微生物学报  2024, Vol. 64 Issue (8): 2731-2751   DOI: 10.13343/j.cnki.wsxb.20240022.
http://dx.doi.org/10.13343/j.cnki.wsxb.20240022
中国科学院微生物研究所,中国微生物学会

文章信息

张小梅, 彭萱, 龙雨欣, 倪海燕, 邹龙, 龙中儿. 2024
ZHANG Xiaomei, PENG Xuan, LONG Yuxin, NI Haiyan, ZOU Long, LONG Zhong'er.
转录组分析揭示盐酸克林霉素胁迫下嗜根考克氏菌DC2201的响应机制
Transcriptome analysis reveals the response mechanism of Kocuria rhizophila DC2201 to clindamycin hydrochloride
微生物学报, 64(8): 2731-2751
Acta Microbiologica Sinica, 64(8): 2731-2751

文章历史

收稿日期:2024-01-08
网络出版日期:2024-04-02
转录组分析揭示盐酸克林霉素胁迫下嗜根考克氏菌DC2201的响应机制
张小梅 , 彭萱 , 龙雨欣 , 倪海燕 , 邹龙 , 龙中儿     
江西师范大学生命科学学院 南昌市鄱阳湖湿地微生物资源开发与利用重点实验室, 江西 南昌 330022
摘要[目的] 研究0.5倍最低抑菌浓度(minimal inhibitory concentration, MIC)的盐酸克林霉素胁迫下,嗜根考克氏菌DC2201的差异表达基因(differentially expressed genes, DEGs),揭示盐酸克林霉素胁迫下嗜根考克氏菌(Kocuria rhizophila) DC2201的响应机制。[方法] 以LB液体培养基培养的嗜根考克氏菌DC2201细胞为对照,采用Illumina HiSeq测序平台进行RNA-seq双端测序,分析0.5 MIC的盐酸克林霉素胁迫下嗜根考克氏菌的基因表达情况,并采用实时荧光定量PCR方法验证。[结果] 从盐酸克林霉素胁迫下的嗜根考克氏菌中共筛选到1 202个显著DEGs,其中显著上调表达基因604个,显著下调表达基因598个。经基因本体论(gene ontology, GO)注释,筛选到分子功能(molecular function, MF)、细胞组分(cellular component, CC)和生物学过程(biological process, BP) 3个一级分类指标,35个二级分类指标共1 041个显著DEGs。经京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)注释,筛选到与DNA修复途径相关的显著DEGs 16个,与核糖体合成途径相关的显著DEGs 43个,与ATP结合盒(ATP-binding cassette, ABC)转运蛋白相关的显著DEGs 28个,与戊糖磷酸途径、糖酵解、三羧酸(tricarboxylic acid, TCA)循环、淀粉与蔗糖、丙酮酸、丁酸等碳水化合物代谢相关的显著DEGs 77个,与肽聚糖合成相关的显著DEGs 5个。[结论] 盐酸克林霉素胁迫下,嗜根考克氏菌DC2201的响应机制是一个全局性反应机制,细菌通过增强多重耐药性(multidrug resistance, MDR)家族的主要促进者超家族(major facilitator superfamily, MFS)转运体的表达来增加对盐酸克林霉素的外排,通过增强DNA修复和RNA代谢途径,以保证基因组的稳定性和RNA的正常功能,通过增强核糖体合成途径来弥补盐酸克林霉素与自身50S核糖体结合后导致的蛋白质合成障碍,以提高蛋白质合成效率。与此同时,减少碳水化合物的吸收和转运,抑制自身的能量代谢途径,以减缓自身的生长速率而降低对能量的需求,相应地,细胞壁的稳定性也受到影响。
关键词嗜根考克氏菌DC2201    盐酸克林霉素    转录组    响应机制    
Transcriptome analysis reveals the response mechanism of Kocuria rhizophila DC2201 to clindamycin hydrochloride
ZHANG Xiaomei , PENG Xuan , LONG Yuxin , NI Haiyan , ZOU Long , LONG Zhong'er     
Nanchang Key Laboratory of Microbial Resources Exploitation & Utilization from Poyang Lake Wetland, College of Life Sciences, Jiangxi Normal University, Nanchang 330022, Jiangxi, China
Abstract: [Objective] To mine the differentially expressed genes (DEGs) of Kocuria rhizophila DC2201 exposed to clindamycin hydrochloride at 0.5 minimum inhibitory concentration (MIC) and reveal the response mechanism of Kocuria rhizophila DC2201 to clindamycin hydrochloride. [Methods] With the Kocuria rhizophila DC2201 cells cultured in LB liquid medium as the control, Illumina HiSeq platform was used for paired-end sequencing to determine the gene expression of Kocuria rhizophila DC2201 cells exposed to clindamycin hydrochloride at 0.5 MIC. Real-time fluorescence quantitative PCR was then conducted for validation. [Results] A total of 1 202 significantly DEGs were screened out from Kocuria rhizophila DC2201 under the stress of clindamycin hydrochloride, including 604 significantly up-regulated genes and 598 significantly down-regulated genes. After gene ontology (GO) annotation, 1 041 significantly DEGs were annotated into 35 GO terms of molecular function (MF), cell composition (CC), and biological process (BP). The Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis predicted 16 significantly DEGs related to DNA repair, 43 significantly DEGs related to ribosomal synthesis, 28 DEGs associated with ATP-binding cassette (ABC) transporters, 77 significantly DEGs associated with the pentose phosphate pathway, glycolysis, tricarboxylic acid (TCA) cycle, starch and sucrose, pyruvate, butyrate and other carbohydrate metabolisms, and 5 significantly DEGs related to peptidoglycan synthesis. [Conclusion] Kocuria rhizophila DC2201 exposed to clindamycin hydrochloride adopts a global response mechanism. It increases the efflux of clindamycin hydrochloride by up-regulating the gene expression of major facilitator superfamily (MFS) transporters in the multidrug resistance (MDR) family. By enhancing DNA repair and RNA metabolism pathways, the strain ensures the genomic stability and normal RNA function. In addition, it enhances the ribosome synthesis pathway to compensate for the protein synthesis barrier caused by the binding of clindamycin hydrochloride with the 50S ribosome. Furthermore, the strain reduces the absorption and transportation of carbohydrates to restrain the energy metabolisms pathways, thus slowing down the growth and reducing the energy demand. Correspondingly, the cell wall stability of Kocuria rhizophila DC2201 is also affected.
Keywords: Kocuria rhizophila DC2201    clindamycin hydrochloride    transcriptome    response mechanism    

质控菌株常用于评价商业培养基和生化试剂盒的质量、检测抗菌剂的抗菌强度[1-2]。由于抗生素等抗菌药物的不当使用或过度使用,质控菌株有时也会对抗生素等抗菌药物产生耐药性,进而影响抗菌药物的抗菌强度测试结果,从而对抗生素的质量控制和效果评估带来挑战。研究质控菌株对抗生素的响应机制,有助于揭示细菌对抗菌药物的适应和抵抗机制。

考克氏菌属是一类革兰氏阳性细菌,属于放线菌目,微球菌科,广泛分布于自然界中[3-6],具有耐盐性、耐有机溶剂和降解酚类化合物的能力[7-9]。嗜根考克氏菌(Kocuria rhizophila) DC2201,原名为藤黄微球菌ATCC 9341,最早从根际样品中分离得到,常作为质控菌株,用于抗生素等抗菌药物抗菌性能的检测[10]。虽然嗜根考克氏菌DC2201是一株常用于抗生素敏感性试验的质控菌株,但有关嗜根考克氏菌DC2201对抗生素的敏感性和响应机制却知之甚少。

