Change of Differentially Expressed Genes and SNP Before or After Pseudomonas aeruginosa Resistance to Tachyplesin I

doi:10.13560/j.cnki.biotech.bull.1985.2020-1445

Abstract

Abstract:

To explore the resistance mechanism of Pseudomonas aeruginosa to antibacterial peptide tachyplesin I,we employed RNA sequencing to analyze the changes of differentially expressed genes,sRNA target genes,and SNP between tachyplesin I-resistant strain and the original strain. By GO function and KEGG pathway enrichment,the results showed that differentially expressed genes were the most related to the functions of integral component of membrane,nucleotide binding,formate dehydrogenase（NAD+）activity,and there was no significant enrichment pathway. SNP of 22 differentially expressed genes changed,which was related to known genes encoding lipid A deacylase,outer membrane protein,cold shock protein,and lipopolysaccharide modification and hypothesized proteins. Further prediction and analysis uncovered 11 differentially expressed sRNAs and corresponding 863 differentially expressed target genes. The functions of these sRNA target genes were mainly related to histidine biosynthetic process,homoserine kinase activity,and 5-carboxymethyl-2-hydroxymuconate delta-isomerase activity. It is referred that the resistance mechanism of P. aeruginosa to tachyplesin I may be regulated by affecting the synthesis and metabolism of amino acids,the formation and modification of membrane proteins,and metabolism of iron ions,as well as by leading to the base mutations of few genes. At the same time,sRNA played its regulatory role by acting these related target genes expression. The SNP mutated genes and its mRNA regulations to their target genes by sRNA remain to be further verified.

Key words: Pseudomonas aeruginosa, RNA sequencing, sRNA, SNP, target genes, enrichment analysis

HONG Jun, WEI Xia-yi, JI Bing-jie, YE Yan-xin, CHENG Tian-ci. Change of Differentially Expressed Genes and SNP Before or After Pseudomonas aeruginosa Resistance to Tachyplesin I[J]. Biotechnology Bulletin, 2021, 37(9): 191-202.

