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. 2021 Nov 20;41(11):1725–1732. [Article in Chinese] doi: 10.12122/j.issn.1673-4254.2021.11.19

Srn821798在变异链球菌变链素IV形成中的作用

Regulatory role of small RNA srn821978 in mutacin IV expression in Streptococcus mutans

Li XU 1, Shanshan LIU 1, Min WANG 2, Fang LIU 1, Rongxiu ZHANG 1, Kai ZHANG 1,*
PMCID: PMC8685694  PMID: 34916201

Abstract

Objective

To analyze the role of small RNA srn821798 in posttranscriptional regulation of mutacin IV expression in Streptococcus mutans.

Methods

The potential target genes of srn821978 were predicted using RNAhybrid, RNAPredator and IntaRNA. We collected 10 Streptococcus mutans (S.muans) strains with high expression of mutacin IV and another 10 S.muans strains that did not express mutacin IV screened by inhibition zone test, and the expression levels of srn821798 and the candidate target genes in these strains were detected by qPCR. Using synthesized mimics and inhibitors of srn821798, we constructed S.muans strains with high or low srn821798 expression via electroporation based on the standard strain of S.muans UA159, and analyzed the expression levels of srn821798 and its candidate target genes in these strains. We also examined the binding ability of srn821798 to its target gene sepM using electrophoresis and a dual- luciferase reporter system.

Results

The expression levels of the candidate target genes of srn821798 including sepM, comD, comE, nlmA and nlmB were significantly higher while the expression level of srn821798 was significantly lower in clinical S.muans strains with high expression of mutacin IV than in those without mutacin IV expression (P < 0.05). Although the expression levels of the candidate target genes in strains with up- regulated or down- regulated srn821798 expression did not differ significantly from those in the standard strain, the expression level of sepM showed a trend of differential distribution, and srn821798 was predicted to have a strong binding ability to sepM action site.

Conclusion

srn821798 may play a regulatory role in the expression of mutacin IV in S.muans, but the underlying mechanism remains to be explored.

Keywords: Streptococcus mutans, mutacin IV, sRNAs, sepM

龋病是最常见的会影响患者身心健康的慢性感染性疾病[1-3]。第四次全国口腔健康流行病学调查结果显示，我国55~64岁年龄组的恒牙患龋率高达95.60%。因此，龋病防治的任务在我国仍很艰巨[4]。变异链球菌（SM）是导致龋病发生的一种重要的微生物[5-7]。戈登链球菌又名格氏链球菌（SG），是一种牙面早期定植的先锋菌，与龋病的形成呈负相关[8, 9]。而变链素Ⅳ是变异链球菌产生的拮抗戈登链球菌的一种重要的细菌素[10]。

近年研究表明，细菌毒力因子的表达受细菌体内小RNA（sRNAs）的广泛调控[11, 12]。细菌sRNAs通常长度小于300个核苷酸，位于基因组基因间区[12]，其主要通过碱基配对与靶基因结合发挥转录后调控作用[13, 14]。1971年，Griffin等[15]通过在大肠埃希菌中首次发现了一些高度表达的sRNAs如4.5S，转运- 信使RNA（tmRNA），RNaseP及Spot42等。随着测序技术和生物信息学的发展，sRNAs在其他多种细菌中也得到广泛研究。例如，Toffano等[16]测序发现灿烂弧菌中存在成百上千种潜在sRNAs，与其他弧菌的保守性分析发现，大部分sRNAs为灿烂弧菌特异性表达，其中有28种sRNAs在所有弧菌中均有所表达；Howden等[17]对暴露于抗生素状态的金黄色葡萄球菌进行RNA测序，结果发现409个潜在的sRNAs。

我们前期研究发现，变异链球菌中亦存在sRNAs的表达[18-20]，srn821798是其中成功鉴定的一种sRNA。然而，sRNAs是否在变异链球菌变链素Ⅳ的转录后调控中发挥关键作用，目前尚不清楚。研究报道sepM在变链素Ⅳ的表达中发挥重要作用[10]，鉴于3种靶基因预测软件均提示sepM是srn821798的靶基因，本文旨在初步探索srn821798在变链素Ⅳ形成中的作用。

