肠毒素、调控蛋白等有关,涉及多种致病因子,其中PhoP-PhoQ对志贺菌毒力的调控起重要作用,但其对毒力的调控机制不清楚,有待深入研究。
本课题的目的为研究福氏志贺菌PhoP-PhoQ双组分信号转导系统对其毒力的调控机制。本研究首先对课题组前期筛选出的PhoQ抑制剂对福氏志贺菌Sf301毒力的影响进行研究：通过HeLa细胞福氏志贺菌侵袭实验和小鼠角结膜志贺菌感染实验,研究PhoQ抑制剂对福氏志贺菌毒力的影响,并利用Real-timePCR比较PhoQ抑制剂作用前后福氏志贺菌相关毒力基因的转录水平。在此基础上,利用同源重组技术构建福氏志贺菌Sf301株phoP-phoQ双组分信号转导系统基因突变株,利用已HeLa细胞侵袭实验及豚鼠角结膜感染实验,比Sf矽301与phoP-phoQ敲除突变株的毒力变化。同时,利用Real-time PCR比较Sf301与phoP-phoQ突变株毒力相关基因的转录水平,探讨PhoP-PhoQ双组分系统对志贺菌毒力的调节作用。
第一章PhoP-PhoQ双组分信号转导系统的生物信息学分析
鉴于PhoP-PhoQ双组分信号转导系统在志贺菌中普遍存在,我们对志贺菌PhoP-PhoQ双组分信号转导系统进行了生物信息学分析。Shigella flexneri2a str.301中phoQ基因与其它6株志贺菌株的同源性均大于99%；在其它革兰阴性菌中,Shigella flexneri2a str.301与Escherichia coli及Salmonella enterica subsp. enterica serovar Typhi的同源性均大于76%。PhoQ在革兰阴性菌中表达双组分系统中的感应蛋白,蛋白全长包含486aa,编码跨膜的组氨酸蛋白激酶,PhoQ的氨基酸序列在志贺菌中的同源性大于99%；在其它革兰阴性菌中的同源性大于85%。Shigella flexneri2a str.301中phoP基因与其它6株志贺菌株的同源性均大于98%；在其它革兰阴性菌中,Shigella flexneri2a str.301与Escherichia coli及Salmonella enterica subsp. enterica serovar Typhi的同源性均大于80%。PhoP在革兰阴性菌中表达双组分系统中的反应调节蛋白,蛋白全长包含223aa,编码胞内反应调节蛋白,PhoP氨基酸序列在志贺菌中的同源性大于98%,在其它革兰阴性菌中的同源性大于80%。而与革兰阳性菌的同源性小于32%。
第二章phoQ抑制剂对福氏志贺菌毒力的影响
PhoQ蛋白具有调控福氏志贺菌毒力的作用,在前期实验中,本课题组以PhoQ为药物靶标筛选获得4个PhoQ抑制剂,在此基础上,本研究通过HeLa细胞志贺菌侵袭实验和小鼠角结膜福氏志贺菌感染实验,研究了PhoQ抑制剂对福氏志贺菌毒力的影响。
首先我们检测了4个PhoQ抑制剂对福氏志贺菌生长的影响：分别将终浓度为100μmol/L的PhoQ抑制剂加入Sf9380培养,每隔1h测定sf9380的CFU,连续测定12h。结果显示,4个PhoQ抑制剂对Sf9380的生长均没有影响。
在此基础上,我们建立了福氏志贺菌感染HeLa细胞模型：将抑制剂(100μmo/L)与福氏志贺菌Sf9380共同培养4h、6h、8h后加入HeLa细胞,侵袭60min后去除细菌,加入庆大霉素以杀灭细胞外的细菌。去除庆大霉素后,加入Triton X-100释放出侵袭入细胞内的细菌,取一定量的Triton X-100涂板后培养,计算出平板上的细菌,得到侵袭入细胞的细菌总数与侵袭细胞的细菌总数的比值来表示侵袭率。结果发现：PhoQ抑制剂与细菌共同培养4h时抑制剂对福氏志贺菌的侵袭力没有抑制作用；与细菌共同培养6h时,PhoQ抑制剂对福氏志贺菌的侵袭力有一定的抑制作用；与细菌共同培养8h时,PhoQ抑制剂对福氏志贺菌的侵袭力有明显的抑制作用。