克林霉素是一种林可酰胺类抗生素,对大多数革兰氏阳性菌具有抑菌作用,通过与50S核糖体亚基结合抑制细菌蛋白质的合成,从而抑制细菌的生长[11]。本研究通过对盐酸克林霉素胁迫下的嗜根考克氏菌DC2201进行转录组测序分析,目的在于揭示嗜根考克氏菌DC2201在抗生素作用下的响应机制,有助于理解嗜根考克氏菌DC2201对抗菌药物的适应性和拮抗机制,对开发更有效的质量控制策略和提高质控菌株的应用价值具有重要意义。

1 材料与方法 1.1 菌株及试剂

嗜根考克氏菌DC2201,由江西师范大学生命科学学院提供,储存于50%甘油中,−80 ℃冷冻保存。

LB培养基的配制参考李舜岩等[12]的方法,MH培养基的配制参考杨秋玲等[13]的方法。

盐酸克林霉素(以下简称“克林霉素”),北京索莱宝科技有限公司;细菌总RNA提取试剂盒,天根生化科技(北京)有限公司;cDNA反转录试剂盒、实时荧光定量PCR试剂盒,宝生物工程(大连)有限公司;其他化学试剂,上海麦克林生化科技股份有限公司。

1.2 主要仪器

测序平台Illumina HiSeq,Illumina公司;NanoDrop 2000、高速离心机、实时荧光定量PCR仪,ThermoFisher Scientific公司;全自动微生物生长曲线测定仪,广州昇锐生物科技有限公司;扫描电子显微镜(scanning electron microscope, SEM),日立公司。

1.3 菌株的活化培养

将−80 ℃冷冻甘油管中保藏的嗜根考克氏菌DC2201菌种划线至LB固体培养基上,37 ℃培养24 h。

1.4 克林霉素对嗜根考克氏菌DC2201的MIC

采用试管二倍稀释法[14]测定克林霉素的最小抑菌浓度(minimal inhibitory concentration, MIC)。

1.5 克林霉素胁迫处理

从活化平板上挑取单克隆接种至装有100 mL LB液体培养基的三角摇瓶中,37 ℃、200 r/min培养18 h。按3%比例将培养物分别转接至两组装有100 mL LB液体培养基的三角摇瓶中,其中一组三角摇瓶中的LB液体培养基中添加终浓度为0.5 MIC的克林霉素(处理组),另一组三角摇瓶中的LB液体培养基未加克林霉素(对照组)。每组设3个生物学重复,其中处理组的3个生物学重复分别记为K1、K2、K3,对照组的3个生物学重复分别记为M1、M2、M3。所有摇瓶于37 ℃、200 r/min培养8 h后,4 ℃、4 000×g离心10 min收集菌体。

1.6 RNA的提取

采用RNA提取试剂盒[天根生化科技(北京)有限公司],按照说明书方法从收集的菌样中提取RNA,使用cDNA反转录试剂盒中的gDNA Eraser去除基因组DNA,对RNA进行质量检测,确保其质量合格后,再用于后续的建库流程。

1.7 转录组测序

将提取得到的合格RNA样品分别去除rRNA,加入fragmentation buffer,将mRNA随机断裂成200 bp左右的小片段,之后以mRNA为模板,利用随机引物反转录合成双链cDNA并进行碱基修饰用于构建cDNA文库。经PCR扩增和微型荧光计定量后,利用Illumina HiSeq测序平台进行RNA-seq双端测序。RNA提取、cDNA文库构建及测序过程均由上海美吉生物医药科技有限公司完成。

1.8 生物信息学分析

利用Illumina平台生成的数据进行生物信息学分析,并对提供的参考基因进行基础的功能注释,基于蛋白序列与非冗余蛋白数据库(non-redundant protein sequence database, NR)、Swiss-Prot数据库(https://web.expasy.org/docs/swiss-prot_guideline.html)、Pfam数据库(http://pfam.xfam.org/)、基因本体论数据库(gene ontology, GO)数据库(http://www.geneontology.org/)、京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes, KEGG)数据库(http://www.genome.jp/kegg/)等五大数据库进行比对,得到相应的功能注释信息。

1.9 基因差异表达分析

实验通过软件RSEM分别对基因的表达水平进行定量分析,定量指标为每百万条reads的转录本(transcript per million, TPM),利用差异分析软件DESeq2、显著性水平P-adjust以及多重检验矫正方法Benjamini-Hochberg (BH)方法三方面对基因的表达量及差异倍数进行统计分析,确定处理组中的显著差异表达基因(differentially expressed genes, DEGs),并对这些显著性DEGs进行GO分析和KEGG代谢途径分析,以确定显著DEGs在功能上的分布与主要涉及的代谢通路。

1.10 实时荧光定量PCR验证差异表达基因

为验证转录组测序数据的准确性,实验通过实时荧光定量PCR验证DEGs的表达水平。首先使用cDNA反转录试剂盒将1.6中获得的RNA反转录成cDNA,反应体系为(20 μL):gDNA Eraser去除基因组DNA后获得的反应液10 μL,RT Primer Mix 1 μL,PrimerScript RT Enzyme Mix Ι 1 μL,5×Primer Script Buffer 2 (for real time) 4 μL,RNase Free ddH2O 4 μL。反应参数设置:37 ℃ 15 min,85 ℃ 5 s,4 ℃ 5 min。再根据转录组测序分析的结果,以16S rRNA基因为内参基因,随机挑选5个DEGs进行实时荧光定量PCR验证,通过Primer5.0软件设计引物,具体序列见表 1,并交由生工生物工程(上海)股份有限公司合成。实时荧光定量PCR的反应体系(20.0 μL):cDNA 4.0 μL,2×TB Green Premix Ex Taq Ⅱ (Tli RnaseH Plus) 10.0 μL,50×ROX Reference Dye Ⅱ 0.4 μL,正向引物(10 µmol/L)和反向引物(10 µmol/L)各0.4 μL,灭菌水4.8 μL。扩增(采用两步法扩增)反应条件:95 ℃预变性30 s;95 ℃变性5 s,60 ℃退火34 s,循环40次。采用2−ΔΔCt的方法计算实时荧光定量PCR的数据,得到基因的相对表达量。

表 1. 实时荧光定量PCR的基因引物序列 Table 1. Primer sequences of the genes for qRT-PCR
Primer name Primer sequence (5′→3′)
KRH_RS02815-F (secE) TTCGGACGCATCTGGCTGTTC
KRH_RS02815-R (secE) TGAATGCCACGAACACCAGCAC
KRH_RS05185-F (lepB) ACGTCAAACGCGTGATGGC
KRH_RS05185-R (lepB) CATCCCCAGGGTAGACGTAGTC
KRH_RS00615-F AGCAGTGGCTCGAGCAGTTC
KRH_RS00615-R GATGTTGTCCACGTAGCGCTC
KRH_RS06330-F (recO) CGCATCGTGATCCTGCTCAG
KRH_RS06330-R (recO) AGACGATGTCCAGCGACCGG
KRH_RS06555-F (efp) CGCAGCGAGTACCAGTACCTGTAC
KRH_RS06555-R (efp) CTGGTTCTCGAGCATGAAGTTGGC
16S-F GGGTTTTACTGGTTTTGGATGGGC
16S-R CGTGTCTCAGTCCCCAGTGTGGG