1. 材料和方法

1.1. srn821798的靶基因预测和通路生物信息学分析

RNAhybrid、RNAPredator和IntaRNA软件预测<italic>srn821798</italic>的候选作用靶基因；DAVID（<a href="http://david.ncifcrf.gov/" target="_blank">http://david.ncifcrf.gov/</a>）用于分析<italic>srn821798</italic>参与的KEGG通路；antiSMASH（bacterial version）（<a href="http://antismash.secondarymetabolites.org/" target="_blank">http://antismash.secondarymetabolites.org/</a>）被用来分析<italic>srn821978</italic>用于预测变异链球菌的次级代谢产物。

1.2. srn821798及其候选靶基因在不同变链素Ⅳ表达组变异链球菌临床菌株中的表达水平检测

在实验室前期保存的变异链球菌临床株中筛选c血清型菌株，包括高变链素Ⅳ表达和无变链素Ⅳ表达的变异链球菌临床菌株各10株。戈登链球菌是评估变链素Ⅳ表达水平的指示菌[21]。变异链球菌临床株在脑心浸液肉汤（BHI）液体培养基中培育过夜，当培养至A600为0.3时，从每个分离菌株中取10 μL加入BHI琼脂板中，37 ℃培育12 h，然后再接种相等量的戈登链球菌，在37 ℃，5% CO2条件下继续培养12 h后，指示菌株清除区代表变链素Ⅳ的活性。

变异链球菌临床株过夜培养后，用miRNA Mini试剂盒（Qiagen）提取细菌总RNA。Mir-XTM miRNA第一链合成试剂盒（Takara）和Mir- XTM miRNA qRT-PCR TB GreenTM试剂盒（Takara）分别被用于srn821798的聚合酶链反应（PCR）和逆转录定量聚合酶链反应（RT- qPCR）。srn8217978的PCR反应条件为95 ℃ 10 s，95 ℃ 5 s 40个循环，50 ℃退火20 s。以16s rRNA的表达水平作为内参。16s rRNA，sepM，comD，comE，nlmA，nlmB的PCR条件同之前研究[22, 23]。

1.3. 标准株中srn821798上、下调对候选靶基因表达水平的影响测定

由上海吉玛生物设计并合成srn821798模拟物（GAUUAGUUAUCAGAUUUU，AAUCUGAUAAC UAAUCUU）和抑制剂（AAAAUCUGAUAACUAAUC）。变异链球菌UA159（ATCC 700610）在37 ℃，5% CO2下1000 μL BHI液体培养基（1∶100体积比）中培育过夜，然后用0.9%生理盐水洗两次，在新鲜的BHI中重悬。将40 μL的样品和模拟物（8 μL模拟物原液和250 μL无酶水的混合物）或抑制剂（10 μL抑制剂原液和625 μL无酶水的混合物）共同加入1 mm间隙的预冷的电转化杯，采用ECM（BTX，USA）进行电转化。模拟物和抑制剂的原液浓度均为20 μmol。电转参数：单脉冲2500 V，电容25 μF，电阻200 Ω。细菌电穿孔后移至新鲜BHI液体培养基中继续培养8 h（指数期中期），然后进行总RNA的提纯，即刻反转录PCR，并检测srn821798和候选靶基因的表达水平。引物序列见表 1。

1.