在确定了抑制剂与细菌相互作用的时间后,我们选取与PhoQ抑制剂作用8h后的福氏志贺菌Sf9380作为实验菌株,侵袭60min后,研究PhoQ抑制剂1、2、3和4对福氏志贺菌Sf9380侵袭力的抑制作用。结果显示：福氏志贺菌Sf9380经抑制剂1(12.5μmol/L)、2(25μmol/L)、3(100μmol/L)作用后,对HeLa细胞的侵袭率均有显著下降(p<0.01);抑制剂4(100μmol/L)不能抑制Sf93S0对HeLa细胞的侵袭力。进一步为了验证筛选出的PhoQ抑制剂对细菌侵袭力的影响,我们选取了与福氏志贺菌PhoQ同源性较高的沙门菌作为实验菌株,研究PhoQ抑制剂对沙门菌侵袭HeLa细胞的影响。结果发现,抑制剂1、2、3均能抑制沙门菌对HeLa细胞的侵袭(p<0.01)侵袭率分别为0.89×10-2、1.564×10-2、0.036×10-2,而抑制剂4则不能抑制沙门菌对HeLa细胞的侵袭,侵袭率为5.7×10-2,沙门菌的侵袭率为6×10-2。
我们采用小鼠角结膜志贺菌感染模型,比较了PhoQ抑制剂1、2、3和4对福氏志贺菌Sf9380毒力的抑制作用：将PhoQ抑制剂与福氏志贺菌Sf9380共同培养8h后,取3×108的细菌滴入小鼠角膜内,于感染后24h、48h、72h、96h分别观察小鼠角结膜感染情况,结果发现：抑制剂1(12.5umol/L)、2(25umol/L)和4(100umol/L)能够明显抑制Sf9380所引起的小鼠角结膜炎症反应,降低24h和48h时的急性角膜结膜炎症程度,并加快炎症恢复速度,72h后炎症明显减弱(P<0.05)；抑制剂3(100umol/L)在24h可降低炎症程度(P<0.05)但不能减弱48h时的炎症程度。
进一步,我们利用Real-time PCR方法研究PhoQ抑制剂对Sf9380毒力相关基因转录水平的影响,结果表明与未经抑制剂处理的Sf9380相比,经抑制剂1(12.5μmol/L)、2(25μmol/L)和3(100μmol/L)处理的Sf9380中,ipaA、 ipaB、ipaC、ipaD、ipaH、mxiA、virB及virF的转录水平均下降,而经抑制剂4(100μmol/L)处理的Sf9380中,ipaA、ipaB、ipaC、ipaD、ipaH、mxiA、virB及virF的转录水平没有明显变化。研究结果提示,PhoQ抑制剂可以一定程度的抑制福氏志贺菌的毒力,且PhoP-PhoQ双组分信号转导系统对福氏志贺菌毒力具有重要的调控作用。
第三章phoP-phoQ对福氏志贺菌毒力的影响
PhoP-PhoQ双组分信号转导系统是沙门菌和大肠杆菌等革兰阴性菌中的一个重要的毒力调控单元,可调控多种毒力基因的转录和表达。在研究PhoQ抑制剂对福氏志贺菌侵袭HeLa细胞的影响中,我们在发现PhoQ抑制剂能够抑制福氏志贺菌对HeLa细胞的侵袭力,同时也发现Sf301野生株对HeLa细胞的侵袭力较低。因此,我们首先对Sf301野生株的毒力进行恢复,以便于下一步毒力的比较：我们利用在易感动物中连续传代以提高细菌毒力的方法,将Sf301野生株在易感动物豚鼠角膜内连续传代,以提高其毒力。经豚鼠角膜内的2次传代,5/301的毒力明显提高：接种细菌48h后出现脓性分泌物,72h后角膜表面混浊,呈灰白色,评分由“++”提高到“+++”。在此基础上,我们比较Sf301与Sf301毒力恢复株(Sf301Fv)的生物学特性。与Sf301相比,Sf301Fv的生长曲线有所改变：Sf301Fv在1-2h为延迟期,3-4h为对数生长早期,5-6h为对数生长中期,7-8h为对数生长晚期,9-12h为平台期。Sf301则在1-2h为对数生长早期,3-6h为对数生长中期,7-8h为对数生长晚期,9h-12h为平台期。通过HeLa细胞侵袭实验比较Sf301与Sf301Fv的毒力：与Sf301株相比,Sf301Fv对HeLa的侵袭力由5×10-6提高到2.6×10-3。