聚磷酸盐激酶1在脑膜炎大肠杆菌K1株致病中的相关功能研究
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摘要
大肠杆菌(Escherichia coli, E. coli) K1株是引发新生儿脑膜炎最常见的革兰阴性杆菌。尽管抗生素和支持疗法已被广泛使用,但由于对大肠杆菌性脑膜炎的致病机制还有许多未知之处,致使其发病率和死亡率仍然居高不下。绝大多数大肠杆菌性的新生儿脑膜炎是血源性播散的结果,其发生的关键步骤为血液循环中的细菌穿越血脑屏障(blood-brain barrier, BBB)。而血脑屏障最主要的组成部分又是脑微血管内皮细胞(brain microvascular endothelial cells, BMECs)。大肠杆菌成功穿越血脑屏障需要达到几个条件,包括严重的菌血症,细菌黏附及侵袭进入脑微血管内皮细胞,宿主细胞的细胞骨架重排,以及相关信号途径的激活等。研究表明,大肠杆菌侵袭脑微血管内皮细胞呈现明显的时期差异性,即稳定生长期的细菌侵袭力要明显高于处于对数生长期的细菌。这也提示大肠杆菌K1株稳定生长期是其侵袭相关的毒力基因表达的主要时期。
聚磷酸盐激酶1(polyphosphate kinase1, PPK1)由聚磷酸盐激酶基因1编码,是大肠杆菌内负责催化ATP脱磷酸残基形成聚磷酸盐的一种主要激酶。多项研究证实,这种激酶或聚磷酸盐(polyphosphate, poly P)与多种细菌稳定生长期的毒力相关。E. coli K-12株缺失ppk1会导致其稳定生长期的压力适应性及存活能力下降。弯曲弧菌ppk1敲除后会表现出生长、运动及表面黏附能力的下降。绿脓杆菌敲除ppk1后会导致其群体感应和菌膜生成能力产生缺陷。志贺氏菌和沙门氏菌ppk1缺失后稳定生长期的存活能力和毒力明显降低。有学者发现,poly P与6因子RpoS的表达存在重要关系。ppk1缺失引发poly P合成减少,会导致RpoS的表达明显下降。RpoS是一种重要的稳定生长期毒力基因调控蛋白,它参与了包括大肠杆菌0157：H7、伤寒沙门氏菌、福氏志贺菌、结肠炎耶尔森氏菌、霍乱弧菌以及伯氏疏螺旋体在内的多种致病原的稳定生长期毒力表达调控。然而值得关注得是,E. coli K1 RS218,(由新生儿脑膜炎患者脑脊液中分离的E. coliK1株)的rpoS基因上却存在可导致调控功能沉默的基因突变。IbeR被证实是该细菌内的一个具有RpoS类似功能的调控蛋白,参与了稳定生长期包括毒力和压力适应性相关的多个基因的表达调控。借助生物信息学手段预测到,在ibeR基因上游的基因间隔区存在cAMP受体蛋白的结合位点。而有学者在伤寒沙门氏菌中发现ppk1参与了cAMP受体蛋白的表达调控,并由此参与了rpoS的表达调控。那么ppk1/poly P在E.coli K1入侵血脑屏障中扮演什么角色,以及ppk1缺失引起poly P的减少,是否会导致ibeR的表达下调,来参与细菌稳定期毒力及压力适应性的调控,都是我们希望探索的内容。围绕以上线索,本文主要开展以下几方面的研究：(1)利用染色体同源重组技术,构建E. coli K1 E44株(具有利福平抗性的RS218株)的ppk1基因缺失株及突变回补株,鉴定其基本属性,并利用glassmilk及甲苯胺蓝染色法提取、比较细菌内的poly P水平；(2)体外比较野生株、突变株及回补株在渗透压及酸性环境压力的条件下,三者间的生存率的差异；(3)利用人BMEC (human brain microvascular endothelial cells, HBMEC)单层细胞,体外比较野生株、突变株及回补株对HBMEC的黏附和侵袭水平。电镜观察黏附和进入HBMEC的细菌。利用血源性乳鼠脑膜炎模型,明确ppk1基因是否影响致病菌穿越血脑屏障的能力。同时利用激光共聚焦技术,比较三者诱导HBMEC肌动蛋白重排的能力；(4)利用荧光定量RT-PCR技术,比较分析野生株、突变株和回补株的ibeR及重要侵袭基因ibeA(与ibeR位于同一操纵子ibeRAT上)的mRNA表达水平。
方法与结果：
一、大肠杆菌K1致病株ppk1基因敲除及特性分析
1、为了研究ppk1基因在大肠杆菌致病中的毒力相关功能,构建ppk1基因敲除株PD44。根据E44染色体上的ppk1基因及其两侧的DNA序列,设计合成2对引物,以E44染色体为模板,用PCR分别扩增ppk1基因两端1.14 kb的A片段和1.28 kb的B片段,分别连接至pGEM-T easy载体上。利用二者带有的共同酶切位点将A、B连接起来形成AB片段。然后将AB片段亚克隆至自杀载体pCVD442,构建自杀重组质粒pCVDPK1,转化到SM10 Xpir,并通过接合性转导的方法将pCVDPK1从SM10λpir转化到E44中,通过氨苄敏感性及PCR方法筛选出ppk1基因缺失株。经过PCR、测序等结果证实成功构建E. coli K1的ppk1基因缺失株PD44。
2、K1株E44的ppk1基因全长2064 bp,编码687个氨基酸,BLAST表明与大肠杆菌K12株PPK1有99%氨基酸序列同源性,将带有完整ORF的ppk1基因序列插入质粒pGEM-T得到重组质粒pGEMPK1。用作回补实验用载体。
3、在营养丰富条件下,E44及PD44菌体的菌落形成和生长能力并无显著差异。因而突变株适用于研究该基因对感染相关毒力的影响。另外通过荚膜染色我们发现,ppk1缺失后细菌仍然可以形成荚膜,这对于细菌在HBMEC的存活非常重要。
4、我们考察了ppk1缺失对E44在培养3小时和18小时的poly P含量。利用glassmilk和甲苯胺蓝法检测,发现突变株的poly P含量明显低于野生株,而回补ppkl基因可能使其基本恢复到原来的水平。
二、ppk1缺失降低了大肠杆菌K1株对环境压力的适应能力
从生长环境以及感染途径来看,E. coli K1引发新生儿脑膜炎可能需要经受一些特殊环境的压力并生存下来。比如粪便内的高渗环境,新生儿胃部和产妇产道的酸性环境(人在通常状况下,胃液的pH值在1-3；阴道内pH值约为3.8-4.5)。为了检测ppk1/poly P在E. coli K1抵抗外界环境压力中的作用,我们通过比较ppk1缺失株、回补株以及野生株三者间在热刺激、高渗透压以及酸性培养基中的生存率情况。结果显示,敲除ppk1基因后,E. coli K1 E44在这些压力条件下的生存率均明显下降。这也表明ppk1Vpoly P在E. coli K1抵抗外界环境压力中扮演了重要的角色。
三、ppkl缺失降低了大肠杆菌K1株跨血脑屏障的能力
1、体外模型采用HBMEC单层细胞,实验前24 h,将BMEC铺至24孔板,待细胞汇合成单层后备用(约105细胞/孔)。待测细菌37℃静置培养24 h至稳定期,PBS适当稀释备用,以OD600估算菌浓,按感染倍数,即细菌数：细胞数约100：1接入待测菌,同时留样涂平板培养计数得确切菌浓。37℃孵育2h后,PBS清洗4次后裂解细胞并涂布LB平板计菌落数,此为黏附的细菌总数,并计算黏附率。侵袭组三个复孔,与100μg/mL庆大霉素37℃孵育1h来杀死所有胞外细菌,PBS清洗4次,裂解细胞涂布LB平板计菌落数,即为胞内菌数,并计算侵袭率。与野生株E441、2和3h的黏附率相比,PD44黏附BMEC单层细胞能力显著降低。庆大霉素保护实验结果表明,突变株在侵袭水平上也表现出明显的降低。PD44转化入回补质粒pGEMPK1后,突变株黏附能力和侵袭能力基本得到恢复。利用电子显微镜,可以观察到黏附、侵入HBMEC的突变株要少于野生株。
2、用罗丹明标记的鬼笔环肽对HBMEC进行染色,用共聚焦荧光显微镜观察发现：未孵育细菌的正常HBMEC组肌动蛋白分布均匀,野生株孵育组胞质肌动蛋白减少,出现边缘聚集以及突触,而突变株孵育组也出现类似情况,但改变程度明显弱于野生株组。回补ppkl基因可以使肌动蛋白重排水平恢复到E44组相当水平。