引物序列

Primer sequence for qPCR of srn821798 and its candidate target genes

ID Primer subsequence
srn821798 GATTAGTTATCAGATTTT
comD (F) CTCTGATTGACCATTCTTCTGG
comD (R) CATTCTGAGTTTATGCCCCTC
nlmA (F) AATGGACAGCCAAACACTTTC
nlmA (R) TAACAAGAGTCGCACCTGCC
nlmB (F) TGTCAGAAGTTTTTGGTGG
nlmB (R) ACTCCAGCACATCCAGCAAG
16S rRNA (F) CTTACCAGGTCTTGACATCCCG
16S rRNA (R) ACCCAACATCTCACGACACGAG
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1.4. srn821798和sepM结合实验

人工合成sepM 1（RNAhybrid）、sepM 2（RNApredator）、sepM 3（IntaRNA）以及srn821798小RNA片段（上海吉玛生物），并对其分别进行琼脂糖凝胶电泳。srn821798作为对照组；同时将（srn821798+ sepM 1）、（srn821798+sepM 2）、（srn821798+sepM 3）进行预混孵育分别电泳，作为实验组。

1.5. 双荧光素酶报告基因验证srn821798对sepM的靶向调控作用

针对RNAhybrid、RNAPredator和IntaRNA软件预测的sepM基因上与srn821798互作的序列，在psiCHECK2质粒上构建sepM野生型和突变型质粒，并将这些质粒和合成的mimic及mimic NC转染至293T细胞，建立如下分组：mimic NC + psiCHECK2- WT、mimic+psiCHECK2-WT组、mimic NC+ psiCHECK2- RNAhybrid -MUT组、mimic + psiCHECK2- RNAhybrid -MUT组、mimic NC + psiCHECK2- RNAPredator -MUT组、mimic + psiCHECK2-RNAPredator -MUT组、mimic NC + psiCHECK2- IntaRNA-MUT组、mimic+psiCHECK2-IntaRNA-MUT组、mimic NC + psiCHECK2组、mimic + psiCHECK2组。采用Dual-Luciferase双荧光素酶检测试剂盒检测各组细胞相对荧光信号强度。

1.6. 统计学分析

用IBM SPSS20.0软件包进行数据分析，定量数据采用t检验方法，P < 0.05为差异具有统计学意义。

2. 结果

2.1. 已鉴定sRNAs的生物信息学分析

2.1.1. 已鉴定sRNAs靶基因预测结果

3种靶基因预测专用软件RNAhybrid、RNAPredator和IntaRNA对srn821798进行靶基因预测，结果提示，sepM，comD，comE，nlmA，nlmB是srn821798的候选作用靶基因（表 2），其中3种软件的预测结果均提示sepM是srn821798的候选靶基因（表 3）。

2.

3种软件靶基因预测的srn821798-mRNA作用位点

srn821798 mRNA action sites predicted using 3 software

Target genes Software Energy (Kcal/mol) mRNA start mRNA end sRNA start sRNA end
RNAhybrid -20.30 15 36 1 18
sepM RNAPredator -10.93 24 35 1 12
IntaRNA -8.92 100 111 1 12
RNAhybrid - - - - -
comD RNAPredator -9.44 138 146 6 14
IntaRNA -5.81 49 62 1 14
RNAhybrid - - - - -
comE RNAPredator -5.68 725 736 1 12
IntaRNA - - - - -
RNAhybrid - - - - -
nlmA RNAPredator -4.12 -156 -149 6 13
IntaRNA -2.09 98 106 8 16
RNAhybrid - - - - -
nlmB RNAPredator -5.18 -190 -182 8 16
IntaRNA -3.41 122 135 2 18
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3.

3种软件靶基因预测的srn821798-sepM作用位点

srn821798-sepM action sites predicted by software

Software Position (start-end) Interaction (5'→3')
sepM srn821798 sepM srn821798
RNAhybrid 15-36 1-18 AAAAUUUAAAUGGUGGCUAAUC GATTAGTTATCAGATTTT
RNAPredator 24-35 1-12 AUGGUGGCUAAU GATTAGTTATCA
IntaRNA 100-111 1-12 AUGCCCGGCGGA GATTAGTTATCA
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2.1.2. srn821798参与的通路分析

KEGG通路分析提示，srn821798参与以次级代谢产物合成为主的多数通路，而次级代谢产物分析软件antiSMASH（bacterial version）显示变链素Ⅳ是变异链球菌中一种重要的次级代谢产物（图 1），这与靶基因预测结果一致。

1.