1.11 菌体形态观察

按照1.5中方法分别收集克林霉素胁迫处理组和对照组细菌,加入2%戊二醛,4 ℃固定3 h。4 ℃、4 000×g离心5 min去除戊二醛,依序用30%、50%、60%、70%、80%和100%浓度的乙醇各脱水10 min。最后,将处理好的细菌样品滴在扫描电镜(scanning electron microscope, SEM)支架上,用Au溅射包覆,SEM观察细菌形态。

1.12 菌体生长曲线测定

按照1.5中方法分别培养对照组和处理组嗜根考克氏菌DC2201细胞,通过全自动微生物生长曲线测定仪分别测量嗜根考克氏菌DC2201的生长曲线。

2 结果与分析 2.1 克林霉素胁迫下的嗜根考克氏菌DC2201生长状况

首先测定了克林霉素对嗜根考克氏菌DC2201的MIC为0.156 25 μg/mL,低于《全国细菌耐药监测网技术方案(2020年版)》的0.5 μg/mL克林霉素耐药标准[15],表明嗜根考克氏菌DC2201对克林霉素敏感,符合质控菌种的质量要求;然后分析了0.5 MIC (0.078 μg/mL)克林霉素胁迫下的嗜根考克氏菌DC2201的生长情况,结果表明克林霉素的胁迫处理会大大降低嗜根考克氏菌DC2201的生长速度(图 1A),同时嗜根考克氏菌DC2201细胞表面出现明显的塌陷现象,细胞形态发生明显的变形(图 1B1C)。

图 1 嗜根考克氏菌DC2201的生长曲线图和细胞SEM图 Figure 1 Growth curves and cellular SEM of Kocuria rhizophila DC2201. A: Growth curves of Kocuria rhizophila DC2201. B: SEM image of cells in the control group. C: SEM image of cells in the treatment group (grown in medium containing clindamycin at 0.5 MIC).

2.2 转录组测序样品

实验分别提取了0.5 MIC的克林霉素处理组和对照组的嗜根考克氏菌DC2201总RNA,其OD260/OD280OD260/OD230以及RQN值分析结果(表 2)表明,所提取RNA质量较高,满足建库需求。

表 2. RNA样品分析结果 Table 2. Analysis of RNA samples
Sample name Concentration (ng/μL) Total amount (μg) OD260/OD280 OD260/OD230 RQN
K1 78.70 2.75 2.15 2.05 8.70
K2 60.90 2.13 2.05 1.82 5.90
K3 134.30 4.70 2.04 1.81 8.40
M1 53.00 1.86 2.10 2.19 10.00
M2 94.70 3.31 2.12 2.17 10.00
M3 93.90 3.29 2.10 2.21 10.00

2.3 转录组测序数据质控

样品测序所得数据的质量评估情况如表 3所示,处理组和对照组分别获得89 636 598和87 144 566个原始序列数据双端reads。测序数据已提交至国家微生物科学数据中心,获登录号NMDC10018677。数据质控后分别计算Phred数值大于20和30的碱基占总碱基的百分比,结果发现数据经过滤后其碱基组成的结果均为Q20大于98.00%,Q30大于95.00%,错误率均小于0.02%,处理组和对照组分别获得88 904 274和85 854 296个质控后双端reads,测序质量合格。

表 3. 样品测序质控统计表 Table 3. Sample sequencing quality control statistics
Sample Raw reads Clean reads Error rate (%) Q20 (%) Q30 (%)
K1 25 421 614 25 224 316 0.020 98.37 95.30
K2 32 451 124 32 179 156 0.020 98.62 95.90
K3 31 763 860 31 500 802 0.020 98.41 95.39
M1 29 617 240 29 209 256 0.020 98.56 95.77
M2 30 275 596 29 812 234 0.020 98.49 95.56
M3 27 251 730 26 832 806 0.020 98.54 95.72

与此同时,实验分析了生物学重复样本之间转录组数据的相关性,结果表明样品K1、K2、K3之间以及M1、M2、M3样品之间的相关性R2均大于0.9,表明基因在样本间的表达量相似性高,转录组测序数据结果可靠。

2.4 差异表达基因的筛选

克林霉素胁迫下,嗜根考克氏菌DC2201中共有1 202个显著DEGs (P-adjust < 0.05,上调log2 (fold change) > 1)或下调log2 (fold change) < −1),其中有604个基因显著上调表达,598个基因显著下调表达。采用火山图和聚类热图进行可视化,展示DEGs在两组样本间表达差异的倍数变化值、统计学检验值以及基因的功能分类(图 2A2B)。

图 2 0.5 MIC克林霉素胁迫下嗜根考克氏菌DC2201的基因表达 Figure 2 Gene expression of Kocuria rhizophila DC2201 exposed to clindamycin at 0.5 MIC. A: Volcano plot shows gene expression. Red, blue, and grey points represent up-regulated, down-regulated, and non-significant genes, respectively. B: Cluster analysis of significantly DEGs.

2.5 实时荧光定量PCR验证差异表达基因

为了验证克林霉素胁迫下,嗜根考克氏菌DC2201转录组分析结果中DEGs表达水平变化分析的可靠性,以16S rRNA为内参基因,随机挑选了5个DEGs进行实时荧光定量PCR分析,比较RT-qPCR与转录组测序结果(图 3)可知,5个基因的表达趋势完全相同,表明转录组测序结果的准确性和可信度较高。

图 3 实时荧光定量PCR验证差异表达基因 Figure 3 Verification of DEGs by qRT-PCR.

2.6 差异表达基因的GO分析

克林霉素胁迫下的嗜根考克氏菌DC2201中筛选得到的显著DEGs经GO注释,结果如图 4所示。

图 4 差异表达基因的GO注释 Figure 4 Distribution of DEGs for GO analysis. A: DEGs of biological process. B: DEGs of cellular component. C: DEGs of molecular function. Red and blue square represent up-regulated and down-regulated genes, respectively.

显著DEGs的GO注释结果分为生物学途径(biological process, BP)、细胞组分(cellular component, CC)和分子功能(molecular function, MF) 3类。在BP中注释到显著DEGs 486个,其中显著上调基因380个,显著下调基因106个。富集程度较高的显著上调表达基因主要被注释在“蛋白代谢过程(protein metabolic process)” “细胞酰胺代谢过程(cellular amide metabolic process)” “肽的代谢过程(peptide metabolic processes)” “酰胺生物合成过程(amide biosynthetic process)” “细胞大分子生物合成过程(cellular macromolecule biosynthetic process)” “肽生物合成(peptide biosynthetic process)” “翻译(translation)”。显著下调表达基因主要富集在“蛋白代谢过程(protein metabolic process)” “跨膜运输(transmembrane transport)”,如图 4A所示。在CC中注释到显著DEGs 335个,其中显著上调表达基因282个,显著下调表达基因53个。富集程度较高的显著上调表达基因主要与“含蛋白复合体(protein-containing complex)” “核糖核蛋白复合体(ribonucleoprotein complex)” “细胞器(organelle)” “核糖体(ribosome)”有关。显著下调表达基因主要与“含蛋白复合体(protein-containing complex)” “转运体复合物(transporter complex)”以及“跨膜转运体复合物(transmembrane transporter complex)”有关,如图 4B所示。此外,在MF中注释到显著DEGs 220个,其中显著上调表达基因176个,显著下调表达基因44个。富集程度较高的显著上调表达基因被注释在“核糖体结构单元(structural constituent of ribosome)” “结构分子活性(structural molecule activity)” “rRNA结合(rRNA binding)” “转录调控因子活性(transcription regulator activity)” “DNA结合转录因子的活性(DNA-binding transcription factor activity)”。显著下调表达基因主要富集在“主要主动跨膜转运体活性(primary active transmembrane transporter activity)”和“主动离子跨膜转运活性(active ion transmembrane transporter activity)”,如图 4C所示。