进一步我们比较Sf301与Sf301Fv在对数生长早期、中期、晚期的毒力相关基因的转录水平,结果显示毒力相关基因的变化较明显：cpxA、icsA (virG)、ipaA、ipaB、ipaC、 mxiA、 phoR、 slyA、virB、virF、wzzE、ycfC转录水平上调明显；envZ、ipaD、ompC、ompF、rep转录水平下调。结果表明Sf301经易感豚鼠传代,毒力相关基因的表达发生了改变,所获得的5/301Fv毒力明显提高,可以用于研究phoP-phoQ对福氏志贺菌毒力影响。
在恢复了5/301野生株毒力的基础上,我们构建了福氏志贺菌phoP-phoQ敲除株。由于对5/301野生株进行毒力恢复后,Sf301的一些毒力基因发生变化,为了明确phoP-phoQ对福氏志贺菌毒力的影响,我们对Sf301及Sf301Fv分别进行phoP-phoQ的敲除。我们利用pSB890质粒构建phoP-phoQ敲除质粒：根据NCBI公布的Sf301的基因组序列(NC_004337),设计ΔphoP-phoQ的上下游同源臂。由于Sf301及Sf301Fv对pSB890携带的Tet抗性基因耐药,故需要在敲除质粒中加入抗性基因。在筛选了抗生素的基础上,我们首先构建了含有phoP-phoQ基因上下游片段(同源臂)的敲除质粒pSB89ΔAphoP-phoQ。在此基础上,将amp抗性基因插入质粒pSB89ΔAphoP-phoQ中,构建质粒pSBS9ΔAphoP-phoQ-amp。进一步通过两次同源重组得至phoP-phoQ敲除株。
在构建了Sf301ΔphoP-phoQ与Sf301Fv ΔphoP-phoQ的基础上,我们比较了Sf301、Sf301ΔphoP-phoQ、Sf301Fv及Sf301Fv ΔphoP-phoQ的生长情况：Sf301Fv与Sf301Fv ΔphoP-phoQ的生长曲线相似,在1-2h为延迟期,3-4h为对数生长早期,5-6h为对数生长中期,7-8h为对数生长晚期,9-12h为平台期；Sf301与Sf301ΔphoP-phoQ的生长曲线相似,均在1-2h为对数生长早期,3-6h为对数生长期中期,7-8h为对数生长晚期,9h-12h为平台期。在此基础上我们比较Sf301、Sf301ΔphoP-phoQ,Sf301Fv及Sf301Fv ΔphoP-phoQ的生化特性：与Sf301相比,Sf301ΔphoP-phoQ的ARA(阿拉伯糖)的反应由阳性变为阴性,同时Sf301Fv与Sf301Fv ΔphoP-phoQ也有相似的改变,其余反应Sf301与Sf301ΔphoP-phoQ, Sf301Fv与Sf301Fv ΔphoP-phoQ均相似。进一步我们比较两对敲除株的耐药性：与Sf301Fv相比,Sf301Fv ΔphoP-phoQ对萘啶酮酸及链霉素的耐药性提高,对头孢他啶、头孢噻肟的耐药性下降,这与Sf301与Sf301Δ Δphop-phoQ的改变相似。
在此基础上利用已建立的HeLa侵袭实验,分别比较Sf301与Sf301Δphop-phoQ及Sf301Fv与Sf301Fv ΔphoP-phoQ对HeLa细胞的侵袭力,结果以侵袭入细胞的细菌总数与侵袭细胞的细菌总数的比值来表示。结果显示,与Sf301Fv相比,Sf301Fv ΔphoP-phoQ对HeLa细胞的侵袭力明显下降,由2.6×10-3下降到4.9×10-5。这与Sf301与Sf301ΔphoP-phoQ改变的趋势一致。同时,我们通过豚鼠角结膜感染实验,观察豚鼠角结膜炎的严重程度,比较Sf301与Sf301ΔphoP-phoQ及Sf301Fv与Sf301Fv ΔphoP-phoQ对豚鼠角结膜的侵袭力。感染过程中分别观察24h、48h及72h豚鼠角结膜感染情况并评分。结果发现,与Sf301Fv相比,Sf301Fv ΔphoP-phoQ对豚鼠角膜的感染力明显下降,Sf301Fv ΔphoP-phoQ没有引起豚鼠的角膜炎症,评分由“+++”下降为“一”。这与Sf301株与Sf301ΔphoP-phoQ改变的趋势一致：与Sf301相比,Sf301ΔphoP-phoQ对豚鼠角膜的感染力明显下降,不能引起豚鼠的角结膜炎,而Sf301引起的豚鼠角结膜炎较严重,有脓性分泌物。