3、五日龄的乳鼠被接种E44及PD44,感染18 h后,血样及CSF样本涂布LB平板计菌落数。接种ppkl基因缺失株PD44可以引起与野生株E44相似水平的菌血症,然而,23只接种了PD44的乳鼠只有9只发生脑膜炎(39%),而同样数量接种了野生株E44的乳鼠当中,17只CSF培养呈阳性,占了74%(P=0.036, Pearson Chi-Square test)。联合体外及体内实验结果,ppkl基因缺失导致大肠杆菌K1株穿越血脑屏障的能力明显降低。
四、ppkl缺失降低了稳定期调节基因ibeR及重要侵袭相关基因ibeA的表达水平
为了检测ppkl敲除是否对稳定期调节基因ibeR表达是否有影响,我们通过实时荧光定量RT-PCR法比较了野生株、突变株及回补株之间的ibeR基因相对拷贝数。与内参基因GAPDH标化后,PD44 ibeR相对拷贝数为(13.35±2.11),明显低于E44的ibeR相对拷贝数(31.10±7.90)及回补株ibeR相对拷贝数(23.76±3.71)。IbeA mRNA荧光定量结果显示,经内参基因GAPDH标化后,PD44ibeA相对拷贝数为(32.12±7.33),明显低于E44的ibeA相对拷贝数(57.6±10.31)及回补株ibeA相对拷贝数(49.71±8.41)。以上结果表明ppkl敲除引发poly P产量减少,可导致稳定期调节基因ibeR及重要毒力基因ibeA的表达水平下调。
统计学分析：
数据均表示为均数±标准差.两组间的比较采用t检验,多重比较采用单因素方差分析。动物实验阳性率的比较采用Pearson卡方检验。P<0.05认为差异有统计学意义,检验水准为a=0.05。数据分析采用SPSS 13.0软件。
结论：
ppkl基因在多种微生物内包括结核分枝杆菌,脑膜炎奈瑟菌,幽门螺杆菌,霍乱弧菌,伤寒沙门氏菌等,都是一个高度保守的基因。由于在哺乳动物细胞内没有发现PPK1的同源物的存在,这就使得PPK1成为潜在的药物开发的一个良好靶标。为了明确poly P在大肠杆菌K1株致脑膜炎中的相关作用,我们构建了ppkl的基因敲除株,并通过体外、体内血脑屏障模型及基因回补实验对相关特性和功能进行了研究。结果显示,在体外实验中,ppkl的基因敲除株PD44对HBMEC的黏附和侵袭水平明显低于野生株E44及回补株PD44(pGEMPKl);与之一致的是,体内实验中PD44穿越血脑屏障的能力也低于E44。同时,我们还检测了三者之间的poly P含量,尤其在稳定期,E44的poly P积累水平明显高于PD44(非放射性法基本检测不到),而ppkl的回补可以使PD44的poly P基本回复到与E44相当的水平。有一点需要提出的是,敲除ppkl基因后,绿脓杆菌和弯曲弧菌依然存在poly P的累积现象,这是由于这两种细菌体内不仅存在ppkl,而且还存在着能使GTP脱磷产生poly P的ppk2。
在许多致病菌入侵宿主细胞时,都会造成宿主细胞骨架如肌动蛋白的重排及下游信号通路的激活。这种情况同样存在于脑膜炎大肠杆菌K1株。为了检测ppkl敲除是否会对E. coli K1诱导的细胞骨架重排产生影响,我们将野生株、突变株及回补株与HBMEC孵育后,用罗丹明-鬼笔环肽对肌动蛋白进行染色,发现在正常的HBMEC内,肌动蛋白排列非常均匀。而野生株及回补株感染的HBMEC,肌动蛋白在胞质内分布减少,而出现边缘聚集现象,且可以观察到突触的形成。突变株处理过的HBMEC虽然也存在肌动蛋白重排现象,但明显弱于前两组。
在E. coli K1引发脑膜炎的过程中,致病菌还需要面对一些环境的压力并存活下来,这也是其致病的机制之一。比如在人体的胃部,致病菌需要抵抗胃酸的刺激。由于产道感染也是新生儿脑膜炎的感染因素之一,因此E. coli K1需面对产妇产道内的低pH值环境。另外粪便内的高渗环境也是致病菌需要抵抗的环境压力。作为PPK1催化的酶反应产物,poly P被证实与稳定期调节子RpoS的表达相关。当敲除ppkl后,poly P减少,导致RpoS的表达下调。然而在E. coli K1 RS218, rpoS基因存在着导致其功能缺失的基因突变。IbeR为作为另外一个稳定期调节子参与了致病菌稳定期的压力适应性及毒力基因表达的调控。通过蛋白质组学分析发现,ibeR的缺失会导致TnaA、LpdAA、OmpC、TufB、GapA、OmpA、AhpC等蛋白的表达发生改变。这些蛋白都与E. coli K1的能量代谢或毒力存在着密切的关系。借助生物信息学的方法,黄胜和等还预测到在ibeR基因上游的基因间隔区有一个cAMP受体蛋白的潜在结合位点。在一项关于沙门氏菌的研究中已证实,ppk1可以通过cAMP受体蛋白对rpoS产生影响(其基因启动子区存在cAMP受体蛋白结合位点)。为了检测ppkl是否与E. coli K1的压力适应性调控有关系,及是否能通过影响稳定期调节基因ibeR来参与对毒力及压力适应性的调控,我们通过体外存活实验检测了在pH2.8及高渗透压的环境下,野生株、突变株及回补株的生存率。另外我们还通过real time RT-PCR法检测了ppk1敲除株、野生株及回补株的IbeR和IbeA的mRNA表达水平。通过上述实验我们发现,ppkl缺失、poly P合成减少,可引起E.coli K1 E44在压力环境下的生存率明显降低。而且可以导致IbeR和IbeA的mRNA表达水平下调。
综合以上结果我们可以得出如下结论：ppk1/poly P与脑膜炎大肠杆菌K1株致病有着重要的关系,并且可以通过影响稳定期调节基因ibeR及重要毒力基因ibeA的表达来参与相关功能的调控。但ppkl究竟是以什么样的机制来调控ibeR及ibeA,是否也如沙门氏菌的rpoS一样通过cAMP受体蛋白来影响ibeR的表达,将是我们后续研究中需要探索的问题。
Escherichia coli (E. coli) Kl is the most common gram-negative organism causing neonatal bacterial meningitis. Due to our incomplete knowledge about the pathogenesis of E. coli meningitis, this disease has remained high mortality and morbidity despite the advances in antimicrobial chemotherapy and supportive care. Most cases of neonatal E. coli meningitis develop as a result of hematogenous spread, and a critical step for the occurrence of E. coli meningitis is the circulating bacteria crossing blood-brain barrier (BBB), which is mainly composed of brain microvascular endothelial cells (BMECs). To across the BBB successfully for E. coli, it needs to meet some certain prerequisites including a high degree of bacteremia, binding to and invasion of HBMEC, host cell cytoskeleton rearrangement and related signal pathway activation.