Figures/Tables 11

References 22

[1]	Nakamura T, Furunaka H, Miyata T, et al. Tachyplesin, a class of antimicrobial peptide from the hemocytes of the horseshoe crab(Tachypleus tridentatus)isolation and chemical structure[J]. Journal of Biological Chemistry, 1988, 263:16709-16713. pmid: 3141410
[2]	Anaya-López1 JL, López-Meza JE. Bacterial resistance to cationic antimicrobial peptides[J]. Critical Reviews in Microbiology, 2013, 39(2):180-195. doi: 10.3109/1040841X.2012.699025 pmid: 22799636
[3]	Cai Y, Chai D, Wang R, et al. Colistin resistance of Acinetobacter baumannii:clinical reports, mechanisms and antimicrobial strategies[J]. Antimicrobial Agents and Chemotherapy, 2012, 67:1607-1615.
[4]	Hong J, Hu JY, Ke F. Experimental induction of bacterial resistance to the antimicrobial peptide tachyplesin I and investigation of the resistance mechanisms[J]. Antimicrobial Agents and Chemotherapy, 2016, 60(10):6067-6075. doi: 10.1128/AAC.00640-16 pmid: 27480861
[5]	洪军, 胡建业, 刘坤, 等. 绿脓杆菌对鲎素耐受性特点及耐受性机制[J]. 微生物学报, 2018, 58(9):1593-1604.
	Hong J, Hu JY, Liu K, et al. Characteristics and resistance of tachyplesin-I resistance in Pseudomonas aeruginosa[J]. Acta Microbiologica Sinica, 2018, 58(9):1593-1604.
[6]	Peng T, Kan J, Lun JS, et al. Identification of novel sRNAs involved in oxidative stress response in the fish pathogen Vibrio alginolyticus by transcriptome analysis[J]. Journal of Fish Diseases, 2019, 42(2):1-15.
[7]	Barnhill EC, Crucello A, Houserova D, et al. Characterization of novel small RNAs(sRNAs)contributing to the desiccation response of Salmonella enterica serovar Typhimurium[J]. RNA Biology, 2019, 16(11):1643-1657. doi: 10.1080/15476286.2019.1653680 URL
[8]	叶中杨, 邱怀雨, 祝丙华, 等. sRNA调控细菌耐药相关基因表达研究进展[J]. 中国生物工程杂志, 2018, 38(7):89-93.
	Ye ZY, Qiu HY, Zhu BH, et al. Research progress of sRNA regulates the expression of genes in related with bacterial resistance[J]. China Biotechnology, 2018, 38(7):89-93.
[9]	Miller CL, Manuel R, Rajasekhar KSL, et al. RsmW, Pseudomonas aeruginosa small non-coding RsmA-binding RNA upregulated in biofilm versus planktonic growth conditions[J]. BMC Microbiology, 2016, 16(1):155. doi: 10.1186/s12866-016-0771-y URL
[10]	刘翠翠. 福氏志贺菌耐药株的转录组测定及sRNA差异分析[D]. 北京:中国农业科学院, 2014.
	Liu CC. The transcriptome sequencing analysis and sRNA difference analysis in drug-resistant Shigella flexneri[D]. Beijing:Chinese Academy of Agricultural Sciences, 2014.
[11]	Zhang YF, Han K, Chandler CE, et al. Probing the sRNA regulatory landscapeof P. aeruginosa:post-transcriptional control of determinants of pathogenicity and antibiotic susceptibility[J]. Molecular Microbiology, 2017, 106(6):919-937. doi: 10.1111/mmi.2017.106.issue-6 URL
[12]	张栋, 高风英, 卢迈新, 等. 尼罗罗非鱼β2 m基因SNP位点和单倍型与无乳链球菌抗性的关联分析[J]. 水生生物学报, 2018, 42(5):903-912.
	Zhang D, Gao FY, Lu MX, et al. Association analysis of SNP locus and haplotype of β2m gene in Nile tilapia(Oreochromis niloticus)with its resistance to Streptococcus agalactiae[J]. Acta Hydrobiologica Sinica, 2018, 42(5):903-912.
[13]	Wang L, Feng Z, Wang X, et al. DEGseq:an R package for identifying differentially expressed genes from RNAseq data[J]. Bioinformatics, 2010, 26:136-138. doi: 10.1093/bioinformatics/btp612 URL
[14]	Ashburner M, Ball CA, Blake JA, et al. Gene ontology:tool for the unification of biology[J]. Nature Genetics, 2000, 25(1):2529.
[15]	Kanehisa M, Goto S, Kawashima S, et al. The KEGG resource for deciphering the genome[J]. Nucleic Acids Research, 2004, 32:D277280.
[16]	Winsor GL, Griffiths EJ, Lo R, et al. Enhanced annotations and features for comparing thousands of Pseudomonas genomes in the Pseudomonas genome database[J]. Nucleic Acids Research, 2016, 44:D646-D653. doi: 10.1093/nar/gkv1227 URL
[17]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))Method[J]. Methods, 2001, 25:402-408. pmid: 11846609
[18]	McPhee JB, Bains M, Winsor G, et al. Contribution of the PhoP-PhoQ and PmrA-PmrB two-component regulatory systems to Mg²⁺-induced gene regulation in Pseudomonas aeruginosa[J]. Journal of Bacteriology, 2006, 188:3995-4006. doi: 10.1128/JB.00053-06 URL
[19]	Moskowitz SM, Brannon MK, Dasgupta N, et al. PmrB mutations promote polymyxin resistance of Pseudomonas aeruginosa isolated from colistin-treated cystic fibrosis patients[J]. Antimicrobial Agents and Chemotherapy, 2012, 56(2):1019-1030. doi: 10.1128/AAC.05829-11 pmid: 22106224
[20]	Shepherd J, Ibba M. Direction of aminoacylated transfer RNAs into antibiotic synjournal and peptidoglycan-mediated antibiotic resistance[J]. FEBS Letters, 2013, 587(18):2895-2904. doi: 10.1016/j.febslet.2013.07.036 URL
[21]	Ernst CM, Peschel A. Broad-spectrum antimicrobial peptide resistance by MprF-mediated aminoacylation and flipping of phospholipids[J]. Molecular Microbiology, 2011, 80:290-299. doi: 10.1111/mmi.2011.80.issue-2 URL
[22]	许达, 宗树成, 胡森, 等. 细菌sRNA代谢调控的研究进展[J]. 中国预防兽医学报, 2017, 39(1):80-84.
	Xu D, Zong SC, Hu S, et al. Research progress on regulation of Bacterial sRNA metabolism[J]. Chinese Journal of Preventive Veterinary Medicine, 2017, 39(1):80-84.

PA-99突变株样品 Sample of PA-99 mutant	Number of SNP	Transition	Transversion	Heterozygosity
T09	139	75.54%	23.74%	0.72%
T10	226	86.73%	12.83%	0.44%
T11	126	72.22%	26.98%	0.79%

PA-99突变株样品 Sample of PA-99 mutant	Number of SNP	Transition	Transversion	Heterozygosity
T09	139	75.54%	23.74%	0.72%
T10	226	86.73%	12.83%	0.44%
T11	126	72.22%	26.98%	0.79%