在观察了Sf301与Sf301ΔphoP-phoQ及Sf301Fv与Sf301Fv ΔphoP-phoQ的生物学特性和毒力变化后,我们分别比较Sf301与Sf301ΔphoP-phoQ及Sf301Fv与Sf301Fv ΔphoP-phoQ在对数生长早期、中期、晚期的毒力相关基因的转录水平。结果表明毒力相关基因的变化较明显,转录水平下调的基因有：icsA(virG)、ipaA、ipaB、ipaC、ipaD、mxiA、ompC、virB,转录水平没有明显变化的基因有：cpxA、ipaH、ompF、ompR、phoB、slyA、sodB、wzzE、ycfC。提示phoP-phoQ通过对一些毒力相关基因的调控来影响志贺菌的毒力。
Shigella is a Gram-negative bacterium responsible for the majority of bacillary dysentery, which can spread to mucous membrane of colon by the fecal-oral route. In China shigellosis is becoming the third infectious disease which only next to virus hepatitis and tuberculosis. The major serological type in China is flexneri2a. Because of the abuse of antibiotic and horizontal transmission of antibiotic resistance genes, Shigella is becoming multidrug resistance in clinic, especially resistance to ampicillin, sulfanilamide and tetracycline, even to carbostyril and cephalosporin, which are usually used in clinic now. New therapeutic targets and drugs are thus urgently needed to decrease the incidence of shigellosis worldwide. Inhibit bacteria virulence can bring theory evidence to treat shigellosis. The main pathopoiesis steps of Shigella are invade host cell, intercellular spread and release anatoxin. Its virulence are relate to its adhesion, invasiveness, enterotoxin, regulate protein and many other pathopoiesis factors. Among these factors, PhoP-PhoQ is important to regulate Shigella virulence, but its mechanism of regulating Shigella virulence is unknown which need in-depth study.