E. coli K1 invasion of human BMEC (HBMEC) has been shown to be significantly higher in the stationary-phase (SP) than in the exponential-phase, suggesting that the SP is important for expression of E. coli Kl invasion-associated virulence genes. Polyphosphate (Poly P) kinasel (PPK1), which is the major enzyme for the synthesis of inorganic polyphosphate (poly P) from ATP and encoded by the ppkl gene, has been identified to be associated with stationary-phase survival in many bacteria species. A ppkl mutant of E. coli K-12 was deficient in responses to stresses and failed to survive in the stationary phase. The Vibrio cholerae ppkl mutants were defective in growth, motility, and surface attachment. Pseudomonas aeruginosa with ppkl knockout was aberrant in quorum sensing and biofilm formation. The ppkl mutants of Shigella and Salmonella spp. were insufficient in long-term survival and virulence. It has been shown that Poly P is associated with the expression of RpoS, the sigma factor responsible for regulating the expression of survival and many virulence genes during the stationary-phase (SP) in various bacteria, such as E. coli O157:H7, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica, Vibrio cholera, and Borrelia burgdorfer. It is worth noting, however, that a loss-of-function mutation was found in the SP regulatory gene rpoS of E. coli K1 strain E44, and IbeR has been identified by us as an RpoS-like regulator contributing to the expression of SP genes (including many virulence and stress adaption related genes) in E44.
Methods and results:
Because the role of ppkl/poly P in the pathogenesis of meningitic Escherichia coli K1, and the connection between the ppk1/poly P and SP regulation gene ibeR are both unkwon, an isogenic in-frame ppkl deletion mutant (PD44) of E. coli K1 strain has been made and characterized. The expression level of the important invasion gene ibeA, which locates in the same operon of ibeRAT with ibeR was also tested in this study.
1. Isogenic deletion of ppkl gene in E. coli K1 strain E44
In order to determine the role of the ppkl gene in the pathogenesis of E. coli meningitis, a ppkl isogenic in-frame deletion mutant was constructed. As shown in Fig 1A, two PCR DNA fragments (1.14 kb and 1.28 kb) flanking the ppkl coding region (2.06 kb) were obtained and ligated together into the suicide plasmid pCVD442 to produce the recombinant plasmid pCVDPKl. Then the plasmid pCVDPK1 was transformed into SM10γpir, and the ppkl deletion mutant was obtained by mating E44 with SM10γpir carrying pCVDPKl as described in Materials and Methods. The deletion of the ppkl gene was confirmed by colony PCR and DNA sequencing.
2. Examination of poly P levels
The growth profiles of E44, PD44 and the ppkl complemented strain in rich medium were identical (data not shown).To examine the poly P levels in the cells, poly P was extrcated with glassmilk and quantitated by using the Toluidine Blue O dye as described in Materials and Methods.
The poly P levels in PD44 were singnifcantly lower than those of the E44 and the ppkl complemented strain at both time point of 3h and 18h. Especially in stationary phase, the poly P level in the E44 and the ppkl complemented strain amounted to 13.3 and 12.6 nmol poly P/mg of total celluar protein respectively. However, the PD44 exhibited barely detectable level of poly P (<1.5 nmol poly P/ mg of total celluar protein). These results show that the ppkl gene plays an important role in the poly P production of E. coli E44.
3. Survival abilities of E44 and PD44 under hypertonic or acid stress condition
To test survival under osmotic stress, bacteria were exposed to 2.5 M NaCl for 2 hours and plated on LB agar plates to determine the survival rates. After 2 hours, the survival rates of E44, PD44 and PD44 carrying ppk1 was 39.62%,9.84% and 38.37% respectively. For acid stress assay, overnight cultures were collected and suspended in acidic LB (pH 2.8) for 60 minutes. Similar with the results of hypertonic stress assay, the ppkl mutant strain PD44 was significantly defective in survival in the acidic LB when compared to E44 and PD44 carrying ppkl. After 1 hour under this condition, the survival rate of PD44 was only about 1/6 of E44 and the ppkl complemented strain. These results suggest that poly P is required for stress tolerance of E44 in stationary phase.
4. Bacteria adhesion and invasion of HBMEC
The in vitro adhesion and invasion assay were performed as described in Materials and Methods. Because the adhesion of E. coli to the HBMEC is the initial step of bacterial crossing of the BBB during the development of meningitis, the effect of ppkl deletion in E. coli E44 adhesion of HBMEC was examined. The HBMEC were incubated with E44 and PD44 for 1,2,3 hours. After three times of washing, the bacteria adhering to HBMEC were enumerated and the adhesion rates were calculated. The adhesion rates of E44 at 1,2, and 3h were 3.97%,9.80% and 26.80% respectively. However, the adhesion rates of PD44 reduced to 1.73%,3.87% and 9.23%. In order to test whether the complementation of ppkl can restore the adhesion phenotype, E44 carrying the plasmid pGEM-T, PD44 carrying either pGEM-T or pGEMPKl that contains the complete coding region of ppkl were incubated with the HBMEC monolayers for 2 h, and the adhesion rates were analyzed. The adhesion rates of PD44 carrying pGEM-T and E44 carrying pGEM-T were 3.20% and 8.43% respectively. The adhesion rate of PD44 complemented wit ppk1was 7.30% which raised to about 90% when compared to that of the parent strain E44. Similarly, the ppkl deletion led to a significant reduction of invasion ability for E44. Following the initial adhesion to HBMEC, part of E. coli will be internalized druing the E. coli accrosing the BBB. And this can be analyzed with the invasion assay as described previously. The in vitro invasion results showed that the invasion rates of PD44 (0.041%) and ZD1 (0.029%) were significantly lower than that of their parent strain E44 (0.137%), and the plasmid pGEMPK1 containing the ppkl gene was able to complement the less invasive phenotype of PD44.