1

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srn821798 KEGG通路分析结果

KEGG pathway analysis of srn821798. A: KEGG analysis of the target genes predicted by RNAPredator suggests that biosynthesis of secondary metabolites is one of the important pathways involving srn821798. B: KEGG analysis of the target genes predicted by IntaRNA suggests that biosynthesis of secondary metabolites is one of the important pathways involving srn821798

2.1.3. 变异链球菌临床菌株中srn821798和靶基因的表达水平分析

高表达变链素Ⅳ组中sepM，comD，comE，nlmA及nlmB均有表达，且表达水平均显著高于无表达变链素Ⅳ组（P < 0.05），而高表达变链素Ⅳ组srn821798的表达水平显著低于无表达变链素Ⅳ组（P < 0.001）。低表达菌株，但两组菌株中候选靶基因的表达水平均未存在显著差异，其中sepM的表达水平在两组间存在差异的趋势，而前期3种靶基因预测软件的交集结果亦提示srn821798可调控sepM的表达，并且RNAhybrid和RNAPredator预测的srn821798-sepM结合位点高度一致。因此我们继而检测了3种软件预测的srn821798- sepM结合能力，结果发现，RNAhybrid和RNAPredator预测的srn821798-sepM位点结合能力高（结合率分别为82.84%和85.19%）（图 3）。

3.

3

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srn821798过、低表达株中候选靶基因表达水平的检测及srn821798-sepM预测结合位点的结合能力分析

Detection of relative expression levels of the candidate target genes in srn821798 high expression and low expression strains and analysis of binding ability of srn821798 with predicted binding sites of sepM. A: High expression and low expression of srn821798 in srn821798 high expression and low expression strains group (*P < 0.05 vs S.mutans). B-F: Relative expression levels of the candidate target genes in srn821798 high expression and low expression strains group. G: Analysis of binding ability of srn821798-sepM; sepM 1, sepM 2 and sepM 3 represent the binding sequences of srn821798 and sepM gene predicted by RNAhybrid, RNAPredator and IntaRNA. H: Binding rates of srn821798 with sepM 1, sepM 2 and sepM 3 analyzed with SEPM 1, SEPM 2 and SEPM 3 as controls (***P < 0.001).

2.

2

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变异链球菌临床株中srn821798和靶基因表达水平分析

Expression analysis of srn821798 and its target gene in clinical strains of Streptococcus mutans. A: Serotype test result. B: Expression map of mutacin IV in clinical strains with high or no mutacin IV expression. C: Relative expression level of srn821798. D-H: Relative expression levels of the candidate target genes (*P < 0.05, **P < 0.01, ***P < 0.001).

2.2. srn821798对sepM靶向调控的双荧光素酶报告基因验证

以mimic NC+psiCHECK2-WT为对照，srn821798模拟物与野生型载体（psiCHECK2-WT）共转染293T细胞后，细胞荧光素酶活性明显下降（RNAhybrid P=0.003；RNAPredator P=0.002）；以mimic NC + psiCHECK2-MUT为对照，srn821798模拟物与突变型载体（psiCHECK2-RNAhybrid-MUT或psiCHECK2- RNAPredator-MUT）共转染293T细胞后，细胞荧光素酶活性未见明显差异（RNAhybrid P=0.800；RNAPredator P=0.292）。针对IntaRNA的预测结果，双荧光素酶报告基因检测结果显示，以mimic NC + psiCHECK2- WT为对照, 以mimic NC + psiCHECK2- MUT为对照，srn821798模拟物（mimic）与突变型载体（psiCHECK2-IntaRNA-MUT）共转染293T细胞后，细胞荧光素酶活性亦明显下降，差异有统计学意义（P=0.014，图 4）。

4.

4

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双荧光素酶报告基因验证srn821798靶向调控sepM的结果

Targeted regulatory role of srn821798 in sepM expression detected by dual-luciferase reporter system. A: Dual-luciferase reporter assay for examining srn821798-sepM interaction according to the RNAhybrid prediction. B: Dual-luciferase reporter assay for examining srn821798-sepM interaction according to the RNAPredator prediction. C: Dual-luciferase reporter assay of srn821798-sepM interaction according to the IntaRNAprediction. *P < 0.05; **P < 0.01.