The aim of this study is to explore the mechanism of regulating virulence by two-component signal transduction systems (TCSTS) PhoP-PhoQ in Shigella flexneri. First, the effects of four PhoQ inhibitors which were found by former experiments were detected:the HeLa cell invasion assay and mouse sereny test were used to detect the effects of four PhoQ inhibitors on the virulence of Shigella flexneri. Real-time PCR was used to compare the transcription level of virulence genes of Shigella flexneri which inoculated with or without PhoQ inhibitors. Base on this, the technique of homologous recombination was used to construct the phoP-phoQ gene deletion mutant of Shigella flexneri301. The HeLa cell invasion test and guinea pig sereny test were used to detect the virulence of Sf301and Sf301ΔphoP-phoQ. Then Real-time PCR was used to compare the transcription level of virulence genes between Sf301 and ΔphoP-phoQ stain, investigate the mechanism of virulence which regulated by PhoP-PhoQ in Shigella.
Chapter1Bioinformatic analysis of PhoP-PhoQ two-component signal transduction systems
Because the PhoP-PhoQ TCSTS play an important role in regulating the virulence of Shigella, we analyzed this TCSTS in Shigella based on bioinformatic analysis. The homo logy of phoQ gene in Shigella flexneri2a str.301is more than99%compare to other six Shigella strains.In other Gram negative bacteria, the homology of phoQ is more than76%compare to Escherichia coli or Salmonella enterica subsp. enterica serovar Typhi. PhoQ in Gram negative bacteria is a sensory protein, contains486aa. It encodes transmembrane histidine protein kinases. The homology of amino acid sequence of PhoQ is more than99%in Shigella, more than85%in other Gram negative bacteria. The homology of phoP gene in Shigella flexneri2a str.301is more than98%compare to other six Shigella strains. In other Gram negative bacteria, the homology of phoP is more than80%compare to Escherichia coli or Salmonella enterica subsp. enterica serovar Typhi. In Gram negative bacteria PhoP is a response regulator protein, contains223aa, encodes intracellular response regulator protein. The homology of amino acid sequence of PhoP is more than98%, more than80%in other Gram negative bacteria, lower than32%in Gram positive bacteria.
Chapter2The effects of the PhoQ histidine kinase inhibitors on the virulence of Shigella flexneri
PhoQ can regulate the virulence of Shigella flexneri, four PhoQ inhibitors which targeting in PhoQ protein were found by former experiments. In this chapter, HeLa cell invasion test and mouse sereny test were used to detect the effects of PhoQ inhibitors on the virulence of Shigella flexneri. First we tested the effects of four PhoQ inhibitors on the growth of Shigella: Sf9380inoculated with PhoQ inhibitors (final concentration100μmol/L), and count CFU of Sf9380every1h, continue12h. The result suggested that four PhoQ inhibitors have no effects on the growth of Sf9380.
The HeLa cell invasion assay was used as a cellular model to evaluate the inhibitory effects of the four potential PhoQ inhibitors:The bacteria were treated with each of the four potential PhoQ inhibitors (100μmo/L) grown for4h,6h,8h. Then the bacteria were inoculated with HeLa cells for60min prior to the addition of gentamicin to kill extracellular bacteria. After the incubation, the Hela cells in each well were lysed in1ml of PBS containing0.1%Triton X-100. The results of the assays were expressed as the number of bacteria recovered from gentamicin-treated cells divided by the number of inoculated bacteria added to the cell. The colonies of lysates containing bacterial cells on LB plates were counted. Result indicated that the PhoQ inhibitors which co-culture with bacterial for4h have no effects on Shigella flexneri invation. PhoQ inhibitors which co-culture with bacterial for6h have no obvious effects on Shigella flexneri invation. PhoQ inhibitors which co-culture with bacterial for8h have obvious effects on Shigella flexneri invation. Based on these results, we used Sf9380which co-culture with bacterial for8h for this research, invitation time is60min. Results shown that the PhoQ inhibitor1(12.5μmo/L),2(25μmo/L),3(100μmo/L) could weaken the HeLa cell invasiveness of Sf9380(p<0.05) while inhibitor4has no effects on Shigella flexneri invation. To future test the PhoQ inhibitors effect on bacteria invation, we used Salmonella which has high homogeneity compare to Shigella flexneri. Result shown that inhibitor1(12.5μmo/L),2(25μmo/L),3(100μmo/L) could reduced invasion of HeLa cells by Salmonella, the invasion rate are0.89×10-2,1.564×10-2,0.036×10-2, respectively. But inhibitor4could not reduced invasion of HeLa cells by Salmonella, the invasion rate is5.7×10-2. The invasion rate of Salmonella is6×10-2.