5. Cytoskeleton changes in HBMEC induced by the bacteria
After treatment with bacteria, the HBMEC were stained with Rhodamine-labelled phalloidin, which makes the the organization of actin be easily and clearly observed under a confocal fluorescence microscope. In the untreated group, the actin filament evenly distributed throughout the internal compartment of the cytoplasm. In the groups incubated with E44 and PD44 (pGEMPK1), the actin filament in the cytoplasm reduced obviously, and it could be observed a denser actin aggregates in the margin of the cells. A long protrusion of actin filament staining in HBMEC also can often be observed in these two groups. In the cells treated with PD44, a less internal reduction and marginal aggregates of actin filament than that of the other two groups treated with E. coli was observed.
6. Neonatal rat model of hematogenous E. coli K1 meningitis
The in vitro experiments demonstrated that the ppkl deletion mutant PD44 was less adherent and invasive than its parent strain E44 in the HBMEC cell culture model. To further investigate the role of ppkl on the development of neonatal meningitis, the infant rat model of experimental hematogenesis meningitis was used to determine the in vivo virulence phenotype. The blood and CSF specimen were cultured for indication of bacteremia and meningitis, respectively. The magnitude of bacteremia induced by the mutant strain was similar to that of the wild-type strain. However, the occurrence of meningitis (defined as positive CSF cultures) in animals receiving PD44 (9 of 23, or 39%), was significantly lower (P< 0.05) than in those receiving the parent strain E44 (17 of 23, or 74%). Our in vitro and in vivo studies demonstrated that the ppkl gene was required for E. coli E44 penetration across the BBB in vitro and in vivo.
7. The mRNA expression levels of IbeR and IbeA in E. coli E44 and PD44
In order to determine whether loss of ppkl modulated the stationary phase regulator IbeR and the major virulence factor IbeA in E. coli E44, real time RT-PCR was used to detect the mRNA expression levels of IbeR and IbeA as described above. After normalization with the copy number of the GAPDH gene, the relative copy number of ibeR in PD44 (13.35±2.11) was significantly less than that in E44 (31.10±7.90), and the relative copy number of ibeR in the ppkl complemented strain raised to 23.76±3.71. The relative copy number of ibeA in PD44 (32.12±7.33) was also less than that in E44(57.60±10.31) and the ppkl complemented strain(49.71±8.41). The result suggested that the ppkl deletion led to a decline expression of ibeR and ibeA in E. coli K1 E44.
Statistical analysis
All values are expressed as mean±standard deviation. Statistical analysis was performed with Student's t-test for comparison of two groups, and with ANOVA for multiple comparisons. In vivo experiments data were analyzed by Pearson Chi-Square test. Differences with P<0.05 were considered to be statistically significant.
Conclusion
The ppkl gene is a highly conserved gene among many bacterial species, including bacterial pathogens, Mycobacterium tuberculosis, Neisseria meningitides, Helicobacter pylori, Vibrio cholerae, Salmonella typhimurium, etc.. Due to the absence of the PPK1 homologue in mammalian cells, PPK1 exhibits potential to be an attractive target for chemotherapy. In order to determine the role of poly P in the pathogenesis of E. coli Kl meningitis, we constructed the ppkl deletion mutants to test its biological functions using the in vitro and in vivo models of the BBB and genetic complementation. We showed that the mutant PD44 was considerably less adherent and invasive for HBMEC, and significantly less virulent in the entry into the CNS in the newborn rat model of experimental hematogenous meningitis. The less adherent and invasive phenotype of the PD44 mutant were complemented by the ppkl gene. Meanwhile, the poly P level of the PD44 in stationary phase was also found to be obviously lower than that of the parent strain E44 and the ppkl complemented strain by using the nonradioactive method. It should also be noticed that, the phenomenon of poly P accumulation still exists in ppkl mutant of P. aeruginosa and Campylobacter jejuni, which may be due to the harbouring of not only ppkl but also ppk2 mediating poly P-driven generation of GTP in these orgnisms.
Remodelling of the host-cell actin cytoskeleton has been observed during pathogenic invasion. The E. coli adhesion and invasion of the HBMEC leading to cytoskeletal reorganization and downstrem signal activiation is important for the bacteria to accross the HBMEC. To test the cytoskeletal reorganization in the HBMEC induced by ppkl deletion mutant and the wild-type strain, the rhodamine-labelled phalloidin was used to make the rearrangement of actin filaments induced by E. coli visible under the confocal fluorescence microscope. Our findings showed that the actin filaments rearrangement in the HBMEC induced by PD44 was significantly weaker than that induced by the parent strain E44 and the ppkl complemented strain.
During the development process of neonatal E. coli meningitis, it's an important pathogenesis for the pathogen to endure the hyperosmotic of fecal in intestine and acid stress in the host's stomach or vagina of puerpera. As a product of the reaction catalyzed by the PPK1, poly P has been reported to be associated with the expression of the SP regulator RpoS, which governs expression of over 50 genes and plays a central role on stationary-phase adaptations in most E. coli strains. The adaptation processes modulated by RpoS is important for the bacteria to survival in kinds of environments. However, the SP regulation in meningitic E. coli K1 carrying the ibeR gene was drastically different from that in many other E. coli strains. There is a nonsense mutation in the rpoS gene in E. coli K1 strain E44, which can keep its stress resistance independent of RpoS. The phenomenon of rpoS mutation is not unique to E. coli E44, but also exists in some other enteric pathogens. We have demonstrated that IbeR is an RpoS-like regulator contributing to the E. coli E44 entry into HBMEC and stress tolerance in the stationary-phase. Some proteins which are related to environmental modifications or central metabolism can be regulated by IbeR in E44, such as TnaA, LpdA, OmpC, TufB, GapA, OmpA, AhpC.
As the product of PPK1, poly P, plays an important role in the RpoS induction in other E. coli strains, we further examined whether PPK1 contributed to regulation of IbeR expression and stress tolerance in E44. The ppkl deletion mutant PD44 was tested for its survival rates under stress conditions in comparison with the parent strain E44. The survival rates were significantly reduced after the ppk1 mutant receiving stress treatments compared to that of E44. These results suggest that ppk1/poly P is important for the E. coli E44 to survival in stress environments. Real time RT-PCR was used to test the mRNA expression of IbeR. The expression levels of the major virulence gene ibeA in PD44 and E44 were also compared. The results showed that the IbeR and IbeA expression levels of ppk1 mutant were both lower than those of the parent strain E44, and the complementation of ppk1 gene could significantly improve the mRNA levels of IbeR and IbeA in PD44.