3. 讨论

龋病是口腔科最常见最高发的疾病之一，是由变异链球菌等细菌为主的多种因素作用下所造成的牙体硬组织分解、破坏[24]。变异链球菌形成龋坏的先决条件是对牙面的黏附，从而促进菌斑生物膜的形成，代谢碳源产生酸性终末产物，进而导致牙齿表面脱矿并最终形成龋洞[25]。戈登链球菌是一种与龋病的形成呈负相关的早期牙面定植菌，阻止龋齿形成的关键可能是一方面通过释放氨平衡变异链球菌产生的酸性环境，另一方面其可直接拮抗变异链球菌的生长[21, 26, 27]。

细菌素是由细菌合成的抗菌肽，为细菌的自身生长提供竞争优势[28]。变链素Ⅳ是由变异链球菌中的nlmA和nlmB编码产生的拮抗多种非变异链球菌如戈登链球菌、血链球菌进而保护变异链球菌生长的一种细菌素[29]。文献报道，ComDE双组份调控系统在变链素Ⅳ的表达中起着重要正调控作用，而该系统又进一步受上游SepM蛋白的影响[10]。且本课题组最近的研究结果表明，变异链球菌临床菌株中SepM蛋白的编码基因sepM中存在与变链素Ⅳ形成相关的突变类型[30]。

既往研究表明，sRNAs主要通过与目标靶mRNAs碱基配在转录后水平调控细菌毒力的表达，并且sRNAs有望成为评估临床疾病状况和严重程度的生物标志物[10, 21, 31]。已经有许多细菌的sRNAs被证实[32]。然而，在龋病方面，关于致龋菌sRNAs的研究较少，Lee等[33]发现变异链球菌中15~26个核苷酸大小的sRNAs可能有数百种；Li等[34]通过生物信息学预测得到变异链球菌中存在sRNA L10-Leader，继而通过实验验证并检测了该sRNA在不同生长环境下的表达改变。我们前期在不同的糖（蔗糖和葡萄糖）浓度和初始PH5.5压力下亦检测发现变异链球菌中可检测到大量的sRNAs[18-20]。但是是否存在调控变链素Ⅳ形成的关键sRNAs尚不清楚。我们针对前期已鉴定的sRNAs进行生物信息学预测，发现多数sRNAs可能参与调控变链素IV的表达，我们随机对其中一种sRNA srn225147进行了分析，发现comD是候选靶标，但是进一步实验检测发现，其虽可双向调控靶基因comD的表达，但是对变链素Ⅳ的调控能力微弱[29, 35]。为了进一步寻找可能在变链素Ⅳ形成中起调控作用的sRNAs，本实验中我们筛选了三种靶基因预测软件均有相同靶标结果的srn821798，并且RNA- hybrid和RNAPredator预测的srn821798和sepM的作用位点高度相似，这加强了srn821798调控变链素Ⅳ的可信度，因此，本文就srn821798在变链素Ⅳ中的作用进行重点分析。