The mouse keratoconjunctivitis model was used to evaluate the effects of these PhoQ inhibitors on the virulence of Shigella flexneri infection. Keratoconjunctivitis in the mice infected with bacteria was observed at24h,48h,72h and96h after inoculation. Result shown that inhibitor1(12.5μmo/L),2(25μmo/L) and inhibitor4(100μmol/L) could greatly reduce the keratoconjunctivitis in24h and48h infected by Sf9380and accelerate the recovery of the inflammation, after72h the inflammation were weaken obviously (P<0.05). Inhibitor3could reduce the inflammation in24h but could not release the inflammation in48h, and also could not accelerate the recovery of the inflammation (P>0.05).
Then we used Real-time PCR to test the PhoQ inhibitors effects on transcription level of Sf9380virulence genes. The results shown that the transpiration level of ipaA, ipaB, ipaC, ipaD, ipaH, mxiA,virB and virF in Sf9380which treated with inhibitor1(12.5μmol/L),2(25μmol/L) or3(100μmol/L) were down regulated. But the transpiration level of ipaA, ipaB, ipaC, ipaD, ipaH, mxiA, virB and virF in Sf9380which treated with inhibitor4(100μmol/L) were not down regulated. The results suggested that PhoQ inhibitors we obtained could affect the virulence of Shigella and PhoP-PhoQ TCSTS could regulate the virulence of Shigella.
Chapter3The effects of the phoP-phoQ on the virulence of Shigella flexneri
PhoP-PhoQ TCSTS is a major regulator of virulence in Gram-negative bacteria such as Salmonella and E.coli. It can mediate transcription and expression of some virulence genes. In the research of PhoQ inhibitors, we found that the PhoQ inhibitors could inhibit the invasion of Shigella flexneri infection. At the same time, we also found that the invasion rate of Sf301wide type were low. So the method of continuous passage in sensitive animals was used to recovery the virulence of Sf301wide type. With continuous passage in corneal of guinea pig, the virulence of Sf301were enhance obvious. After48h purulent secretion were found and after72h the corneal of guinea pig were cloudiness and become off-white besides purulence, score were increase from "++" to "+++". Base on this, the biology characteristics of Sf301and Sf301Full virulence stain (Sf301Fv) were detected. Compare to Sf301, the growth curve of Sf301Fv was changed:for Sf301Fv,3-4h was early stage of logarithmic growth phase,5-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9h-12h was plateau phase; for Sf301,1-2h was early stage of logarithmic growth phase;3-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9-12h was plateau phase. By using HeLa cell invasion test, it was found that invasion ability of the Sf301Fv was enhanced compared to Sf301, which from5×10-6to2.6×10-3. Real-time PCR was used to compare the transcription level of virulence genes in early stage, intermediate stage and late stage of logarithmic growth phase. The results shown that the transcription level of cpxA, icsA(virG), ipaA, ipaB, ipaC, mxiA, phoR, sly A, virB, virF, wzzE and ycfC were up-regulation and envZ, ipaD, ompC, ompF and rep were down regulation. It was suggested that by continuous passage in sensitive animals, Sf301could recovery its virulence by induce some virulence genes. The Sf301Fv we obtained in this chapter could be used to study the effects of phoP-phoQ on regulating virulence in Shigella flexneri.