Taken together, our findings suggest that the ppk1/poly P plays an important role in stress resistance and virulence in E. coli K1 E44. And the ppk1 deletion will result in a significant decline expression of the SP-regulator IbeR and important virulence factor IbeA, which is crucial for the E44 to survival in stress environment and cross the blood brain barrier.
聚磷酸盐激酶1(polyphosphate kinase1, PPK1)由聚磷酸盐激酶基因1编码,是大肠杆菌内负责催化ATP脱磷酸残基形成聚磷酸盐的一种主要激酶。多项研究证实,这种激酶或聚磷酸盐(polyphosphate, poly P)与多种细菌稳定生长期的毒力相关。E. coli K-12株缺失ppk1会导致其稳定生长期的压力适应性及存活能力下降。弯曲弧菌ppk1敲除后会表现出生长、运动及表面黏附能力的下降。绿脓杆菌敲除ppk1后会导致其群体感应和菌膜生成能力产生缺陷。志贺氏菌和沙门氏菌ppk1缺失后稳定生长期的存活能力和毒力明显降低。有学者发现,poly P与6因子RpoS的表达存在重要关系。ppk1缺失引发poly P合成减少,会导致RpoS的表达明显下降。RpoS是一种重要的稳定生长期毒力基因调控蛋白,它参与了包括大肠杆菌0157：H7、伤寒沙门氏菌、福氏志贺菌、结肠炎耶尔森氏菌、霍乱弧菌以及伯氏疏螺旋体在内的多种致病原的稳定生长期毒力表达调控。然而值得关注得是,E. coli K1 RS218,(由新生儿脑膜炎患者脑脊液中分离的E. coliK1株)的rpoS基因上却存在可导致调控功能沉默的基因突变。IbeR被证实是该细菌内的一个具有RpoS类似功能的调控蛋白,参与了稳定生长期包括毒力和压力适应性相关的多个基因的表达调控。借助生物信息学手段预测到,在ibeR基因上游的基因间隔区存在cAMP受体蛋白的结合位点。而有学者在伤寒沙门氏菌中发现ppk1参与了cAMP受体蛋白的表达调控,并由此参与了rpoS的表达调控。那么ppk1/poly P在E.coli K1入侵血脑屏障中扮演什么角色,以及ppk1缺失引起poly P的减少,是否会导致ibeR的表达下调,来参与细菌稳定期毒力及压力适应性的调控,都是我们希望探索的内容。围绕以上线索,本文主要开展以下几方面的研究：(1)利用染色体同源重组技术,构建E. coli K1 E44株(具有利福平抗性的RS218株)的ppk1基因缺失株及突变回补株,鉴定其基本属性,并利用glassmilk及甲苯胺蓝染色法提取、比较细菌内的poly P水平；(2)体外比较野生株、突变株及回补株在渗透压及酸性环境压力的条件下,三者间的生存率的差异；(3)利用人BMEC (human brain microvascular endothelial cells, HBMEC)单层细胞,体外比较野生株、突变株及回补株对HBMEC的黏附和侵袭水平。电镜观察黏附和进入HBMEC的细菌。利用血源性乳鼠脑膜炎模型,明确ppk1基因是否影响致病菌穿越血脑屏障的能力。同时利用激光共聚焦技术,比较三者诱导HBMEC肌动蛋白重排的能力；(4)利用荧光定量RT-PCR技术,比较分析野生株、突变株和回补株的ibeR及重要侵袭基因ibeA(与ibeR位于同一操纵子ibeRAT上)的mRNA表达水平。
方法与结果：
一、大肠杆菌K1致病株ppk1基因敲除及特性分析
1、为了研究ppk1基因在大肠杆菌致病中的毒力相关功能,构建ppk1基因敲除株PD44。根据E44染色体上的ppk1基因及其两侧的DNA序列,设计合成2对引物,以E44染色体为模板,用PCR分别扩增ppk1基因两端1.14 kb的A片段和1.28 kb的B片段,分别连接至pGEM-T easy载体上。利用二者带有的共同酶切位点将A、B连接起来形成AB片段。然后将AB片段亚克隆至自杀载体pCVD442,构建自杀重组质粒pCVDPK1,转化到SM10 Xpir,并通过接合性转导的方法将pCVDPK1从SM10λpir转化到E44中,通过氨苄敏感性及PCR方法筛选出ppk1基因缺失株。经过PCR、测序等结果证实成功构建E. coli K1的ppk1基因缺失株PD44。
2、K1株E44的ppk1基因全长2064 bp,编码687个氨基酸,BLAST表明与大肠杆菌K12株PPK1有99%氨基酸序列同源性,将带有完整ORF的ppk1基因序列插入质粒pGEM-T得到重组质粒pGEMPK1。用作回补实验用载体。
3、在营养丰富条件下,E44及PD44菌体的菌落形成和生长能力并无显著差异。因而突变株适用于研究该基因对感染相关毒力的影响。另外通过荚膜染色我们发现,ppk1缺失后细菌仍然可以形成荚膜,这对于细菌在HBMEC的存活非常重要。
4、我们考察了ppk1缺失对E44在培养3小时和18小时的poly P含量。利用glassmilk和甲苯胺蓝法检测,发现突变株的poly P含量明显低于野生株,而回补ppkl基因可能使其基本恢复到原来的水平。
二、ppk1缺失降低了大肠杆菌K1株对环境压力的适应能力
从生长环境以及感染途径来看,E. coli K1引发新生儿脑膜炎可能需要经受一些特殊环境的压力并生存下来。比如粪便内的高渗环境,新生儿胃部和产妇产道的酸性环境(人在通常状况下,胃液的pH值在1-3；阴道内pH值约为3.8-4.5)。为了检测ppk1/poly P在E. coli K1抵抗外界环境压力中的作用,我们通过比较ppk1缺失株、回补株以及野生株三者间在热刺激、高渗透压以及酸性培养基中的生存率情况。结果显示,敲除ppk1基因后,E. coli K1 E44在这些压力条件下的生存率均明显下降。这也表明ppk1Vpoly P在E. coli K1抵抗外界环境压力中扮演了重要的角色。
三、ppkl缺失降低了大肠杆菌K1株跨血脑屏障的能力
1、体外模型采用HBMEC单层细胞,实验前24 h,将BMEC铺至24孔板,待细胞汇合成单层后备用(约105细胞/孔)。待测细菌37℃静置培养24 h至稳定期,PBS适当稀释备用,以OD600估算菌浓,按感染倍数,即细菌数：细胞数约100：1接入待测菌,同时留样涂平板培养计数得确切菌浓。37℃孵育2h后,PBS清洗4次后裂解细胞并涂布LB平板计菌落数,此为黏附的细菌总数,并计算黏附率。侵袭组三个复孔,与100μg/mL庆大霉素37℃孵育1h来杀死所有胞外细菌,PBS清洗4次,裂解细胞涂布LB平板计菌落数,即为胞内菌数,并计算侵袭率。与野生株E441、2和3h的黏附率相比,PD44黏附BMEC单层细胞能力显著降低。庆大霉素保护实验结果表明,突变株在侵袭水平上也表现出明显的降低。PD44转化入回补质粒pGEMPK1后,突变株黏附能力和侵袭能力基本得到恢复。利用电子显微镜,可以观察到黏附、侵入HBMEC的突变株要少于野生株。
2、用罗丹明标记的鬼笔环肽对HBMEC进行染色,用共聚焦荧光显微镜观察发现：未孵育细菌的正常HBMEC组肌动蛋白分布均匀,野生株孵育组胞质肌动蛋白减少,出现边缘聚集以及突触,而突变株孵育组也出现类似情况,但改变程度明显弱于野生株组。回补ppkl基因可以使肌动蛋白重排水平恢复到E44组相当水平。