2.7 重要差异表达基因的功能分析

在注释到的2 283个DEGs中,除了271个为编码未知功能蛋白(hypothetical protein)的基因之外,其余DEGs都注释为有明确功能的基因,其产物中包含许多对细菌生命活动有重要功能的蛋白质,如细菌转录因子、MFS转运体、DNA损伤修复等。本节仅介绍显著DEGs中注释到的转录因子、MFS转运体和毒素-抗毒素(toxin-antitoxin, TA)系统,其他结合KEGG注释进行介绍。

2.7.1 转录因子

从克林霉素胁迫下的嗜根考克氏菌DC2201细胞中筛选得到的1 202个显著DEGs中共注释到23类,合计52个转录因子(表 4),其中只有2个显著下调表达,其他50个均显著上调表达,显著上调表达的转录因子基因中有10个属于TetR/AcrR家族,9个属于MarR家族转录因子。

表 4. 差异表达基因中注释到的转录因子统计表 Table 4. Statistics of significantly DEGs encoding transcription factors
Transcription factor family Number of DEGs
Up-regulated Down-regulated
TetR/AcrR 10
MarR 9
IclR 3
ArsR/SmtB 2
WhiB 2
Metal-dependent 2
Fur 2
GntR 2
Transcriptional regulator 2
TetR 2
Lrp/AsnC 2
LysR 2
Metal-sensitive 1
LacI 1
ROK 1
FmdB 1
DeoR/GlpR 1
XRE 1
CarD 1
Rrf2 1
PucR 1
Crp/Fnr 1
Response regulator 2
− means no data.

2.7.2 MFS转运体

克林霉素胁迫下,嗜根考克氏菌DC2201中MFS转运体相关的显著DEGs 19个(表 5),包括显著上调表达基因13个,显著下调表达基因6个。其中,显著上调表达的基因中包括编码MDR家族的MFS转运体的基因KRH_RS05890 [log2 (fold change)为1.17]和编码DHA2 (drug: H+ antiporter 2, DHA2)家族外排MFS转运体渗透酶亚基的基因KRH_RS07690 [log2 (fold change)为1.67]。

表 5. 编码MFS转运体相关的差异表达基因 Table 5. Significantly DEGs encoding MFS transporters
Gene ID Gene name log2 (fold change) Regulation Product
KRH_RS00075 KRH_RS00075 4.41 Up MFS transporter
KRH_RS11475 KRH_RS11475 2.19 Up MFS transporter
KRH_RS00690 KRH_RS00690 2.18 Up MFS transporter
KRH_RS03495 KRH_RS03495 2.02 Up MFS transporter
KRH_RS11030 KRH_RS11030 1.88 Up MFS transporter
KRH_RS09005 KRH_RS09005 1.77 Up MFS transporter
KRH_RS07690 KRH_RS07690 1.67 Up DHA2 family efflux MFS transporter permease subunit
KRH_RS08120 KRH_RS08120 1.67 Up Sugar porter family MFS transporter
KRH_RS12630 KRH_RS12630 1.62 Up MFS transporter
KRH_RS08365 KRH_RS08365 1.54 Up MFS transporter
KRH_RS01445 KRH_RS01445 1.52 Up MFS transporter
KRH_RS00100 KRH_RS00100 1.35 Up MFS transporter
KRH_RS05890 KRH_RS05890 1.17 Up MDR family MFS transporter
KRH_RS00905 KRH_RS00905 −1.56 Down Multidrug efflux MFS transporter
KRH_RS01120 KRH_RS01120 −1.63 Down MFS transporter
KRH_RS01170 KRH_RS01170 −1.67 Down MFS transporter
KRH_RS01835 KRH_RS01835 −2.32 Down MFS transporter
KRH_RS00350 KRH_RS00350 −3.15 Down MFS transporter
KRH_RS03220 KRH_RS03220 −3.24 Down MFS transporter

2.7.3 TA系统

TA系统是一种广泛分布于各类细菌中的细菌防御系统[16-17]。在克林霉素的胁迫作用下,嗜根考克氏菌DC2201细胞中与TA系统相关的显著DEGs有4个(表 6),而且全为显著上调表达基因,其中包括上调最为显著、编码RelE/ParE家族毒素的基因KRH_RS00650 [log2 (fold change)为4.79]。

表 6. 编码毒素-抗毒素系统的差异表达基因 Table 6. Significantly DEGs encoding toxin-antitoxin system
Gene ID Gene name log2 (fold change) Regulation Product
KRH_RS00650 KRH_RS00650 4.79 Up Type II toxin-antitoxin system RelE/ParE family toxin
KRH_RS10555 KRH_RS10555 3.67 Up Type IV toxin-antitoxin system AbiEi family antitoxin
KRH_RS00645 KRH_RS00645 3.17 Up Type II toxin-antitoxin system prevent-host-death family antitoxin
KRH_RS12145 KRH_RS12145 1.76 Up Type II toxin-antitoxin system death-on-curing family toxin

2.8 差异表达基因的KEGG分析

将克林霉素胁迫下嗜根考克氏菌DC2201细胞中的显著DEGs进行KEGG注释。结果表明,在克林霉素胁迫下,嗜根考克氏菌DC2201细胞的DNA损伤修复系统、核糖体、ABC转运蛋白、碳水化合物代谢、肽聚糖的生物合成等代谢途径均发生变化。

2.8.1 DNA损伤修复

克林霉素胁迫处理下嗜根考克氏菌DC2201细胞显著DEGs中有16个与DNA的错配修复、碱基切除修复、核苷酸切除修复和同源重组修复等DNA修复途径相关(表 7),其中13个基因显著上调,3个基因显著下调。显著上调表达的基因包括编码同时具有DNA-糖基化酶和DNA-AP裂解酶双功能的基因mutM [log2 (fold change)为1.58]、编码核酸内切酶III的基因nth [log2 (fold change)为1.41]、编码3′→5′核酸外切酶的基因KRH_RS04860 [log2 (fold change)为2.72]、编码DNA糖基化酶的基因KRH_RS01670 [log2 (fold change)为1.18]和KRH_RS07615 [log2 (fold change)为1.14]、编码DNA修复蛋白的基因recO [log2 (fold change)为1.11]。

表 7. 与DNA损伤修复相关的显著差异表达基因 Table 7. Significantly DEGs associated with DNA damage repair
Gene ID Gene name log2 (fold change) Regulation Product
KRH_RS11540 KRH_RS11540 4.20 Up Single-stranded DNA-binding protein
KRH_RS01360 KRH_RS01360 2.85 Up DNA polymerase III subunit gamma and tau
KRH_RS04165 KRH_RS04165 2.77 Up ATP-dependent helicase
KRH_RS04860 KRH_RS04860 2.72 Up 3′→5′ exonuclease
KRH_RS06410 holA 2.35 Up DNA polymerase III subunit delta
KRH_RS05310 ssb 1.87 Up Single-stranded DNA-binding protein
KRH_RS05090 mutM 1.58 Up Bifunctional DNA-formamidopyrimidine glycosylase/DNA-(apurinic or apyrimidinic site) lyase
KRH_RS02015 KRH_RS02015 1.44 Up DNA-3-methyladenine glycosylase I
KRH_RS02215 nth 1.41 Up Endonuclease III
KRH_RS03810 KRH_RS03810 1.19 Up Mismatch-specific DNA-glycosylase
KRH_RS01670 KRH_RS01670 1.18 Up DNA-3-methyladenine glycosylase I
KRH_RS07615 KRH_RS07615 1.14 Up DNA-3-methyladenine glycosylase
KRH_RS06330 recO 1.11 Up DNA repair protein RecO
KRH_RS01355 recR −1.37 Down Recombination mediator RecR
KRH_RS06680 ruvB −1.47 Down Holliday junction branch migration DNA helicase RuvB
KRH_RS06685 ruvA −1.99 Down Holliday junction branch migration protein RuvA