To further verify the effects of PhoP-PhoQ TCSTS on regulating the Shigella flexneri virulence, the vecter pSB890was used to construct a phoP-phoQ knockout stain of Shigella flexneri. Because the transcription level of some virulence genes in Sf301Fv were changed, so sf301ΔphoP-phoQ and Sf301Fv ΔphoP-phoQ were constructed. Two homologous arms were designed base on the genome sequence of Sf301which were published by NCBI (NC_004337). Because sf301and sf301Fv are resistance to Tet which pSB890contains, so we should inserted an antibiotic resistant gene into vecter pSB890. After screen the antibiotic, we constructed plasmid pSB890ΔphoP-phoQ which contains two homologous arms of phoP-phoQ. Then an ampicillinum resistant gene(amp) was inserted into pSB890ΔphoP-phoQ and plasmid pSBS90ΔphoP-phoQ-amp was obtained. With conjugation and twice homologous recombinations, two phoP-phoQ knockout strains of Shigella flexneri were obtained. Base on the Sf301ΔphoP-phoQ and Sf301Fv ΔphoP-phoQ we obtained, the growth curve of Sf301,Sf301ΔphoP-phoQ,Sf301Fv and Sf301Fv ΔphoP-phoQ were detected. The growth curve of Sf301Fv and Sf301Fv ΔphoP-phoQ were similar:3-4h was early stage of logarithmic growth phase,5-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9h-12h was plateau phase. The growth curve of Sf301and Sf301ΔphoP-phoQ were similar:1-2h was early stage of logarithmic growth phase;3-6h was intermediate stage of logarithmic growth phase,7-8h was late stage of logarithmic growth phase and9-12h was plateau phase. Then biochemistry characteristic of these stains were tesed. Results shown that the ARA (Arabinose) of Sf301ΔphoP-phoQ was negative while Sf301was positive which was similar to the change between Sf301Fv and Sf301Fv ΔphoP-phoQ. The results of drug-resistance test were shown that, for Sf301Fv AphoP-phoQ, the sensibility of NAL and STR were increased compare to Sf301Fv, while sensibility of CAZ and CFT were decreased which were similar to the change between Sf301and Sf301ΔphoP-phoQ.
By using HeLa cell invasion test, the virulence of Sf301,Sf301ΔphoP-phoQ, Sf301Fv and Sf301Fv ΔphoP-phoQ were detected. The results of these assays were expressed as the number of bacteria recovered from gentamicin-treated cells divided by the number of inoculated bacteria added to the cell. It was found that invasion ability of the Sf301Fv ΔphoP-phoQ was decreased compared to Sf301Fv, from2.6×10-3to4.9×10-5, which was similar to the change between Sf301and Sf301ΔphoP-phoQ. Guinea pig sereny test was used to detected infectivity of Sf301,Sf301 AphoP-phoQ, Sf301Fv and Sf301Fv AphoP-phoQ. Keratoconjunctivitis in the guinea pig infected with bacteria was observed at24h,48h and72h after inoculation. The results of guinea pig sereny test shown that the infectivity of mutant was reduced compared to Sf301Fv, and could not cause inflammation in guinea pig. The score were decrease from "+++" to "-" which was similar to the change between Sf301and Sf301AphoP-phoQ. Real-time PCR was used to compare the transcription level of virulence genes between Sf301and Sf301AphoP-phoQ or Sf301Fv and Sf301Fv AphoP-phoQ in early stage, intermediate stage and late stage of logarithmic growth phase. The results shown that the transcription level of icsA(virG), ipaA, ipaB, ipaC, ipaD, mxiA, ompC and virB were down regulation and cpxA, ipaH, ompF, ompR,phoB, slyA, sod,B wzzE and ycfC were have no change. These results suggested that phoP-phoQ could regulate some virulence genes to affect the virulence of Shigella.
引文
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