耻垢分枝杆菌精氨酸代谢变化对细菌毒力及肺上皮细胞线粒体功能的影响

doi:10.3969/j.issn.1673-8640.2022.05.015

摘要/Abstract

摘要： 目的探讨精氨酸代谢变化对耻垢分枝杆菌（M.smeg）毒力、致病性及宿主生理的影响。方法通过构建M.smeg argR敲除株,以野生型M.smeg为对照,测定细菌体外生长能力变化;采用实时荧光定量聚合酶链反应（RT-qPCR）检测细菌脂类代谢相关基因fadD2、fadA5、kstR等的表达。用M.smeg侵染人非小细胞肺癌上皮细胞系A549,检测CCK8细胞活力,采用RT-qPCR检测细胞线粒体功能相关基因UCP1和DNM1L,观察细菌对宿主细胞生理的影响。结果敲除argR可提高细菌脂质储存能力,在以葡萄糖为单一碳源的培养基中更具生长优势（P<0.001）;同时,M.smeg分枝菌酸合成上调;侵染A549细胞2 h后,入胞率显著增加（P<0.001）,细菌毒力增强,对A549细胞的侵染能力提高,并通过影响线粒体功能影响宿主细胞的活力。结论 M.smeg精氨酸代谢负转录调控因子argR的敲除提升了细菌毒力,并可抑制宿主细胞线粒体功能。

关键词: 耻垢分枝杆菌, A549细胞, argR, 线粒体

Abstract: Objective To investigate the influence of arginine metabolism on the virulence,pathogenicity and host physiology of Mycolicibacterium smegmatis（M. smeg）. Methods A M. smeg knockout isolate of argR was constructed. Wild-type M. smeg was used as control,and the growth ability of bacteria in vitro was determined. Real-time fluorescence quantitative polymerase chain reaction （RT-qPCR） was used to determine the expressions of fadD2,fadA5 and kstR. Epithelial cells A549 were infected with M. smeg. Cell activity was determined as well. UCP1 and DNM1L were determined by RT-qPCR. The physiological effect on host cells was observed. Results It was found that argR knockout increased the lipid storage capacity of bacteria and had a growth advantage in the medium with glucose as a single carbon source（P<0.001）. The synthesis of M. smeg mycoacid was up-regulated,and the virulence of epithelial cells A549 was increased. After the infection of epithelial cells A549 2 h,the cell entry rate was increased（P<0.001）,and the infection ability of epithelial cells A549 was improved. The deletion of argR affected cell viability by affecting its mitochondrial function. Conclusions The knockout of argR,a negative transcriptional regulator of arginine metabolism in M. smeg,enhances bacterial virulence and inhibits mitochondrial function in alveolar epithelial cells.

Key words: Mycolicibacterium smegmatis, A549, argR, Mitochondrion

周紫薇, 林晨, 李烨雨, 王玉臣, 张鹭. 耻垢分枝杆菌精氨酸代谢变化对细菌毒力及肺上皮细胞线粒体功能的影响[J]. 检验医学, 2022, 37(5): 472-476.

ZHOU Ziwei, LIN Chen, LI Yeyu, WANG Yuchen, ZHANG Lu. Influence of arginine metabolism of Mycolicibacterium smegmatis on the virulence of bacteria and mitochondrial function of alveolar epithelial cells[J]. Laboratory Medicine, 2022, 37(5): 472-476.

图/表 8

表1 精氨酸代谢相关基因引物序列

引物名称	引物序列（5'~3'）	靶基因
argC	F：TGTCGTTCACTCCGGTTCTC	MSMEG_
	R：GTTGCTACCGATCACCGAAC	3776
argB	F：TCGCTGCTGGATCTCATTGC	MSMEG_
	R：GACCTTCGACATCGGTGAGC	3774
argJ	F：TGTCGTTCAACGGATCTCCC	MSMEG_
	R：TTCTCTTCGACGTAGGCGTG	3775
argF	F：CATGGCGCACTCGTTGATG	MSMEG_
	R：TTCGCATCCTTCGTGAGAGC	3772
argR	F：GCCGAAGGTATCGACGTCAC	MSMEG_
	R：GCAAGGTTAGCGCTGGCATC	3771

表2 M.smeg脂类代谢相关基因及引物

引物名称	引物序列（5'~3'）	靶基因
fadD2	F：CAGAAACCCGACCTCTCGTC	MSMEG_
	R：GGATCTTGACCGTGATCCCC	0599
fadA5	F：GCCGAGACCTACTACCACCT	MSMEG_
	R：CGTTGACGTTCACCTTGTCC	5996
kstR	F：GCGGCTGAACTTTATGGTCG	MSMEG_
	R：ATGTGGTACTGGTCCTCGGT	6042
MSMEG_	F：AGTACCTGATGGCGCTGTTC	MSMEG_
2695	R：CACGTTCACCTGGAGCTTCT	2695
pks2	F：CAATCAACTCGCCACAGCAG	MSMEG_
	R：CGGTGATGCCGAAGAACTCC	4727

表3 细胞线粒体功能相关基因及引物

引物名称	前向引物序列（5'~3'）	反向引物序列（5'~3'）
UCP1	CGCAGGGAAAGAAACAGCAC	CGTCAAGCCTTCGGTTGTTG
DNM1L	CTCGGGAACAGCGAGATTGT	GCTGTGCCATGTCCTCAGAT