2.8.2 核糖体

克林霉素的胁迫处理导致嗜根考克氏菌DC2201中核糖体途径相关的显著DEGs达到43个(表 8),其中42个基因显著上调,1个基因显著下调。其中,编码50S核糖体蛋白L19和编码30S核糖体蛋白S12的两个基因rplS [log2 (fold change)为4.79]和rpsL [log2 (fold change)为4.38]的上调效果最为显著,而显著下调的基因为ykgO [log2 (fold change)为−1.55],编码50S核糖体蛋白L36。

表 8. 核糖体代谢相关的显著差异表达基因 Table 8. Significantly DEGs related to ribosomal metabolic pathway
Gene ID Gene name log2 (fold change) Regulation Product
KRH_RS05180 rplS 4.79 Up 50S ribosomal protein L19
KRH_RS02950 rpsL 4.38 Up 30S ribosomal protein S12
KRH_RS03095 rpmJ 3.73 Up 50S ribosomal protein L36
KRH_RS03125 rplM 3.70 Up 50S ribosomal protein L13
KRH_RS03025 rplN 3.64 Up 50S ribosomal protein L14
KRH_RS11635 rpmH 3.64 Up 50S ribosomal protein L34
KRH_RS03105 rpsK 3.34 Up 30S ribosomal protein S11
KRH_RS03100 rpsM 3.26 Up 30S ribosomal protein S13
KRH_RS06405 rpsT 3.02 Up 30S ribosomal protein S20
KRH_RS02875 rplJ 2.73 Up 50S ribosomal protein L10
KRH_RS03130 rpsI 2.70 Up 30S ribosomal protein S9
KRH_RS05080 rpmF 2.66 Up 50S ribosomal protein L32
KRH_RS02955 rpsG 2.57 Up 30S ribosomal protein S7
KRH_RS07710 rpmI 2.48 Up 50S ribosomal protein L35
KRH_RS02970 rpsJ 2.44 Up 30S ribosomal protein S10
KRH_RS01735 rpmB 2.38 Up 50S ribosomal protein L28
KRH_RS08000 rpsB 2.32 Up 30S ribosomal protein S2
KRH_RS05505 rplU 2.25 Up 50S ribosomal protein L21
KRH_RS01740 rpmG 2.16 Up 50S ribosomal protein L33
KRH_RS02975 rplC 2.11 Up 50S ribosomal protein L3
KRH_RS08465 KRH_RS08465 2.09 Up 50S ribosomal protein L25/general stress protein Ctc
KRH_RS03030 rplX 2.05 Up 50S ribosomal protein L24
KRH_RS03020 rpsQ 1.98 Up 30S ribosomal protein S17
KRH_RS07705 rplT 1.98 Up 50S ribosomal protein L20
KRH_RS11545 rpsF 1.96 Up 30S ribosomal protein S6
KRH_RS03060 rpmD 1.91 Up 50S ribosomal protein L30
KRH_RS06605 rpsD 1.88 Up 30S ribosomal protein S4
KRH_RS05160 rpsP 1.87 Up 30S ribosomal protein S16
KRH_RS03115 rplQ 1.86 Up 50S ribosomal protein L17
KRH_RS05510 rpmA 1.77 Up 50S ribosomal protein L27
KRH_RS03065 rplO 1.76 Up 50S ribosomal protein L15
KRH_RS03035 rplE 1.70 Up 50S ribosomal protein L5
KRH_RS03015 rpmC 1.68 Up 50S ribosomal protein L29
KRH_RS03050 rplR 1.56 Up 50S ribosomal protein L18
KRH_RS03000 rplV 1.48 Up 50S ribosomal protein L22
KRH_RS03045 rplF 1.45 Up 50S ribosomal protein L6
KRH_RS01745 rpsN 1.43 Up 30S ribosomal protein S14
KRH_RS03010 rplP 1.41 Up 50S ribosomal protein L16
KRH_RS02995 rpsS 1.30 Up 30S ribosomal protein S19
KRH_RS05825 rpsA 1.30 Up 30S ribosomal protein S1
KRH_RS03040 rpsH 1.21 Up 30S ribosomal protein S8
KRH_RS03055 rpsE 1.21 Up 30S ribosomal protein S5
KRH_RS11430 ykgO −1.55 Down Type B 50S ribosomal protein L36

另外,在显著DEGs中发现编码核糖体亚基组装以及起始翻译新的蛋白质所必需的3个转录起始因子的基因infAinfBinfC也显著上调。

2.8.3 ABC转运蛋白

克林霉素的胁迫处理导致嗜根考克氏菌DC2201的ABC转运蛋白相关的显著DEGs有28个(表 9),其中7个基因显著上调,21个基因显著下调。上调表达最为显著的2个基因为KRH_RS00615 [log2 (fold change)为3.97]和KRH_RS00605 [log2 (fold change)为2.68],分别编码金属ABC转运蛋白底物结合蛋白和金属ABC转运蛋白渗透酶。同时,编码铁转运体渗透酶的基因KRH_RS05450 [log2 (fold change)为1.18]也显著上调。下调最为显著的3个基因分别是ugpC [log2 (fold change)为−5.83]、KRH_RS01800 [log2 (fold change)为−5.46]和KRH_RS01805 [log2 (fold change)为−4.89],它们分别编码sn-甘油-3-磷酸ABC转运蛋白ATP结合蛋白、碳水化合物ABC转运蛋白渗透酶和糖ABC转运蛋白渗透酶,下调基因中还包括磷酸盐特殊转运系统(phosphate specific transport, Pst)中的3个关键基因pstA [log2 (fold change) 为−1.45],pstB [log2 (fold change)为−1.61]和pstS [log2 (fold change)为−1.25]。