#ID or Gene name	Protein name	log₂FC	Regulated	Start	End	SNP number	SNP
pvdL	Peptide synthase	1.15621	Up	2707666	2720694	2	2712019C>A;2715424T>C
Novel_244	—	-1.71631	Down	1720641	1732256	1	1732254T>C
PA1938	Hypothetical protein	-1.95428	Down	2118926	2119747	1	2119153T>C
Novel_497	—	1.95302	Up	3672989	3674050	1	3673743G>A
Novel_745	—	2.58303	Up	5130955	5135726	1	5130955T>C
Novel_797	—	-1.77794	Down	5343486	5344904	1	5344709G>A
Novel_840	—	-2.52117	Down	5621609	5624734	1	5621617G>A
PA1428a	Hypothetical protein	1.07562	Up	1552566	1554155	1	1553722G>A
PA0193	Hypothetical protein	1.186966	Up	221585	222487	1	222358C>T
exoY	Adenylate cyclase	-1.75024	Down	2410344	2411480	1	2411150G>T
PA2451	Hypothetical protein	1.56984	Up	2752866	2754445	1	2754392G>A
cspD	Cold-shock protein CspD	-1.0514	Down	2965201	2965473	1	2965461C>T
PA2712	Hypothetical protein	-1.01673	Down	3066296	3067159	1	3066971A>G
PA3380	Hypothetical protein	2.27038	Up	3787021	3787479	1	3787410G>A
opr86	Outer membrane protein Opr86	-1.51418	Down	4085062	4087455	1	4085961C>G
cupB3	Usher CupB3	-1.59417	Down	4565421	4567955	1	4565913A>G
fepD	Ferric enterobactin transporter FepD	1.26932	Up	4655368	4656390	1	4656097T>C
pagL	Lipid A 3-O-deacylase	-2.39699	Down	5229459	5229980	1	5229837C>T
pmrB	Two-component regulator system Signal sensor kinase PmrB	1.00016	Up	5364760	5366193	1	5364817G>T
PA4837	Hypothetical protein	1.411356	Up	5427716	5429842	1	5428274G>A
bioD	ATP-dependent dethiobiotin Synthetase BioD	1.47708	Up	563549	564235	1	563798C>T
vreR	Sigma factor regulator VreR	1.68468	Up	735487	736446	1	735564C>T

#ID or Gene name	Protein name	log₂FC	Regulated	Start	End	SNP number	SNP
pvdL	Peptide synthase	1.15621	Up	2707666	2720694	2	2712019C>A;2715424T>C
Novel_244	—	-1.71631	Down	1720641	1732256	1	1732254T>C
PA1938	Hypothetical protein	-1.95428	Down	2118926	2119747	1	2119153T>C
Novel_497	—	1.95302	Up	3672989	3674050	1	3673743G>A
Novel_745	—	2.58303	Up	5130955	5135726	1	5130955T>C
Novel_797	—	-1.77794	Down	5343486	5344904	1	5344709G>A
Novel_840	—	-2.52117	Down	5621609	5624734	1	5621617G>A
PA1428a	Hypothetical protein	1.07562	Up	1552566	1554155	1	1553722G>A
PA0193	Hypothetical protein	1.186966	Up	221585	222487	1	222358C>T
exoY	Adenylate cyclase	-1.75024	Down	2410344	2411480	1	2411150G>T
PA2451	Hypothetical protein	1.56984	Up	2752866	2754445	1	2754392G>A
cspD	Cold-shock protein CspD	-1.0514	Down	2965201	2965473	1	2965461C>T
PA2712	Hypothetical protein	-1.01673	Down	3066296	3067159	1	3066971A>G
PA3380	Hypothetical protein	2.27038	Up	3787021	3787479	1	3787410G>A
opr86	Outer membrane protein Opr86	-1.51418	Down	4085062	4087455	1	4085961C>G
cupB3	Usher CupB3	-1.59417	Down	4565421	4567955	1	4565913A>G
fepD	Ferric enterobactin transporter FepD	1.26932	Up	4655368	4656390	1	4656097T>C
pagL	Lipid A 3-O-deacylase	-2.39699	Down	5229459	5229980	1	5229837C>T
pmrB	Two-component regulator system Signal sensor kinase PmrB	1.00016	Up	5364760	5366193	1	5364817G>T
PA4837	Hypothetical protein	1.411356	Up	5427716	5429842	1	5428274G>A
bioD	ATP-dependent dethiobiotin Synthetase BioD	1.47708	Up	563549	564235	1	563798C>T
vreR	Sigma factor regulator VreR	1.68468	Up	735487	736446	1	735564C>T

sRNA ID	Gene length/nt	log₂FC	Regulated	sRNA ID	Gene length/nt	log₂FC	Regulated
Novel_33	188	-3.101050802	Down	Novel_346	136	-1.138061426	Down
Novel_377	378	-2.065876992	Down	Novel_593	127	-1.434477351	Down
Novel_583	78	-3.461144761	Down	Novel_683	153	-1.371013476	Down
Novel_584	116	-2.305895532	Down	Novel_344	60	1.764469442	Up
Novel_6	94	-1.576755833	Down	Novel_5	284	-1.660811503	Down
Novel_853	117	-1.507421364	Down