3、五日龄的乳鼠被接种E44及PD44,感染18 h后,血样及CSF样本涂布LB平板计菌落数。接种ppkl基因缺失株PD44可以引起与野生株E44相似水平的菌血症,然而,23只接种了PD44的乳鼠只有9只发生脑膜炎(39%),而同样数量接种了野生株E44的乳鼠当中,17只CSF培养呈阳性,占了74%(P=0.036, Pearson Chi-Square test)。联合体外及体内实验结果,ppkl基因缺失导致大肠杆菌K1株穿越血脑屏障的能力明显降低。
四、ppkl缺失降低了稳定期调节基因ibeR及重要侵袭相关基因ibeA的表达水平
为了检测ppkl敲除是否对稳定期调节基因ibeR表达是否有影响,我们通过实时荧光定量RT-PCR法比较了野生株、突变株及回补株之间的ibeR基因相对拷贝数。与内参基因GAPDH标化后,PD44 ibeR相对拷贝数为(13.35±2.11),明显低于E44的ibeR相对拷贝数(31.10±7.90)及回补株ibeR相对拷贝数(23.76±3.71)。IbeA mRNA荧光定量结果显示,经内参基因GAPDH标化后,PD44ibeA相对拷贝数为(32.12±7.33),明显低于E44的ibeA相对拷贝数(57.6±10.31)及回补株ibeA相对拷贝数(49.71±8.41)。以上结果表明ppkl敲除引发poly P产量减少,可导致稳定期调节基因ibeR及重要毒力基因ibeA的表达水平下调。
统计学分析：
数据均表示为均数±标准差.两组间的比较采用t检验,多重比较采用单因素方差分析。动物实验阳性率的比较采用Pearson卡方检验。P<0.05认为差异有统计学意义,检验水准为a=0.05。数据分析采用SPSS 13.0软件。
结论：
ppkl基因在多种微生物内包括结核分枝杆菌,脑膜炎奈瑟菌,幽门螺杆菌,霍乱弧菌,伤寒沙门氏菌等,都是一个高度保守的基因。由于在哺乳动物细胞内没有发现PPK1的同源物的存在,这就使得PPK1成为潜在的药物开发的一个良好靶标。为了明确poly P在大肠杆菌K1株致脑膜炎中的相关作用,我们构建了ppkl的基因敲除株,并通过体外、体内血脑屏障模型及基因回补实验对相关特性和功能进行了研究。结果显示,在体外实验中,ppkl的基因敲除株PD44对HBMEC的黏附和侵袭水平明显低于野生株E44及回补株PD44(pGEMPKl);与之一致的是,体内实验中PD44穿越血脑屏障的能力也低于E44。同时,我们还检测了三者之间的poly P含量,尤其在稳定期,E44的poly P积累水平明显高于PD44(非放射性法基本检测不到),而ppkl的回补可以使PD44的poly P基本回复到与E44相当的水平。有一点需要提出的是,敲除ppkl基因后,绿脓杆菌和弯曲弧菌依然存在poly P的累积现象,这是由于这两种细菌体内不仅存在ppkl,而且还存在着能使GTP脱磷产生poly P的ppk2。
在许多致病菌入侵宿主细胞时,都会造成宿主细胞骨架如肌动蛋白的重排及下游信号通路的激活。这种情况同样存在于脑膜炎大肠杆菌K1株。为了检测ppkl敲除是否会对E. coli K1诱导的细胞骨架重排产生影响,我们将野生株、突变株及回补株与HBMEC孵育后,用罗丹明-鬼笔环肽对肌动蛋白进行染色,发现在正常的HBMEC内,肌动蛋白排列非常均匀。而野生株及回补株感染的HBMEC,肌动蛋白在胞质内分布减少,而出现边缘聚集现象,且可以观察到突触的形成。突变株处理过的HBMEC虽然也存在肌动蛋白重排现象,但明显弱于前两组。
在E. coli K1引发脑膜炎的过程中,致病菌还需要面对一些环境的压力并存活下来,这也是其致病的机制之一。比如在人体的胃部,致病菌需要抵抗胃酸的刺激。由于产道感染也是新生儿脑膜炎的感染因素之一,因此E. coli K1需面对产妇产道内的低pH值环境。另外粪便内的高渗环境也是致病菌需要抵抗的环境压力。作为PPK1催化的酶反应产物,poly P被证实与稳定期调节子RpoS的表达相关。当敲除ppkl后,poly P减少,导致RpoS的表达下调。然而在E. coli K1 RS218, rpoS基因存在着导致其功能缺失的基因突变。IbeR为作为另外一个稳定期调节子参与了致病菌稳定期的压力适应性及毒力基因表达的调控。通过蛋白质组学分析发现,ibeR的缺失会导致TnaA、LpdAA、OmpC、TufB、GapA、OmpA、AhpC等蛋白的表达发生改变。这些蛋白都与E. coli K1的能量代谢或毒力存在着密切的关系。借助生物信息学的方法,黄胜和等还预测到在ibeR基因上游的基因间隔区有一个cAMP受体蛋白的潜在结合位点。在一项关于沙门氏菌的研究中已证实,ppk1可以通过cAMP受体蛋白对rpoS产生影响(其基因启动子区存在cAMP受体蛋白结合位点)。为了检测ppkl是否与E. coli K1的压力适应性调控有关系,及是否能通过影响稳定期调节基因ibeR来参与对毒力及压力适应性的调控,我们通过体外存活实验检测了在pH2.8及高渗透压的环境下,野生株、突变株及回补株的生存率。另外我们还通过real time RT-PCR法检测了ppk1敲除株、野生株及回补株的IbeR和IbeA的mRNA表达水平。通过上述实验我们发现,ppkl缺失、poly P合成减少,可引起E.coli K1 E44在压力环境下的生存率明显降低。而且可以导致IbeR和IbeA的mRNA表达水平下调。
综合以上结果我们可以得出如下结论：ppk1/poly P与脑膜炎大肠杆菌K1株致病有着重要的关系,并且可以通过影响稳定期调节基因ibeR及重要毒力基因ibeA的表达来参与相关功能的调控。但ppkl究竟是以什么样的机制来调控ibeR及ibeA,是否也如沙门氏菌的rpoS一样通过cAMP受体蛋白来影响ibeR的表达,将是我们后续研究中需要探索的问题。
Escherichia coli (E. coli) Kl is the most common gram-negative organism causing neonatal bacterial meningitis. Due to our incomplete knowledge about the pathogenesis of E. coli meningitis, this disease has remained high mortality and morbidity despite the advances in antimicrobial chemotherapy and supportive care. Most cases of neonatal E. coli meningitis develop as a result of hematogenous spread, and a critical step for the occurrence of E. coli meningitis is the circulating bacteria crossing blood-brain barrier (BBB), which is mainly composed of brain microvascular endothelial cells (BMECs). To across the BBB successfully for E. coli, it needs to meet some certain prerequisites including a high degree of bacteremia, binding to and invasion of HBMEC, host cell cytoskeleton rearrangement and related signal pathway activation.