表 9. 编码ABC转运蛋白相关的显著差异表达基因 Table 9. Significantly DEGs encoding ABC transporters
Gene ID Gene name log2 (fold change) Regulation Product
KRH_RS00615 KRH_RS00615 3.97 Up Metal ABC transporter substrate-binding protein
KRH_RS00605 KRH_RS00605 2.68 Up Metal ABC transporter permease
KRH_RS07950 KRH_RS07950 2.19 Up MetQ/NlpA family ABC transporter substrate-binding protein
KRH_RS07945 KRH_RS07945 1.41 Up Methionine ABC transporter ATP-binding protein
KRH_RS07940 KRH_RS07940 1.35 Up ABC transporter permease
KRH_RS05440 KRH_RS05440 1.30 Up ABC transporter substrate-binding protein
KRH_RS05450 KRH_RS05450 1.18 Up Iron ABC transporter permease
KRH_RS09440 pstS −1.25 Down Phosphate ABC transporter substrate-binding protein PstS
KRH_RS09430 pstA −1.45 Down Phosphate ABC transporter permease PstA
KRH_RS06050 KRH_RS06050 −1.52 Down Biotin transporter BioY
KRH_RS09425 pstB −1.61 Down Phosphate ABC transporter ATP-binding protein PstB
KRH_RS00505 KRH_RS00505 −1.70 Down Amino acid ABC transporter ATP-binding protein
KRH_RS00180 KRH_RS00180 −1.90 Down ABC transporter permease
KRH_RS00510 KRH_RS00510 −2.21 Down Amino acid ABC transporter permease
KRH_RS09115 KRH_RS09115 −2.24 Down ABC transporter ATP-binding protein
KRH_RS03895 KRH_RS03895 −2.50 Down ABC transporter permease
KRH_RS00185 KRH_RS00185 −2.61 Down ABC transporter permease
KRH_RS01810 KRH_RS01810 −2.79 Down Extracellular solute-binding protein
KRH_RS10405 KRH_RS10405 −3.54 Down ABC transporter ATP-binding protein
KRH_RS03890 KRH_RS03890 −3.57 Down ABC transporter permease
KRH_RS10395 KRH_RS10395 −3.63 Down ABC transporter permease
KRH_RS03885 KRH_RS03885 −3.65 Down ABC transporter family substrate-binding protein
KRH_RS00190 KRH_RS00190 −3.94 Down ABC transporter substrate-binding protein
KRH_RS10390 KRH_RS10390 −4.00 Down ABC transporter substrate-binding protein
KRH_RS10400 KRH_RS10400 −4.18 Down ABC transporter permease
KRH_RS01805 KRH_RS01805 −4.89 Down Sugar ABC transporter permease
KRH_RS01800 KRH_RS01800 −5.46 Down Carbohydrate ABC transporter permease
KRH_RS01795 ugpC −5.83 Down sn-glycerol-3-phosphate ABC transporter ATP-binding protein UgpC

2.8.4 碳水化合物代谢

克林霉素的胁迫处理导致嗜根考克氏菌DC2201中与戊糖磷酸途径、糖酵解、三羧酸(tricarboxylic acid, TCA)循环、淀粉与蔗糖、丙酮酸、丁酸等碳水化合物代谢相关的显著DEGs达到77个,其中仅有5个基因显著上调,72个基因显著下调(表 10)。例如,编码糖酵解途径中限速酶6-磷酸果糖激酶和丙酮酸激酶的基因KRH_RS02780 [log2 (fold change)为−2.21]和pyk [log2 (fold change)为−1.75]以及编码TCA循环中限速酶柠檬酸合酶基因KRH_RS03990 [log2 (fold change)为−1.38]都表现为显著下调。同时,编码琥珀酸脱氢酶基因KRH_RS03635 [log2 (fold change)为−4.90]也显著下调。

表 10. 碳水化合物代谢相关的显著差异表达基因 Table 10. Significantly DEGs related to carbohydrate metabolism pathway
Gene ID Gene name log2 (fold change) Regulation Product
KRH_RS12530 KRH_RS12530 4.72 Up Pyruvate kinase
KRH_RS12535 KRH_RS12535 3.31 Up Hypothetical protein
KRH_RS04945 KRH_RS04945 1.52 Up Acetolactate synthase large subunit
KRH_RS06335 leuA 1.39 Up 2-isopropylmalate synthase
KRH_RS01730 malQ 1.34 Up 4-alpha-glucanotransferase
KRH_RS01960 KRH_RS01960 −1.01 Down d-hexose-6-phosphate mutarotase
KRH_RS00255 KRH_RS00255 −1.08 Down Glucose-6-phosphate dehydrogenase
KRH_RS09625 KRH_RS09625 −1.11 Down Thiamine pyrophosphate-binding protein
KRH_RS09340 deoC −1.18 Down Deoxyribose-phosphate aldolase
KRH_RS05935 KRH_RS05935 −1.19 Down Phosphoglycerate kinase
KRH_RS08815 KRH_RS08815 −1.20 Down Succinate dehydrogenase hydrophobic membrane anchor subunit
KRH_RS09140 otsB −1.26 Down Trehalose-phosphatase
KRH_RS09820 KRH_RS09820 −1.27 Down NAD(P)-dependent alcohol dehydrogenase
KRH_RS01140 KRH_RS01140 −1.36 Down CoA transferase subunit B
KRH_RS03990 KRH_RS03990 −1.38 Down Citrate synthase
KRH_RS08655 KRH_RS08655 −1.44 Down Pyruvate dehydrogenase
KRH_RS02470 KRH_RS02470 −1.45 Down 3-hydroxyacyl-CoA dehydrogenase family protein
KRH_RS09135 KRH_RS09135 −1.49 Down Trehalose-6-phosphate synthase
KRH_RS00105 KRH_RS00105 −1.50 Down Thiamine pyrophosphate-binding protein
KRH_RS05275 KRH_RS05275 −1.52 Down ROK family protein
KRH_RS08820 sdhA −1.57 Down Succinate dehydrogenase flavoprotein subunit
KRH_RS05940 tpiA −1.65 Down Triose-phosphate isomerase
KRH_RS05930 gap −1.71 Down Type I glyceraldehyde-3-phosphate dehydrogenase
KRH_RS10705 KRH_RS10705 −1.72 Down NAD-dependent succinate-semialdehyde dehydrogenase
KRH_RS04505 KRH_RS04505 −1.74 Down Sugar phosphate nucleotidyltransferase
KRH_RS05800 pyk −1.75 Down Pyruvate kinase
KRH_RS07190 gndA −1.82 Down NADP-dependent phosphogluconate dehydrogenase
KRH_RS00715 pta −1.89 Down Phosphate acetyltransferase
KRH_RS06235 glgA −1.98 Down Glycogen synthase
KRH_RS03335 galU −2.00 Down UTP-glucose-1-phosphate uridylyltransferase GalU
KRH_RS09210 glgP −2.03 Down Alpha-glucan family phosphorylase
KRH_RS09205 KRH_RS09205 −2.03 Down Alpha-1, 4-glucan-maltose-1-phosphate maltosyltransferase
KRH_RS04565 KRH_RS04565 −2.04 Down Multifunctional oxoglutarate decarboxylase/oxoglutarate dehydrogenase thiamine pyrophosphate-binding subunit/ dihydrolipoyllysine-residue succinyltransferase subunit
KRH_RS00820 KRH_RS00820 −2.06 Down Enoyl-CoA hydratase
KRH_RS09750 KRH_RS09750 −2.08 Down Glyceraldehyde-3-phosphate dehydrogenase
KRH_RS07060 lpdA −2.18 Down Dihydrolipoyl dehydrogenase
KRH_RS03875 KRH_RS03875 −2.18 Down NADP-dependent isocitrate dehydrogenase
KRH_RS02780 KRH_RS02780 −2.21 Down 6-phosphofructokinase
KRH_RS09640 KRH_RS09640 −2.22 Down NAD-dependent succinate-semialdehyde dehydrogenase
KRH_RS02430 KRH_RS02430 −2.31 Down L-lactate dehydrogenase
KRH_RS05975 tkt −2.32 Down Transketolase
KRH_RS08320 KRH_RS08320 −2.38 Down Aldehyde dehydrogenase family protein
KRH_RS01580 KRH_RS01580 −2.41 Down Phosphoenolpyruvate carboxykinase (GTP)
KRH_RS08940 sucD −2.49 Down Succinate-CoA ligase subunit alpha
KRH_RS05950 pgl −2.50 Down 6-phosphogluconolactonase
KRH_RS03670 adhP −2.52 Down Alcohol dehydrogenase AdhP
KRH_RS06230 glgC −2.59 Down Glucose-1-phosphate adenylyltransferase
KRH_RS08480 KRH_RS08480 −2.62 Down Ribose-phosphate diphosphokinase
KRH_RS04910 KRH_RS04910 −2.67 Down Malate: quinone oxidoreductase
KRH_RS05965 KRH_RS05965 −2.72 Down Glucose-6-phosphate isomerase
KRH_RS01145 KRH_RS01145 −2.75 Down Acetyl-CoA C-acetyltransferase
KRH_RS05960 zwf −2.79 Down Glucose-6-phosphate dehydrogenase
KRH_RS03180 treZ −2.82 Down Malto-oligosyltrehalose trehalohydrolase
KRH_RS07065 sucB −2.84 Down 2-oxoglutarate dehydrogenase, E2 component, dihydrolipoamide succinyltransferase
KRH_RS01285 KRH_RS01285 −2.89 Down Alcohol dehydrogenase
KRH_RS05255 aceE −2.92 Down Pyruvate dehydrogenase (acetyl-transferring), homodimeric type
KRH_RS05970 tal −2.95 Down Transaldolase
KRH_RS08650 KRH_RS08650 −3.15 Down Class II fumarate hydratase
KRH_RS09645 gabT −3.17 Down 4-aminobutyrate-2-oxoglutarate transaminase
KRH_RS09195 glgB −3.18 Down 1, 4-alpha-glucan branching protein GlgB
KRH_RS07385 KRH_RS07385 −3.20 Down Pyruvate carboxylase
KRH_RS00710 KRH_RS00710 −3.22 Down Acetate kinase
KRH_RS06935 acnA −3.28 Down Aconitate hydratase AcnA
KRH_RS02210 acs −3.29 Down Acetate-CoA ligase
KRH_RS08825 KRH_RS08825 −3.34 Down Succinate dehydrogenase iron-sulfur subunit
KRH_RS00880 KRH_RS00880 −3.34 Down AMP-binding protein
KRH_RS09660 pgm −3.37 Down Alpha-D-glucose phosphate-specific phosphoglucomutase
KRH_RS11240 KRH_RS11240 −3.73 Down ATP-grasp domain-containing protein
KRH_RS11150 KRH_RS11150 −3.88 Down 2, 3-butanediol dehydrogenase
KRH_RS00935 fbaA −3.94 Down Class II fructose-bisphosphate aldolase
KRH_RS09200 treS −3.97 Down Maltose alpha-d-glucosyltransferase
KRH_RS08770 eno −4.04 Down Phosphopyruvate hydratase
KRH_RS03630 KRH_RS03630 −4.09 Down Fumarate reductase/succinate dehydrogenase flavoprotein subunit
KRH_RS09265 KRH_RS09265 −4.17 Down Phosphoglyceromutase
KRH_RS03625 KRH_RS03625 −4.33 Down Succinate dehydrogenase/fumarate reductase iron-sulfur subunit
KRH_RS03635 KRH_RS03635 −4.90 Down Succinate dehydrogenase cytochrome b subunit
KRH_RS09635 KRH_RS09635 −5.37 Down NAD-dependent succinate-semialdehyde dehydrogenase