图1 Arg从头合成通路组成基因表达转录水平验证结果注：每株细菌3个单克隆进行PCR检测,误差线显示为3个单克隆的标准差; WT; ΔargR。

图2 含有不同营养成分的7H9中M.smeg生长情况注：（a）M.smeg生长曲线,24~30 h A值经2倍稀释测得,曲线绘制为拟合后数值,拟合方程为A600 nm=A稀释$\frac{5}{4}$; ● WT; ■ ΔargR;（b）在10%ADC中WT与ΔargR生长情况;（c）在10%ADC与甘油中WT与ΔargR生长情况;每株菌挑取3个单克隆进行RNA抽提并进行PCR验证;误差线表示为3组重复间的标准差;■ WT;■ ΔargR。

图3 细菌脂类代谢相关基因表达变化注：每株细菌3个单克隆进行RT-qPCR,误差线显示为3个单克隆的标准差。

图4 ΔargR入胞率及细胞活力注：（a）细菌CFU差异倍数为2 h时的CFU变化,每株菌3个重复孔,误差线显示为3个复孔的标准差;（b）CCK8细胞活力检测,每株细菌3个单克隆进行RT-qPCR,误差线显示为3个单克隆的标准差;■ WT;■ ΔargR。

图5 细胞线粒体功能相关基因表达变化注：每株细菌侵染3个复孔的细胞进行RT-qPCR,误差线显示为3个单克隆的标准差;■ WT;■ ΔargR。

参考文献 18

[1]	WHO. Global tuberculosis report 2020[Z]. 2020.
[2]	CHARLIER D, BERVOETS I. Regulation of arginine biosynthesis,catabolism and transport in Escherichia coli[J]. Amino Acids, 2019, 51(8):1103-1127. DOI URL
[3]	TIWARI S, VAN TONDER A J, VILCHÈZE C, et al. Arginine-deprivation-induced oxidative damage sterilizes Mycobacterium tuberculosis[J]. Proc Natl Acad Sci U S A, 2018, 115(39):9779-9784. DOI URL
[4]	NAMOUCHI A, CIMINO M, FAVRE-ROCHEX S, et al. Phenotypic and genomic comparison of Mycobacterium aurum and surrogate model species to Mycobacterium tuberculosis:implications for drug discovery[J]. BMC Genomics, 2017, 18(1):530. DOI URL
[5]	TYAGI J S, SHARMA D. Mycobacterium smegmatis and tuberculosis[J]. Trends Microbiol, 2002, 10(2):68-69. DOI URL
[6]	XIONG L, TENG J L, BOTELHO M G, et al. Arginine metabolism in bacterial pathogenesis and cancer therapy[J]. Int J Mol Sci, 2016, 17(3):363. DOI URL
[7]	BARRIOS-PAYÁN J, SAQUI-SALCES M, JEYANATHAN M, et al. Extrapulmonary locations of mycobacterium tuberculosis DNA during latent infection[J]. J Infect Dis, 2012, 206(8):1194-1205. DOI URL
[8]	MEHTA P K, KING C H, WHITE E H, et al. Comparison of in vitro models for the study of Mycobacterium tuberculosis invasion and intracellular replication[J]. Infect Immun, 1996, 64(7):2673-2679. DOI URL
[9]	DOBOS K M, SPOTTS E A, QUINN F D, et al. Necrosis of lung epithelial cells during infection with Mycobacterium tuberculosis is preceded by cell permeation[J]. Infect Immun, 2000, 68(11):6300-6310. DOI URL
[10]	MCDONOUGH K A, KRESS Y. Cytotoxicity for lung epithelial cells is a virulence-associated phenotype of Mycobacterium tuberculosis[J]. Infect Immun, 1995, 63(12):4802-4811. DOI URL
[11]	KIM S Y, SOHN H, CHOI G E, et al. Conversion of Mycobacterium smegmatis to a pathogenic phenotype via passage of epithelial cells during macrophage infection[J]. Med Microbiol Immunol, 2011, 200(3):177-191. DOI URL
[12]	YAN M Y, YAN H Q, REN G X, et al. CRISPR-Cas12a-assisted recombineering in bacteria[J]. Appl Environ Microbiol, 2017, 83(17):e00947-17.
[13]	AGUILAR-AYALA D A, TILLEMAN L, VAN NIEUWERBURGH F, et al. The transcriptome of Mycobacterium tuberculosis in a lipid-rich dormancy model through RNAseq analysis[J]. Sci Rep, 2017, 7(1):17665. DOI URL
[14]	GANAPATHY U, MARRERO J, CALHOUN S, et al. Two enzymes with redundant fructose bisphosphatase activity sustain gluconeogenesis and virulence in Mycobacterium tuberculosis[J]. Nat Commun, 2015, 6:7912. DOI URL
[15]	ARMSTRONG R M, ADAMS K L, ZILISCH J E, et al. Rv2744c is a PspA ortholog that regulates lipid droplet homeostasis and nonreplicating persistence in Mycobacterium tuberculosis[J]. J Bacteriol, 2016, 198(11):1645-1661. DOI URL
[16]	MOHAREER K, MEDIKONDA J, VADANKULA G R, et al. Mycobacterial control of host mitochondria:bioenergetic and metabolic changes shaping cell fate and infection outcome[J]. Front Cell Infect Microbiol, 2020, 10:457. DOI URL
[17]	SOULTAWI C, FORTIER Y, SOUNDARAMOURTY C, et al. Mitochondrial bioenergetics and dynamics during infection[J]. Exp Suppl, 2018, 109:221-233.
[18]	KHAN M, SYED G H, KIM S J, et al. Mitochondrial dynamics and viral infections:a close nexus[J]. Biochim Biophys Acta, 2015, 1853(10 Pt B):2822-2833.