E. coli K1 invasion of human BMEC (HBMEC) has been shown to be significantly higher in the stationary-phase (SP) than in the exponential-phase, suggesting that the SP is important for expression of E. coli Kl invasion-associated virulence genes. Polyphosphate (Poly P) kinasel (PPK1), which is the major enzyme for the synthesis of inorganic polyphosphate (poly P) from ATP and encoded by the ppkl gene, has been identified to be associated with stationary-phase survival in many bacteria species. A ppkl mutant of E. coli K-12 was deficient in responses to stresses and failed to survive in the stationary phase. The Vibrio cholerae ppkl mutants were defective in growth, motility, and surface attachment. Pseudomonas aeruginosa with ppkl knockout was aberrant in quorum sensing and biofilm formation. The ppkl mutants of Shigella and Salmonella spp. were insufficient in long-term survival and virulence. It has been shown that Poly P is associated with the expression of RpoS, the sigma factor responsible for regulating the expression of survival and many virulence genes during the stationary-phase (SP) in various bacteria, such as E. coli O157:H7, Salmonella typhimurium, Shigella flexneri, Yersinia enterocolitica, Vibrio cholera, and Borrelia burgdorfer. It is worth noting, however, that a loss-of-function mutation was found in the SP regulatory gene rpoS of E. coli K1 strain E44, and IbeR has been identified by us as an RpoS-like regulator contributing to the expression of SP genes (including many virulence and stress adaption related genes) in E44.
Methods and results:
Because the role of ppkl/poly P in the pathogenesis of meningitic Escherichia coli K1, and the connection between the ppk1/poly P and SP regulation gene ibeR are both unkwon, an isogenic in-frame ppkl deletion mutant (PD44) of E. coli K1 strain has been made and characterized. The expression level of the important invasion gene ibeA, which locates in the same operon of ibeRAT with ibeR was also tested in this study.
1. Isogenic deletion of ppkl gene in E. coli K1 strain E44
In order to determine the role of the ppkl gene in the pathogenesis of E. coli meningitis, a ppkl isogenic in-frame deletion mutant was constructed. As shown in Fig 1A, two PCR DNA fragments (1.14 kb and 1.28 kb) flanking the ppkl coding region (2.06 kb) were obtained and ligated together into the suicide plasmid pCVD442 to produce the recombinant plasmid pCVDPKl. Then the plasmid pCVDPK1 was transformed into SM10γpir, and the ppkl deletion mutant was obtained by mating E44 with SM10γpir carrying pCVDPKl as described in Materials and Methods. The deletion of the ppkl gene was confirmed by colony PCR and DNA sequencing.
2. Examination of poly P levels
The growth profiles of E44, PD44 and the ppkl complemented strain in rich medium were identical (data not shown).To examine the poly P levels in the cells, poly P was extrcated with glassmilk and quantitated by using the Toluidine Blue O dye as described in Materials and Methods.
The poly P levels in PD44 were singnifcantly lower than those of the E44 and the ppkl complemented strain at both time point of 3h and 18h. Especially in stationary phase, the poly P level in the E44 and the ppkl complemented strain amounted to 13.3 and 12.6 nmol poly P/mg of total celluar protein respectively. However, the PD44 exhibited barely detectable level of poly P (<1.5 nmol poly P/ mg of total celluar protein). These results show that the ppkl gene plays an important role in the poly P production of E. coli E44.
3. Survival abilities of E44 and PD44 under hypertonic or acid stress condition
To test survival under osmotic stress, bacteria were exposed to 2.5 M NaCl for 2 hours and plated on LB agar plates to determine the survival rates. After 2 hours, the survival rates of E44, PD44 and PD44 carrying ppk1 was 39.62%,9.84% and 38.37% respectively. For acid stress assay, overnight cultures were collected and suspended in acidic LB (pH 2.8) for 60 minutes. Similar with the results of hypertonic stress assay, the ppkl mutant strain PD44 was significantly defective in survival in the acidic LB when compared to E44 and PD44 carrying ppkl. After 1 hour under this condition, the survival rate of PD44 was only about 1/6 of E44 and the ppkl complemented strain. These results suggest that poly P is required for stress tolerance of E44 in stationary phase.
4. Bacteria adhesion and invasion of HBMEC
The in vitro adhesion and invasion assay were performed as described in Materials and Methods. Because the adhesion of E. coli to the HBMEC is the initial step of bacterial crossing of the BBB during the development of meningitis, the effect of ppkl deletion in E. coli E44 adhesion of HBMEC was examined. The HBMEC were incubated with E44 and PD44 for 1,2,3 hours. After three times of washing, the bacteria adhering to HBMEC were enumerated and the adhesion rates were calculated. The adhesion rates of E44 at 1,2, and 3h were 3.97%,9.80% and 26.80% respectively. However, the adhesion rates of PD44 reduced to 1.73%,3.87% and 9.23%. In order to test whether the complementation of ppkl can restore the adhesion phenotype, E44 carrying the plasmid pGEM-T, PD44 carrying either pGEM-T or pGEMPKl that contains the complete coding region of ppkl were incubated with the HBMEC monolayers for 2 h, and the adhesion rates were analyzed. The adhesion rates of PD44 carrying pGEM-T and E44 carrying pGEM-T were 3.20% and 8.43% respectively. The adhesion rate of PD44 complemented wit ppk1was 7.30% which raised to about 90% when compared to that of the parent strain E44. Similarly, the ppkl deletion led to a significant reduction of invasion ability for E44. Following the initial adhesion to HBMEC, part of E. coli will be internalized druing the E. coli accrosing the BBB. And this can be analyzed with the invasion assay as described previously. The in vitro invasion results showed that the invasion rates of PD44 (0.041%) and ZD1 (0.029%) were significantly lower than that of their parent strain E44 (0.137%), and the plasmid pGEMPK1 containing the ppkl gene was able to complement the less invasive phenotype of PD44.