2.8.5 肽聚糖合成

KEGG注释发现,克林霉素的胁迫处理导致嗜根考克氏菌DC2201肽聚糖合成相关的显著DEGs有5个(表 11),其中显著上调基因2个,显著下调基因3个。显著下调基因都是肽聚糖生物合成的关键基因,分别为编码磷酸-N-乙酰胞壁氨酰五肽转移酶基因mraY [log2 (fold change)为−1.09]、编码d-丙氨酰-d-丙氨酸羧肽酶基因KRH_RS11780 [log2 (fold change)为−1.06]、编码含青霉素结合转肽酶结构域的蛋白质的基因KRH_RS10210 [log2 (fold change)为−3.48]。

表 11. 肽聚糖合成相关的显著差异表达基因 Table 11. Significantly DEGs related to peptidoglycan biosynthesis
Gene ID Gene name log2 (fold change) Regulation Product
KRH_RS07305 KRH_RS07305 2.49 Up Penicillin-binding protein 2
KRH_RS07300 KRH_RS07300 1.56 Up UDP-N-acetylmuramoyl-l-alanyl-d-glutamate-2, 6-diaminopimelate ligase
KRH_RS11780 KRH_RS11780 −1.06 Down d-alanyl-d-alanine carboxypeptidase family protein
KRH_RS07290 mraY −1.09 Down Phospho-N-acetylmuramoyl-pentapeptide-transferase
KRH_RS10210 KRH_RS10210 −3.48 Down Penicillin-binding transpeptidase domain-containing protein

3 讨论与结论

细菌在DNA损伤、强酸强碱、极端高温、营养饥饿、强氧化还原电势、高渗透以及抗生素等胁迫条件下,会被诱导发生特定的应激反应,如改变细胞的特定结构、调节自身的代谢、激活毒力潜力和激发抗生素耐药性(诱导耐药突变或刺激耐药机制)等,以降低对不利环境的敏感性[18-19]。抗生素等抗菌药物的微生物检验通常依赖于质控菌株对药物的敏感性,而细菌对抗生素等抗菌药物的应激反应对抗生素检验的准确性、治疗的疗效以及临床应用都有重要影响[19]。因此,药物对微生物生长的影响,以及微生物对抗生素等抗菌药物的耐药性研究一直都是药物微生物学研究领域的重点和热点[20-22]。嗜根考克氏菌DC2201是一株常用于抗生素药效试验的质控菌株,本研究以克林霉素为例,采用转录组学方法揭示了嗜根考克氏菌DC2201对抗生素胁迫的响应机制,其结果对揭示嗜根考克氏菌DC2201对抗菌药物的敏感性和耐药机制都具有重要意义。

转录因子在细菌的生长发育以及响应逆境胁迫中具有重要作用,它通过传递和放大胁迫信号,介导相关基因的表达调控[23-24]。本研究通过转录组学方法筛选到抗生素胁迫下嗜根考克氏菌DC2201细胞中合计52个转录因子家族基因表达水平发生显著变化(表 4),最终导致1 202个基因显著差异表达(P-adjust < 0.05,上调log2 (fold change) > 1或下调log2 (fold change) < −1),其中有604个基因显著上调表达,598个基因显著下调表达,这一结果表明抗生素克林霉素的胁迫可导致嗜根考克氏菌DC2201基因表达的全局调控作用。

MFS转运体是一类广泛存在于生物界的膜蛋白,主要负责包括糖、氨基酸、离子、药物等物质的跨膜转运;研究结果表明,MDR家族和DHA2家族都可通过偶联质子转运来驱动多种抗菌药物的排出[25-26]。另外,大肠埃希氏菌中TetR/AcrR家族转录因子可调节编码MDR外排泵acrAB*基因的表达,MarR家族转录因子则是通过调节MarA的表达来调节编码MDR外排泵基因acrAB*tolC的表达水平[27]。本研究发现,克林霉素胁迫下嗜根考克氏菌DC2201细胞中包括10个TetR/AcrR家族和9个MarR家族转录因子均显著上调表达(表 4),由此可以推断,在克林霉素的作用下,嗜根考克氏菌DC2201中TetR/AcrR家族转录因子和MarR家族转录因子的显著上调表达,可能通过调节编码MDR外排泵相关基因的表达,增加药物外排家族MFS转运体对克林霉素的外排来响应克林霉素的胁迫。