本课题组利用生物信息学预测srn821798在变链素Ⅳ中的潜在作用后，进而在20株临床分离菌株（高变链素Ⅳ表达组n=10；变链素Ⅳ无表达组n=10）中进一步检测了srn821798和sepM，comD，comE，nlmA，nlmB候选靶基因的表达水平。我们前期研究发现，蔗糖可明显影响变异链球菌小RNA的表达[10]。本次研究中，考虑到临床菌株之间蔗糖利用率和蔗糖诱导毒力的差异尚不清楚，因此为了排除蔗糖可能引起的影响，我们在本次实验中均未加入蔗糖。结果发现，在高变链素Ⅳ表达组中，sepM，comD，comE，nlmA，nlmB表达水平显著高于无变链素Ⅳ表达组；高表达变链素Ⅳ组srn821798的表达水平显著低于无表达变链素Ⅳ组。靶基因在高、低变链素Ⅳ表达组中呈正向表达，而srn821798在高、低变链素Ⅳ表达组中呈负向表达，这提示，srn821798可能通过调控候选靶基因在变链素Ⅳ中起重要作用，而且可能为负反馈调节。为进一步验证我们的设想，我们进而在变异链球菌UA159标准株上成功构建了srn821798过、低表达菌株，并发现当srn821978的表达水平上调到约100倍时，候选靶基因在srn821798上调组和对照组中的表达均无明显差异。电泳结果提示，RNAhybrid和RNAPredator预测的srn821798-sepM位点结合能力高，这与双荧光素酶报告基因的检测结果一致。双荧光素酶报告基因检测实验发现，mimic NC+野生质粒为对照，srn821798 mimic与野生型载体共转染293T细胞后，细胞荧光素酶活性明显下降；以mimic NC+突变质粒为对照，srn821798 mimic与RNAhybrid或RNAPredator突变型载体共转染293T细胞后，细胞荧光素酶活性未见明显差异。这验证了RNAhybrid和RNAPredator软件预测的srn821798-sepM作用位点的有效性。然而，srn821798 mimic与IntaRNA突变型载体共转染293T细胞后，细胞荧光素酶活性亦明显下降，并且差异倍数与mimic NC+野生质粒组和mimic+野生质粒组的区别差异不大，这主要是因为，mimic与RNAhybrid和RNAPredator预测的位点有结合，但与IntaRNA预测的位点无结合能力或者结合效果微弱。因此，sepM确实是srn821798的直接作用靶标，主要作用位点为RNAhybrid和RNAPredator预测的区域。

综上所述，我们的研究为变异链球菌在转录后水平上产生变链素Ⅳ提供了新的视角，结果提示srn821798通过调控靶基因sepM的表达进而调节变链素Ⅳ的形成。然而，srn821798介导的变链素Ⅳ产生的调节作用较弱。本结果虽与srn225147的研究结论相似，但存在明显不同的地方。首先，srn821798在临床菌株中高变异链球菌表达组中的表达水平约是低变异链球菌表达组的1万倍，而srn225147在两组的表达差异仅约4倍，这提示我们，srn821798对变链素Ⅳ的调控能力可能明显优于srn225147。其次，以往我们关于srn225147在变链素Ⅳ中的研究，仅针对两个靶基因预测软件（RNAhybrid和RNAPredator）得出的单一靶基因comD进行探索，而本次研究中，为了得到更精确的预测结果，我们采用三种靶基因预测软件进行（RNAhybrid、RNAPredator及IntaRNA）分析，对得出的所有已报道的与变链素Ⅳ形成相关的靶基因（sepM, comD, comE, nlmA, nlmB）进行了表达水平的探索，并对三种靶基因预测得到较为一致的sepM结果进行了小RNA-sepM结合片段的靶点验证分析。故，本研究是我们既往研究的扩展和深入。虽然得到的结果也是srn821798介导变链素Ⅳ形成的能力微弱。但是，这可能与本实验标准菌株中srn821798高、低表达组中srn821798的表达差异没有达到临床菌株的差异水平。因此，在后续的研究中，我们需进一步改善实验条件，尝试电转得到更高的srn821798上调倍数来验证我们的假设。此外，在本研究中，我们只关注srn821978，而其他未鉴定的sRNAs是否通过靶向sepM-ComDE通路相关基因在变链素Ⅳ的调控中发挥重要作用尚不清楚，还需进一步研究。

Biography

徐丽，硕士研究生，主治医师，E-mail: kqxuli@163.com

Funding Statement

蚌埠医学院自然科学基金重点项目（BYKY1841ZD）；安徽省教育厅重点项目（KJ2018A0223）; 安徽省重点研究与开发计划项目（1804h08020290）；蚌埠医学院第一附属医院优秀青年项目（2019byyfyq07）

Contributor Information

徐 丽 (Li XU), Email: kqxuli@163.com.

张 凯 (Kai ZHANG), Email: zk29788@163.com.

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