5. Cytoskeleton changes in HBMEC induced by the bacteria
After treatment with bacteria, the HBMEC were stained with Rhodamine-labelled phalloidin, which makes the the organization of actin be easily and clearly observed under a confocal fluorescence microscope. In the untreated group, the actin filament evenly distributed throughout the internal compartment of the cytoplasm. In the groups incubated with E44 and PD44 (pGEMPK1), the actin filament in the cytoplasm reduced obviously, and it could be observed a denser actin aggregates in the margin of the cells. A long protrusion of actin filament staining in HBMEC also can often be observed in these two groups. In the cells treated with PD44, a less internal reduction and marginal aggregates of actin filament than that of the other two groups treated with E. coli was observed.
6. Neonatal rat model of hematogenous E. coli K1 meningitis
The in vitro experiments demonstrated that the ppkl deletion mutant PD44 was less adherent and invasive than its parent strain E44 in the HBMEC cell culture model. To further investigate the role of ppkl on the development of neonatal meningitis, the infant rat model of experimental hematogenesis meningitis was used to determine the in vivo virulence phenotype. The blood and CSF specimen were cultured for indication of bacteremia and meningitis, respectively. The magnitude of bacteremia induced by the mutant strain was similar to that of the wild-type strain. However, the occurrence of meningitis (defined as positive CSF cultures) in animals receiving PD44 (9 of 23, or 39%), was significantly lower (P< 0.05) than in those receiving the parent strain E44 (17 of 23, or 74%). Our in vitro and in vivo studies demonstrated that the ppkl gene was required for E. coli E44 penetration across the BBB in vitro and in vivo.
7. The mRNA expression levels of IbeR and IbeA in E. coli E44 and PD44
In order to determine whether loss of ppkl modulated the stationary phase regulator IbeR and the major virulence factor IbeA in E. coli E44, real time RT-PCR was used to detect the mRNA expression levels of IbeR and IbeA as described above. After normalization with the copy number of the GAPDH gene, the relative copy number of ibeR in PD44 (13.35±2.11) was significantly less than that in E44 (31.10±7.90), and the relative copy number of ibeR in the ppkl complemented strain raised to 23.76±3.71. The relative copy number of ibeA in PD44 (32.12±7.33) was also less than that in E44(57.60±10.31) and the ppkl complemented strain(49.71±8.41). The result suggested that the ppkl deletion led to a decline expression of ibeR and ibeA in E. coli K1 E44.
Statistical analysis
All values are expressed as mean±standard deviation. Statistical analysis was performed with Student's t-test for comparison of two groups, and with ANOVA for multiple comparisons. In vivo experiments data were analyzed by Pearson Chi-Square test. Differences with P<0.05 were considered to be statistically significant.
Conclusion
The ppkl gene is a highly conserved gene among many bacterial species, including bacterial pathogens, Mycobacterium tuberculosis, Neisseria meningitides, Helicobacter pylori, Vibrio cholerae, Salmonella typhimurium, etc.. Due to the absence of the PPK1 homologue in mammalian cells, PPK1 exhibits potential to be an attractive target for chemotherapy. In order to determine the role of poly P in the pathogenesis of E. coli Kl meningitis, we constructed the ppkl deletion mutants to test its biological functions using the in vitro and in vivo models of the BBB and genetic complementation. We showed that the mutant PD44 was considerably less adherent and invasive for HBMEC, and significantly less virulent in the entry into the CNS in the newborn rat model of experimental hematogenous meningitis. The less adherent and invasive phenotype of the PD44 mutant were complemented by the ppkl gene. Meanwhile, the poly P level of the PD44 in stationary phase was also found to be obviously lower than that of the parent strain E44 and the ppkl complemented strain by using the nonradioactive method. It should also be noticed that, the phenomenon of poly P accumulation still exists in ppkl mutant of P. aeruginosa and Campylobacter jejuni, which may be due to the harbouring of not only ppkl but also ppk2 mediating poly P-driven generation of GTP in these orgnisms.
Remodelling of the host-cell actin cytoskeleton has been observed during pathogenic invasion. The E. coli adhesion and invasion of the HBMEC leading to cytoskeletal reorganization and downstrem signal activiation is important for the bacteria to accross the HBMEC. To test the cytoskeletal reorganization in the HBMEC induced by ppkl deletion mutant and the wild-type strain, the rhodamine-labelled phalloidin was used to make the rearrangement of actin filaments induced by E. coli visible under the confocal fluorescence microscope. Our findings showed that the actin filaments rearrangement in the HBMEC induced by PD44 was significantly weaker than that induced by the parent strain E44 and the ppkl complemented strain.
During the development process of neonatal E. coli meningitis, it's an important pathogenesis for the pathogen to endure the hyperosmotic of fecal in intestine and acid stress in the host's stomach or vagina of puerpera. As a product of the reaction catalyzed by the PPK1, poly P has been reported to be associated with the expression of the SP regulator RpoS, which governs expression of over 50 genes and plays a central role on stationary-phase adaptations in most E. coli strains. The adaptation processes modulated by RpoS is important for the bacteria to survival in kinds of environments. However, the SP regulation in meningitic E. coli K1 carrying the ibeR gene was drastically different from that in many other E. coli strains. There is a nonsense mutation in the rpoS gene in E. coli K1 strain E44, which can keep its stress resistance independent of RpoS. The phenomenon of rpoS mutation is not unique to E. coli E44, but also exists in some other enteric pathogens. We have demonstrated that IbeR is an RpoS-like regulator contributing to the E. coli E44 entry into HBMEC and stress tolerance in the stationary-phase. Some proteins which are related to environmental modifications or central metabolism can be regulated by IbeR in E44, such as TnaA, LpdA, OmpC, TufB, GapA, OmpA, AhpC.
As the product of PPK1, poly P, plays an important role in the RpoS induction in other E. coli strains, we further examined whether PPK1 contributed to regulation of IbeR expression and stress tolerance in E44. The ppkl deletion mutant PD44 was tested for its survival rates under stress conditions in comparison with the parent strain E44. The survival rates were significantly reduced after the ppk1 mutant receiving stress treatments compared to that of E44. These results suggest that ppk1/poly P is important for the E. coli E44 to survival in stress environments. Real time RT-PCR was used to test the mRNA expression of IbeR. The expression levels of the major virulence gene ibeA in PD44 and E44 were also compared. The results showed that the IbeR and IbeA expression levels of ppk1 mutant were both lower than those of the parent strain E44, and the complementation of ppk1 gene could significantly improve the mRNA levels of IbeR and IbeA in PD44.
Taken together, our findings suggest that the ppk1/poly P plays an important role in stress resistance and virulence in E. coli K1 E44. And the ppk1 deletion will result in a significant decline expression of the SP-regulator IbeR and important virulence factor IbeA, which is crucial for the E44 to survival in stress environment and cross the blood brain barrier.
引文
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