DNA损伤修复是生物细胞内的DNA分子受到损伤以后恢复结构的现象。本研究发现,在克林霉素的胁迫下,嗜根考克氏菌DC2201细胞显著差异表达基因中有16个与DNA的错配修复、碱基切除修复、核苷酸切除修复和同源重组修复等DNA修复途径相关(表 7)。其中,MutM是原核生物碱基切除修复系统中的双功能酶,不但可以识别DNA损伤,而且能切除损伤的碱基,从而参与到许多损伤修复过程,防止基因突变和基因组不稳定[28-29]。Nth酶能够修复DNA中由于嘧啶氧化而引起的碱基损伤,它通过切割损伤的DNA链,移除氧化碱基,在修复过程中合成新的DNA链[30]。3ʹ→5ʹ核酸外切酶在DNA合成质量控制中具有重要的作用,此外,它们还在DNA复制、修复和重组过程中作为DNA聚合酶的校对外切酶发挥作用[31]。DNA糖基化酶可以去除细胞毒性和诱导DNA碱基变性,从而启动碱基修复途径[32]。RecO是一种重组介质蛋白,对细菌的同源重组、复制修复和脱氧核糖核酸退火都很重要[33]。上述基因上调表达的结果表明,克林霉素的胁迫会导致嗜根考克氏菌DC2201的DNA损伤,嗜根考克氏菌DC2201通过上调与DNA修复相关的基因,可以加速DNA损伤的修复过程。

RelE/ParE家族属于Ⅱ型TA系统。ParE是DNA促旋酶,会导致DNA断裂的积累从而抑制DNA的复制,而RelE具有核糖体依赖性mRNA内切酶活性,可通过切割mRNA来阻止核糖体的翻译[34-35]。研究表明在稳定状态下,II型TA系统中抗毒素通过蛋白质—蛋白质相互作用中和毒素[36]。在环境胁迫下,抗毒素被ATP依赖性蛋白酶选择性降解,从而导致细胞生长停滞和死亡[37]

另外,除编码假定蛋白的基因外,本研究的显著DEGs中上调最为显著的基因编码核糖核酸酶HII [log2 (fold change)为5.38]。研究表明,极端嗜热古菌Pyrococcus abyssi中的核糖核酸酶HII可能是完成DNA复制和修复所必需的酶[38]。我们推测,核糖核酸酶HII的显著上调可能与RelE/ParE家族毒素过表达有关,因为它可能在避免由TA系统介导的潜在程序性DNA断裂和mRNA修复中发挥作用,并且核糖核酸酶HII可能作为间接的克林霉素诱导的应激反应决定因子。

核糖体是细胞中蛋白质生物合成的场所,原核生物的核糖体由30S和50S两个亚基组成,30S亚基在蛋白质的合成中起着至关重要的作用,而50S亚基在蛋白质的合成延伸阶段发挥着核心作用。克林霉素属于林可酰胺类抗生素,能够与核糖体的50S亚基结合,从而阻止翻译[11]。克林霉素胁迫下,KEGG注释嗜根考克氏菌DC2201细胞中核糖体合成相关差异表达基因(表 8)的结果表明,嗜根考克氏菌DC2201需要增加核糖体蛋白的合成以及核糖体组装来弥补克林霉素与核糖体的50S亚基结合后导致的蛋白质合成障碍,从而响应克林霉素的胁迫。

ABC转运蛋白是一类位于膜内的蛋白质超家族,可利用ATP水解的能量在细胞膜上运输各种底物,参与细胞营养吸收、解毒和免疫反应[39-40]等重要生物学过程。本文经KEGG注释克林霉素胁迫下,嗜根考克氏菌DC2201细胞中ABC转运蛋白相关差异表达基因(表 9)的结果表明,嗜根考克氏菌DC2201对金属转运能力会增强,而对碳水化合物和磷的转运会受到抑制。研究结果表明,细菌中大约30%的蛋白质需要相应金属离子的参与才能发挥诸如酶促催化、抗氧化应激和生长代谢等生物学功能[41]。碳水化合物是细菌的主要能量来源,细菌通过代谢碳水化合物来产生能量,并用于细胞的生长和繁殖,它不仅提供细菌生长所需的能量,还为细菌合成细胞壁、核酸和蛋白质等细胞组分提供碳源。Pst系统是磷吸收和转运的关键系统[42],磷的吸收和转运在生物体能量代谢和细胞膜的完整性中发挥重要作用。另外,抑制细菌蛋白质翻译的抗生素也会影响细菌中心代谢和细胞呼吸[43-45]。上述结果表明,在克林霉素胁迫下,嗜根考克氏菌DC2201可通过增强自身的氧化应激来响应克林霉素的胁迫。同时,通过减少碳水化合物和磷的吸收和转运来抑制自身的能量代谢。上述分析结果解释了克林霉素胁迫下的嗜根考克氏菌DC2201的生长繁殖速度减缓的现象(图 1A)。

肽聚糖是革兰氏阳性细菌细胞壁的重要组成成分,对于维持细胞完整性和细胞存活都至关重要。本研究经KEGG注释克林霉素胁迫下的嗜根考克氏菌DC2201细胞中显著差异表达基因的结果(表 11)发现,与肽聚糖合成相关的显著下调基因中,磷酸-N-乙酰胞壁酰胺五肽转移酶负责形成细胞壁肽聚糖合成中的脂质I[46],d-丙氨酰-d-丙氨酸羧肽酶和含青霉素结合转肽酶结构域的蛋白质都是青霉素结合蛋白家族的成员,催化聚糖链的聚合和聚糖链之间的交联,是肽聚糖生物合成中的关键酶[47-48]。本研究表明在克林霉素的胁迫下,嗜根考克氏菌DC2201细胞的肽聚糖生物合成会受到抑制,这也为克林霉素胁迫下的嗜根考克氏菌DC2201细胞表面出现明显的塌陷,以及细胞形态发生明显的形态变化(图 1B1C)提供了依据。

总之,本研究采用转录组测序方法研究了克林霉素胁迫下嗜根考克氏菌DC2201的响应机制。结果表明,0.5 MIC克林霉素的胁迫处理对嗜根考克氏菌DC2201的转录谱具有显著影响。嗜根考克氏菌DC2201通过全局性的代谢调节以响应克林霉素的胁迫。具体而言,在克林霉素胁迫下,嗜根考克氏菌DC2201中有604个基因显著上调表达,598个基因显著下调表达。显著性DEGs涉及细菌转录因子、MFS转运体等重要功能的蛋白基因,以及ABC转运蛋白、核糖体、碳水化合物、肽聚糖和DNA的损伤修复等代谢过程。在综合分析上述显著DEGs功能的基础上,揭示了克林霉素的胁迫下,嗜根考克氏菌DC2201的响应机制是一个全局性反应机制,通过增强MDR家族的MFS转运体的表达从而增加对克林霉素的外排;增强DNA修复和RNA代谢途径,以保证基因组的稳定性和RNA的正常功能;增强核糖体合成途径以弥补克林霉素与自身50S核糖体结合后导致蛋白质合成障碍,提高蛋白质合成效率。与此同时,减少碳水化合物的吸收和转运,以抑制自身的糖酵解途径、TCA循环和戊糖磷酸途径等能量代谢途径,减缓自身的生长速率而降低对能量的需求,相应地,嗜根考克氏菌DC2201的细胞壁的稳定性也受到影响。

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转录组分析揭示盐酸克林霉素胁迫下嗜根考克氏菌DC2201的响应机制
张小梅 , 彭萱 , 龙雨欣 , 倪海燕 , 邹龙 , 龙中儿