Co-expression Mechanism Analysis of Different Tachyplesin I-Resistant Strains in Pseudomonas aeruginosa Based on Transcriptome Sequencing

Abstract

Tachyplesin I is a cationic antimicrobial peptide with 17 amino acids. The long-term continuous exposure to increased concentrations of tachyplesin I induced resistance in Pseudomonas aeruginosa. The global gene expression profiling of tachyplesin I-resistant P. aeruginosa strains PA-60 and PA-99 and the sensitive strain P. aeruginosa CGMCC1.2620 (PA1.2620) were conducted by transcriptome sequencing to analyze the common underlying mechanism of resistance to tachyplesin I in low- or high-resistance mutants. The co-expression patterns, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, sRNA target genes, and single-nucleotide polymorphism (SNP) change were analyzed for the co-expressed genes in this study. A total of 661 differentially co-expressed genes under treatments of PA1.2620 vs. PA-99 and PA1.2620 vs. PA-60 (HL) were divided into 12 kinds of expression patterns. GO and KEGG pathway enrichment analyses indicated that the enrichment of co-expressed genes was mainly associated with oxidoreductase activity, mismatched DNA binding, mismatch repair, RNA degradation of GO terms, aminoacyl-tRNA biosynthesis, and aminobenzoate degradation pathways, and so forth. The co-expressed resistance-related genes were mainly involved in antibiotic efflux and antibiotic inactivation. Seven co-expressed genes had SNP changes. Some co-expressed sRNAs were involved in P. aeruginosa resistance to tachyplesin I by regulating target genes and pathways related to resistance. The common resistance mechanism of P. aeruginosa among different mutants to tachyplesin I was mainly associated with the expression alteration of several genes and sRNA-regulated target genes related to resistance; few genes had base mutations. The findings of this study might provide guidance for understanding the resistance mechanism of P. aeruginosa to tachyplesin I.

Keywords: P. aeruginosa; RNA-Seq; SNP; co-expressed genes; sRNA; tachyplesin I.

FIGURE 1

Volcano plot of differential expression in PA1.2620 different strain. (A) PA1.2620 original strain vs. PA–60 mutant. (B) PA-60 strain vs. PA–99 mutant. Each point in volcano plot represents one gene, and the abscissa represents the logarithm of the expression difference fold of one gene. The vertical axis represents the negative logarithm of the error detection rate. The green dots represent down-regulated genes, the red dots represent up-regulated genes and the black dots represent unchanged genes.

FIGURE 2

Venn diagram of DGEs in different treatments. Venn diagram represents the number of DGEs and overlap between comparison groups. The sum of the numbers in each large circle represents the total number of differential genes in the comparison. The overlapped part of the circle represents the common differential genes between the combinations.

FIGURE 3

Analysis of co-expressed patterns of HL treatments. (A) Subcluster_01. (B) Subcluster_05. (C) Subcluster_08. (D) Subcluster_09. The abscissa represents different sample, the vertical axis represents centered log2 (fpkm + 1). Four clusters were identified based on expression levels in seven different samples. T04–T05 are the two biological duplicates of the original strain PA1.2620; T07–T08 are the two biological duplicates of the PA-60 strain; T09–T11 are the three biological duplicates of PA-99 strain.

FIGURE 4

GO and KEGG pathway enrichment analysis of DEGs between HL treatments. (A) GO enrichment. The bigger the value of –log10 (p-value), the more enriched. p-value < 0.05 is the significant difference, and p-value < 0.01 is the most significant difference. The ordinate is “GO term.” (B) KEGG pathway enrichment. Enrichment factor = Amount of all genes/Amount of DEGs enriched in the pathway in the background gene set. The smaller the enrichment factor, the more significant. The same below.

FIGURE 5

Annotation and analysis of co-expressed resistant genes between HL. (A) AMR genes. ARO represents antibiotic resistance ontology. The outer ring represents antibiotic resistance genes in the figure. (B) AMR gene family. The inner ring represents the anti-microbial resistance (AMR) gene families and the outer ring represents hits the numbers of resistance genes predicted.

FIGURE 6

GO enrichment and KEGG pathway analysis of co-expressed sRNA target genes. (A) GO enrichment. (B) KEGG pathway enrichment.